NZ619314B2 - Culture media for stem cells - Google Patents
Culture media for stem cells Download PDFInfo
- Publication number
- NZ619314B2 NZ619314B2 NZ619314A NZ61931412A NZ619314B2 NZ 619314 B2 NZ619314 B2 NZ 619314B2 NZ 619314 A NZ619314 A NZ 619314A NZ 61931412 A NZ61931412 A NZ 61931412A NZ 619314 B2 NZ619314 B2 NZ 619314B2
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- New Zealand
- Prior art keywords
- cells
- culture medium
- organoid
- inhibitor
- medium
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Abstract
Discloses a culture medium for expanding or differentiating a population of adult stem cells, wherein said culture medium comprises: i. an agonist of Lgr5; and ii. one or more TGF-beta inhibitor, wherein the inhibitor is a TGF-beta inhibitor if it can inhibit TGF-beta signalling in a cellular assay in which cells are stably transfected with a reporter construct comprising the human plasminogen activator inhibitor-1 (PAI-1) promoter. y in which cells are stably transfected with a reporter construct comprising the human plasminogen activator inhibitor-1 (PAI-1) promoter.
Description
CULTURE MEDIA FOR STEM CELLS
All documents cited herein are incorporated by reference in their entirety.
TECHNICAL FIELD
The invention is generally in the field of stem cell culture media and methods, in particular culture
media and methods for expanding populations of stem cells, e.g. human epithelial stem cells.
BACKGROUND
There is great interest in culture media and methods for expanding populations of stem cells.
Populations of stem cells have many uses. For example, stem cells and their differentiated progeny
can be used in cellular assays, drug screening, and toxicity assays. Stem cells also show promise
for cell-based therapies, such as in regenerative medicine for the treatment of damaged tissue.
They can also act as a source of differentiated cells for transplantation purposes e.g. transplantation
of pancreatic beta-cells for treatment of diabetes etc. Furthermore, efficient cell culture media are
important for providing and maintaining populations of cells for research purposes.
There is also interest in culture media and methods for culturing stem cells for the formation,
maintenance and expansion of organoids, such as intestinal crypt-villus, gastric or pancreatic
organoids. An organoid comprises stem cells, such as epithelial stem cells, which retain their
undifferentiated phenotype and self-renewal properties but also have differentiating progeny that
grow into tissue-like structures. Similarly to populations of related or identical cells, crypt-villus,
gastric or pancreatic organoids, which more closely mimic the basic physiology of their tissue of
origin, may be used in toxicity assays, or assays for drugs or food supplements. They may also be
useful for culturing pathogens which currently lack suitable tissue culture or animal models.
Furthermore, such organoids may be useful in regenerative medicine, for example in post-radiation
and/or post-surgery repair of the intestinal epithelium, or in the repair of the intestinal epithelium
in patients suffering from inflammatory bowel disease.
It is clear that there are many clinical and research applications for stem cells and their
differentiated progeny. For all these applications, reproducible stem cell culture methods are of the
utmost importance for providing adequate numbers of cells of suitable quality. For example, for
effective drug screening, conditions must be carefully controlled requiring precise culture methods
for controlling differentiation and proliferation of cells, so that pure populations of phenotypically
and karyotypically identical cells can be generated. Similarly, for cell-based therapies, wherein
cultured cells may be directly provided to patients, the cells must be genetically and
phenotypically sound so as to avoid undesirable immune responses or cell fates when provided to
the patient.
Although a variety of culture systems have been described for culturing primary epithelial stem
cells, including intestinal epithelial stem cells (Bjerknes and Cheng, 2006. Methods Enzymol. 419:
337-83), to date, no long-term culture system has been established which maintains the
differentiation potential and phenotypic and genomic integrity of human epithelial stem cells.
International patent application WO2010/090513 discloses a method for culturing epithelial stem
cells or isolated tissue fragments. The method is optimised for the culturing of human colon and
intestinal crypts by the addition of Wnt-3a to the medium. This was the first time that human
intestinal stem cell cultures had been cultured for a prolonged period of time (up to 3 months) and
provided the first reproducible human intestinal stem cell culture system. However, there is still a
need for improved stem cell culture media and methods, in particular human stem cell culture
media and methods, that improve proliferation rates, survival time and phenotypic and genomic
integrity of stem cells grown in culture.
SUMMARY OF THE INVENTION
The invention provides improved culture media and methods for stem cells, in particular human
epithelial stem cells, and organoids comprising said stem cells, which provide significant
advantages over known culture media and methods. The invention also provides related culture
medium supplements, compositions and uses.
Accordingly, in one aspect the present invention relates to a culture medium for expanding or
differentiating a population of adult stem cells, wherein said culture medium comprises:
i. an agonist of Lgr5; and
ii. one or more TGF-beta inhibitor, wherein the inhibitor is a TGF-beta inhibitor if it can
inhibit TGF-beta signalling in a cellular assay in which cells are stably transfected with a reporter
construct comprising the human plasminogen activator inhibitor-1 (PAI-1) promoter.
In another aspect the invention relates to a composition comprising a culture medium of the
invention and an extracellular matrix or a 3D matrix that mimics the extracellular matrix by its
interaction with the cellular membrane proteins.
In another aspect the invention relates to a hermetically-sealed vessel containing a culture medium
or composition of the invention.
In another aspect the invention relates to the use of a culture medium of the invention for
expanding or differentiating a stem cell, population of stem cells, tissue fragment or organoid.
In another aspect the invention relates to a method for expanding a single adult stem cell, a
population of adult stem cells or a tissue fragment, wherein the method comprises culturing the
single stem cell or population of stem cells in a culture medium of the invention.
In another aspect the invention relates to an organoid or population of cells obtained by the method
of the invention.
In another aspect the invention relates to a composition comprising:
i) one or more organoids or population of cells of the invention; and
ii) a culture medium of the invention and/or an extracellular matrix.
In another aspect the invention relates to an organoid of the invention or a population of cells of
the invention or a composition of the invention for use in drug screening, target validation, target
discovery, toxicology, toxicology screens, personalized medicine, regenerative medicine or ex vivo
cell/organ models.
In another aspect the invention relates to the use of an organoid of the invention or a population of
cells of the invention as a disease model.
In another aspect the invention relates to a method for screening for a therapeutic or prophylactic
drug or cosmetic, wherein the method comprises: culturing an organoid or population of cells of
the invention; exposing said organoid or population of cells to one or a library of candidate
molecules; evaluating said organoid or population of cells for any effects; and identifying the
candidate molecule that causes said effects as a potential drug or cosmetic.
Certain statements that appear below are broader than what appears in the statements of the
invention above. These statements are provided in the interests of providing the reader with a
better understanding of the invention and its practice. The reader is directed to the accompanying
claim set which defines the scope of the invention.
Described herein is a culture medium for expanding a population of stem cells, wherein the culture
medium comprises at least one or more inhibitors that bind to and reduce the activity of one or
more serine/threonine protein kinase targets. This has the effect of allowing continual growth for
at least 3 months at an expansion rate of approximately five-fold expansion per week. The
serine/threonine protein kinase is preferably selected from the group comprising: TGFbeta receptor
kinase 1, ALK4, ALK5, ALK7, p38. Surprisingly, the inventors have found that the inclusion of
inhibitors of certain serine/threonine kinases in culture media significantly improved the
performance of the culture media in expanding a population of stem cells. The population of stem
cells may be normal (healthy) cells or diseased cells (for example, cancer stem cells). Specifically,
inhibitors of p38 and ALK were shown to provide the greatest improvement out of all the
compounds tested. This is unexpected because there is no known mechanism predicting how these
particular inhibitors might work. Indeed, several of the small molecule inhibitors that were chosen
to be tested and function in similar pathways, had no effect on the method. Therefore, the skilled
person could not have predicted that inhibitors of these particular kinases would have such a
marked improvement on the culture medium. A still further improvement was observed when two
inhibitors, for example a p38 inhibitor, such as SB202190 and an ALK inhibitor, such as A83-01,
were added to the culture medium together.
To arrive at this realisation, the inventors investigated signalling pathways that are known to be
subverted in certain cancers e.g. colorectal cancer. They hypothesised that these pathways, which
affect cell fate in cancer, may also play a role in determining cell fate in culture conditions. It
should be emphasised, however, that this hypothesis was entirely new; given the state of the art,
there was no way to predict the effect of any of these additional compounds on the culture
medium, and no particular expectation that any of these compounds might in fact have a beneficial
effect.
In a first screening experiment, a series of vitamins, hormones and growth factors were tested in
combination with standard stem cell culture media. Gastrin and nicotinamide were initially
identified as resulting in significantly improved culture conditions. Incorporating these factors into
the standard culture conditions, a second screening experiment was performed, in which small
molecule inhibitors related to relevant signalling pathways, such as ERK, p38, JNK, PTEN,
ROCK, and Hedgehog, were tested. These pathways were chosen because they were known to be
subverted in certain cancers.
Previous attempts to culture human intestinal stem cells with previously described stem cell
culture medium (comprising Epidermal Growth Factor (EGF or (“E”), Noggin (“N”) and R-
spondin (“R”), referred to herein as “ENR” medium) optimised with Wnt-3A (“W”) (referred to
herein as “WENR” medium), have resulted in the disintegration of most cells within 7 days, with
very few cells surviving beyond 1 month. Such attempts have also been subject to slow
proliferation times, chromosome irregularities and morphological changes from budding to cystic
structures. By “cystic” it is meant that the organoid is mostly spherical. By “budding” it is meant
that the organoid has multiple regions growing out of the basic structure. It is not necessarily
always an advantage to have budding structures, although budding structures typically have a
larger surface area and typically resemble the corresponding in vivo tissue more closely.
The inventors showed that the improved method allowed continual growth of the stem cells for at
least seven months.
The new method also increased the speed of proliferation of the cells in the expanded population.
This is clearly of great utility when growing cells for commercial and therapeutic purposes.
The new method also increased the quality of the cells in the expanded population. This is a great
advantage because clinical and research applications for stem cells and their differentiated progeny
require reproducible stem cell culture methods that provide populations of cells of high quality.
Generally, in vitro expansion of stem cells aims to provide a population of cells which resemble
their in vivo counterparts as closely as possible. This property is herein referred to as the “genomic
and phenotypic integrity” of the cells.
For the first time, the inventors have discovered that it is possible to expand human epithelial stem
cells in culture, without loss of genomic and phenotypic integrity, for at least 7 months (see
Example 1). Under the improved culture conditions described herein, human intestinal organoids
displayed budding organoid structures, rather than the cystic structures seen under previous culture
conditions. Metaphase spreads of organoids more than 3 months old consistently revealed 46
chromosomes in each of the 20 cells taken from three different donors. Furthermore, microarray
analysis revealed that the stem cells in culture possessed similar molecular signatures to intestinal
crypt cells including intestinal stem cell genes.
The inventors also demonstrated that the human intestinal organoids generated by media and
methods of the present invention, mimicked in vivo cell fate decisions in response to external
factors. For example, it has previously been shown that Notch inhibition in intestinal stem cells,
terminates intestinal epithelial proliferation and induces goblet cell hyperplasia in vivo. The
inventors were able to show that the intestinal organoids described herein, when treated with a
Notch inhibitor, ceased proliferation and most cells converted into goblet cells within 3 days.
Similar advantages were observed when including a TGF-beta inhibitor and/or a p38 inhibitor in
culture media for expanding stem cells or organoids from other epithelial tissues, such as stomach,
pancreas, liver and prostate (see the Examples). The tissues may be normal (healthy) tissues or
diseased tissues, for example cancerous tissues or tissues showing a cystic fibrosis phenotype.
These results show the dramatic improvement in the genomic and phenotypic integrity of the stem
cells and organoids produced by the methods and media of the present invention compared to
previous methods and media.
Thus, described herein is a culture medium for expanding and/or differentiating a population of
adult stem cells, wherein said culture medium comprises:
i. any one of Rspondin 1-4 and/or an Rspondin mimic; and
ii. one or more inhibitor that directly or indirectly negatively regulates TGF-beta
signalling.
The invention also provides a composition comprising a culture medium according to the
invention and an extracellular matrix or a 3D matrix that mimics the extracellular matrix by its
interaction with the cellular membrane proteins such as integrins, for example, a laminin-
containing extracellular matrix such as Matrigel (BD Biosciences).
The invention also provides a hermetically-sealed vessel containing a culture medium or
composition according to the invention.
The invention also provides the use of a culture medium according to the invention for expanding
and/or differentiating a stem cell, population of stem cells, tissue fragment or organoid.
The invention also provides methods for expanding a single stem cell, a population of stem cells or
a tissue fragment, preferably to generate an organoid, wherein the method comprises culturing the
single stem cell or population of stem cells in a culture medium according to the invention.
Also described are organoids or populations of cells obtainable by the methods of the invention.
Also described is an organoid, preferably obtainable by the methods of the invention, which is a
three-dimensional organoid comprising epithelial cells surrounding a central lumen, wherein
optionally the epithelial cells exist in distinct dividing domains and differentiating domains.
Also described is an organoid, preferably obtainable by the methods of the invention, which is a
three-dimensional organoid comprising epithelial cells arranged in regions of monolayers,
optionally folded monolayers and regions of stratified cells, and preferably which is a three-
dimensional organoid comprising epithelial cells surrounding a central lumen, wherein optionally
the epithelial cells exist in distinct dividing domains and differentiating domains.
The invention also provides a composition comprising:
i) one or more organoids or population of cells of the invention; and
ii) a culture medium of the invention and/or an extracellular matrix.
The invention also provides an organoid, a population of cells or a composition according to the
invention for use in drug screening, target validation, target discovery, toxicology, toxicology
screens, personalized medicine, regenerative medicine or ex vivo cell/organ models, for example
for use as a disease model.
Also described is an organoid, a population of cells or a composition according to the invention,
for use in transplantation of said organoid, population of cells or composition into a mammal,
preferably into a human.
Also described is a population of stem cells, or organoids comprising said stem cells, that have
been obtained or are obtainable using the culture medium of the invention. The stem cells or
organoids comprising said stem cells may be used, for example, for transplantation purposes or
other therapeutic applications. For example,the stem cells or organoids comprising said stem cells
may be used for drug screening, target validation, target discovery, toxicology and toxicology
screens, personalized medicine, regenerative medicine and ex vivo cell/organ models, for example
disease models.
Also described are compositions comprising a culture medium of the invention.
Also described are culture medium supplements comprising an inhibitor as described.
Also described is a hermetically-sealed vessel comprising a culture medium and/or a culture
medium supplement as described.
The specific ingredients of the culture media, supplements and compositions described herien can
be varied according to particular needs and applications. Likewise, the precise steps of the methods
of the invention can vary according to particular needs and applications.
The culture media, supplements, methods, compositions and uses as described may also be
optimised by routine experimentation. For example, if a culture medium, supplement or
composition fails to give the desired level of stem cell expansion, variables such as the amount of
each ingredient in the culture medium or supplement, seeding densities, culture conditions, culture
periods, etc. can be altered in further experiments. The amount of each of the ingredients described
herein can be optimised independently of the other ingredients by routine optimisation or one or
more ingredients can be added or removed. A culture medium can be tested for its ability to
support expansion of stem cells by testing it alongside or in place of a known culture medium or
method.
The culture media, supplements, methods, compositions and uses as described are described in
more detail below. The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of cell culture, molecular biology and microbiology, which are within the
skill of those working in the art.
Numerous textbooks are available that provide guidance on mammalian cell culture media and
methods, including textbooks dedicated to culture media and methods for culturing stem cells.
Such textbooks include ‘Basic Cell Culture Protocols’ by J. Pollard and J. M. Walker (1997),
‘Mammalian Cell Culture: Essential Techniques’ by A. Doyle and J. B. Griffiths (1997), ‘Culture
of Animal Cells: A Manual of Basic Technique’ by R. I. Freshney (2005), ‘Basic Cell Culture
Protocols’ by C. Helgason and C. L. Miller (2005), ‘Stem Cells: From Bench to Bedside’ by A.
Bongso (2005), ‘Human Stem Cell Manual: A Laboratory Guide’ by J. F. Loring, R. L.
Wesselschmidt and P. H. Schwartz (2007).
Stem cells and cell culture reagents and apparatus for use in the invention are available
commercially, e.g. from Cellartis AB (Göteborg, Sweden), VitroLife AB (Kungsbacka, Sweden),
GIBCO (Invitrogen), Millipore Corporation (Billerica, Massachusetts), Sigma (St. Louis,
Missouri) and Biomol International L.P. (Exeter, UK).
DETAILED DESCRIPTION
Disclosed herein is a culture medium for expanding a population of stem cells, wherein the culture
medium comprises at least one or more inhibitors that bind to and reduce the activity of one or
more serine/threonine protein kinase targets, wherein the culture medium has the effect of allowing
continual growth of the population of stem cells for at least 3 months, preferably at least 4 months,
at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months or
more.
Inhibitors
A culture medium used according to one aspect of the invention comprises any inhibitor that,
directly or indirectly, negatively regulates TGF-beta or p38 signalling. In a preferred embodiment
the culture medium of the invention comprises an inhibitor that directly or indirectly negatively
regulates TGF-beta signalling. In some embodiments the culture medium of the invention
comprises an inhibitor that directly or indirectly negatively regulates TGF-beta and an inhibitor
that directly or indirectly negatively regulates p38 signalling. In a further embodiment, the culture
medium of the invention additionally comprises Rspondin or an Rspondin mimic.
The one or more inhibitor preferably targets a serine/threonine protein kinase selected from the
group comprising: TGF-beta receptor kinase 1, ALK4, ALK5, ALK7, p38. An inhibitor of any one
of these kinases is one that effects a reduction in the enzymatic activity of any one (or more) of
these molecules. Inhibition of ALK and p38 kinase has previously been shown to be linked in B-
cell lymphoma (Bakkebø M Huse K, Hilden VI, Smeland EB, Oksvold MP, “TGF-beta-induced
growth inhibition in B-cell lymphoma correlates with Smad1/5 signalling and constitutively active
p38 MAPK”, BMC Immunol. 11:57, 2010). In this publication, it was found that TGF-beta
sensitive cell lines expressed higher cell surface levels of ALK-5 and that constitutive
phosphorylation of p38 was restricted to the TGF-beta sensitive cell lines. Inhibition of p38
MAPK led to reduced sensitivity to TGF-beta suggesting that phosphorylation of Smad1/5 is
important for the anti-proliferative effects of TGF-beta in B-cell lymphoma. The results indicate a
role for p38 MAPK in the regulation of TGF-beta-induced anti-proliferative effects.
Without wishing to be bound by theory, the present inventors propose that ALK and p38 belong to
a pathway that negatively regulates long-term maintenance of stem cells, in particular, human
epithelial stem cells. The inventors hypothesise that inhibitors that act at any level on this pathway,
including, for example, by inhibiting Smad1/5 signalling, would also be beneficial for stem cell
culture. Smads play a key role in TGF-beta signalling.
In some embodiments an inhibitor of the invention binds to and reduces the activity
serine/threonine protein kinase selected from the group comprising: TGF-beta receptor kinase 1,
ALK4, ALK5, ALK7, p38.
In some embodiments of the invention, the culture medium comprises a TGF-beta inhibitor,
meaning any inhibitor that, directly or indirectly, negatively regulates TGF-beta signalling. In
some embodiments, a culture medium of the invention comprises one or more TGF-beta inhibitor
that binds to and reduces the activity of one or more serine/threonine protein kinases selected from
the group consisting of ALK5, ALK4, TGF-beta receptor kinase 1 and ALK7.
ALK4, ALK5 and ALK7 are all closely related receptors of the TGF-beta superfamily. ALK4 has
GI number 91; ALK5 (also known as TGF-beta receptor kinase 1) has GI number 7046; and ALK7
has GI number 658. In one embodiment, an inhibitor as described herein binds to and reduces the
activity of ALK4, ALK5 (TGF-beta receptor kinase 1) and/or ALK7. In another embodiment, the
TGF-beta receptor binds to and reduces the activity of a Smad protein, for example R-SMAD or
SMAD1-5 (i.e. SMAD 1, SMAD 2, SMAD 3, SMAD 4 or SMAD 5). In a preferred embodiment,
the culture medium of the invention comprises an inhibitor of ALK5.
Various methods for determining if a substance is a TGF-beta inhibitor are known. For example, a
cellular assay may be used, in which cells are stably transfected with a reporter construct
comprising the human PAI-1 promoter or Smad binding sites, driving a luciferase reporter gene.
Inhibition of luciferase activity relative to control groups can be used as a measure of compound
activity (De Gouville et al., Br J Pharmacol. 2005 May; 145(2): 166–177). Another example is the
AlphaScreen® phosphosensor assay for measurement of kinase activity (Drew A E et al.,
Comparison of 2 Cell-Based Phosphoprotein Assays to Support Screening and Development of an
ALK Inhibitor J Biomol Screen. 16(2) 164-173, 2011).
Various TGF-beta inhibitors are known in the art (for example, see Table 1). In some
embodiments the inhibitor that directly or indirectly negatively regulates TGF-beta signalling is
selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-
36494 and SJN-2511.
In some embodiments of the invention, the culture medium comprises a p38 inhibitor, meaning
any inhibitor that, directly or indirectly, negatively regulates p38 signalling. In some embodiments,
an inhibitor as described herein binds to and reduces the activity of p38 (GI number 1432). p38
protein kinases are part of the family of mitogen-activated protein kinases (MAPKs). MAPKs are
serine/threonine-specific protein kinases that respond to extracellular stimuli, such as
environmental stress and inflammatory cytokines, and regulate various cellular activities, such as
gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis. The p38
MAPKs exist as α, β, β2, γ and δ isoforms. A p38 inhibitor is an agent that binds to and reduces
the activity of at least one p38 isoform. Various methods for determining if a substance is a p38
inhibitor are known, and might be used in conjunction with the invention. Examples include:
phospho-specific antibody detection of phosphorylation at Thr180/Tyr182, which provides a well-
established measure of cellular p38 activation or inhibition; biochemical recombinant kinase
assays; tumor necrosis factor alpha (TNFα) secretion assays; and DiscoverRx high throughput
screening platform for p38 inhbitors (see
http://www.discoverx.com/kinases/literature/biochemical/collaterals/DRx_poster_p38%20KBA.pd
f). Several p38 activity assay kits also exist (e.g. Millipore, Sigma-Aldrich).
The inventors hypothesise that in some embodiments, high concentrations (e.g. more than 100 nM,
or more than 1uM, more than 10 uM, or more than 100 uM) of a p38 inhibitor may have the effect
of inhibiting TGF-beta. However, the inventors do not wish to be constrained by this hypothethis
and in other emobodiments, the p38 inhibitor does not inhibit TGF-beta signalling.
Various p38 inhibitors are known in the art (for example, see Table 1). In some embodiments, the
inhibitor that directly or indirectly negatively regulates p38 signalling is selected from the group
consisting of SB-202190, SB-203580, VX-702, VX-745, PD-169316, RO-4402257 and BIRB-
796.In a further embodiment, the culture medium comprises both: a) an inhibitor that binds to and
reduces the activity of any one or more of the kinases from the group consisting of: ALK4, ALK5
and ALK7; and b) an inhibitor that binds to and reduces the activity of p38. In a preferred
embodiment, the culture medium comprises an inhibitor that binds to and reduces the activity of
ALK5 and an inhibitor that binds to and reduces the activity of p38.
In one embodiment, an inhibitor as described herein binds to and reduces the activity of its target
(for example, TGF-beta or p38) by more than 10%; more than 30%; more than 60%; more than
80%; more than 90%; more than 95%; or more than 99% compared to a control, as assessed by a
cellular assay. Examples of cellular assays for measuring target inhibition are well known in the art
as described above.
An inhibitor as described herein may have an IC50 value equal to or less than 2000nM; less than
1000nM; less than 100nM; less than 50nM; less than 30nM; less than 20nM or less than 10nM.
The IC50 value refers to the effectiveness of an inhibitor in inhibiting its target’s biological or
b b bi i io o oc c ch h he e em m mi i ic c ca a al l l f f fu u un n nc c ct t ti i io o on n n... T T Th h he e e IC IC IC5 5 50 0 0 i i in n nd d di i ic c ca a at t te e es s s h h ho o ow w w m m mu u uc c ch h h o o of f f a a a p p pa a ar r rt t ti i ic c cu u ul l la a ar r r i i in n nh h hi i ib b bi i it t to o or r r i i is s s r r re e eq q qu u ui i ir r re e ed d d t t to o o i i in n nh h hi i ib b bi i it t t a
k k ki i in n na a as s se e e b b by y y 5 5 50 0 0% % %... IC IC IC5 5 50 0 0 v v va a al l lu u ue e es s s c c ca a an n n b b be e e c c ca a al l lc c cu u ul l la a at t te e ed d d i i in n n a a ac c cc c co o or r rd d da a an n nc c ce e e w w wi i it t th h h t t th h he e e a a as s ss s sa a ay y y m m me e et t th h ho o od d ds s s s s se e et t t o o ou u ut t t a a ab b bo o ov v ve e e...
An inhibitor a as s d de es sc cr ri ib be ed d h he er re ei in n may act competitively, non-c co om mp pe et ti it ti iv ve el ly y,, u un nc co om mp pe et ti it ti iv ve el ly y o or r b by y
m mi ix xe ed d i in nh hi ib bi it ti io on n.. Fo For r e ex xa am mp pl le e,, i in n c ce er rt ta ai in n e em mb bo od di im me en nt ts s,, a an n i in nh hi ib bi it to or r m ma ay y b be e a a c co om mp pe et ti it ti iv ve e i in nh hi ib bi it to or r
o of f t th he e A AT TP P b bi in nd di in ng g p po oc ck ke et t o of f t th he e t ta ar rg ge et t k ki in na as se e..
Inhibitors a as s d de es sc cr ri ib be ed d h he er re ei in n m ma ay y e ex xi is st t i in n v va ar ri io ou us s f fo or rm ms s,, i in nc cl lu ud di in ng g n na at tu ur ra al l o or r m mo od di if fi ie ed d s su ub bs st tr ra at te es s,,
enzymes, recept t to o or r rs s s,,, s s sm m ma a al l ll l l o o or r rg g ga a an n ni i ic c c m m mo o ol l le e ec c cu u ul l le e es s s,,, s s su u uc c ch h h a a as s s s s sm m ma a al l ll l l n n na a at t tu u ur r ra a al l l o o or r r s s sy y yn n nt t th h he e et t ti i ic c c o o or r rg g ga a an n ni i ic c c m m mo o ol l le e ec c cu u ul l le e es s s
o o of f f u u up p p t t to o o 2 2 20 0 00 0 00 0 0D D Da a a,,, p p pr r re e ef f fe e er r ra a ab b bl l ly y y 8 8 80 0 00 0 0D D Da a a o o or r r l l le e es s ss s s,,, p p pe e ep p pt t ti i id d do o om m mi i im m me e et t ti i ic c cs s s,,, i i in n no o or r rg g ga a an n ni i ic c c m m mo o ol l le e ec c cu u ul l le e es s s,,, p p pe e ep p pt t ti i id d de e es s s,,,
p p po o ol l ly y yp p pe e ep p pt t ti i id d de e es s s,,, a a an n nt t ti i is s se e en n ns s se e e o o ol l li i ig g go o on n nu u uc c cl l le e eo o ot t ti i id d de e es s s a a ap p pt t ta a am m me e er r rs s s,,, a a an n nd d d s s st t tr r ru u uc c ct t tu u ur r ra a al l l o o or r r f f fu u un n nc c ct t ti i io o on n na a al l l m m mimetics of these
i in nc cl lu ud di in ng g s sm ma al ll l m mo ol le ec cu ul le es s.. T Th he e i in nh hi ib bi it to or r as described herein m ma ay y a al ls so o b be e a an n a ap pt ta am me er r.. A As s u us se ed d
h h he e er r re e ei i in n n,,, t t th h he e e t t te e er r rm m m “ “ “a a ap p pt t ta a am m me e er r r” ” ” r r re e ef f fe e er r rs s s t t to o o s s st t tr r ra a an n nd d ds s s o o of f f o o ol l li i ig g go o on n nu u uc c cl l le e eo o ot t ti i id d de e es s s ( ( (D D DN N NA A A o o or r r R R RN N NA A A) ) ) t t th h ha a at t t c c ca a an n n a a ad d do o op p pt t t
highly specific three-dime e en n ns s si i io o on n na a al l l c c co o on n nf f fo o or r rm m ma a at t ti i io o on n ns s s... A A Ap p pt t ta a am m me e er r rs s s a a ar r re e e d d de e es s si i ig g gn n ne e ed d d t t to o o h h ha a av v ve e e h h hi i ig g gh h h b b bi i in n nd d di i in n ng g g
a a af f ff f fi i in n ni i it t ti i ie e es s s a a an n nd d d s s sp p pe e ec c ci i if f fi i ic c ci i it t ti i ie e es s s t t to o ow w wa a ar r rd d ds s s c c ce e er r rt t ta a ai i in n n t t ta a ar r rg g ge e et t t m m mo o ol l le e ec c cu u ul l le e es s s,,, i i in n nc c cl l lu u ud d di i in n ng g g e e ex x xt t tr r ra a ac c ce e el l ll l lu u ul l la a ar r r a a an n nd d d
intracellular proteins.
Fo For r e ex xa am mp pl le e,, t th he e i in nh hi ib bi it to or r m ma ay y b be e a a s sm ma al ll l s sy yn nt th he et ti ic c m mo ol le ec cu ul le e w wi it th h a a m mo ol le ec cu ul la ar r w we ei ig gh ht t o of f b be et tw we ee en n
5 50 0 0 a a an n nd d d 8 8 80 0 00 0 0 D D Da a a,,, b b be e et t tw w we e ee e en n n 8 8 80 0 0 a a an n nd d d 7 7 70 0 00 0 0 D D Da a a,,, b b be e et t tw w we e ee e en n n 1 1 10 0 00 0 0 a a an n nd d d 6 6 60 0 00 0 0 D D Da a a o o or r r b b be e et t tw w we e ee e en n n 1 1 15 5 50 0 0 a a an n nd d d 5 5 50 0 00 0 0 D D Da a a...
In In s so om me e e em mb bo od di im me en nt ts s,, t th he e s sm ma al ll l-m mo ol le ec cu ul le e i in nh hi ib bi it to or r c co om mp pr ri is se es s a a p py yr ri id di in ny yl li im mi id da azo zol le e o or r a a 2 2,,4 4-
d di is su ub bs st ti it tu ut te ed d p pt te er ri id di in ne e o or r a a q qu ui in na azo zol li in ne e,, f fo or r e ex xa am mp pl le e c comprises:
OR OR
Pa Pa Par r rt t ti i ic c cu u ul l la a ar r r e e ex x xa a am m mp p pl l le e es s s o o of f f i i in n nh h hi i ib b bi i it t to o or r rs s s t t th h ha a at t t m m ma a ay y y b b be e e u u us s se e ed d d i i in n n a a ac c cc c co o or r rd d da a an n nc c ce e e w w wi i it t th h h t t th h he e e i i in n nv v ve e en n nt t ti i io o on n n i i in n nc c cl l lu u ud d de e e,,, b b bu u ut t t
are not limited to: SB-2 20 02 21 19 90 0,, SB SB-203580, SB-206718, SB-227931, VX-7 70 02 2,, V VX X-745, PD-
169316, RO-4402257, BIRB- -796, A83-01 SB-431542, SB-505124, SB-5 52 25 53 33 34 4,, L LY Y 3 36 64 4947, SD-
2 2 20 0 08 8 8,,, S S SJ J JN N N 2 2 25 5 51 1 11 1 1 ( ( (s s se e ee e e t t ta a ab b bl l le e e 1 1 1) ) )... A A A c c cu u ul l lt t tu u ur r re e e m m me e ed d di i iu u um m m o o of f f t t th h he e e i i in n nv v ve e en n nt t ti i io o on n n m m ma a ay y y c c co o om m mp p pr r ri i is s se e e o o on n ne e e o o or r r m m mo o or r re e e o o of f f a a an n ny y y
o o of f f t t th h he e e i i in n nh h hi i ib b bi i it t to o or r rs s s l l li i is s st t te e ed d d i i in n n t t ta a ab b bl l le e e 1 1 1... A A A c c cu u ul l lt t tu u ur r re e e m m me e ed d di i iu u um m m o o of f f t t th h he e e i i in n nv v ve e en n nt t ti i io o on n n m m ma a ay y y c c co o om m mp p pr r ri i is s se e e a a an n ny y y
c co om mb bi in na at ti io on n o of f o on ne e i in nh hi ib bi it to or r w wi it th h a an no ot th he er r i in nh hi ib bi it to or r l li is st te ed d.. Fo For r e ex xa am mp pl le e,, a a c cu ul lt tu ur re e m me ed di iu um m o of f t th he e
invention may comprise SB-2 20 02 21 19 90 0 o or r SB SB-203580 or A83-0 01 1; ; o or r a a c cu ul lt tu ur re e m me ed di iu um m o of f t th he e
invention may comprise SB-2 20 02 21 19 90 0 a an nd d A A8 83 3-0 01 1; ; o or r a a c cu ul lt tu ur re e m me ed di iu um m o of f t th he e i in nv ve en nt ti io on n m ma ay y
comprise SB-2 20 03 35 58 80 0 a an nd d A A8 83 3-01. The skilled person will l a ap pp pr re ec ci ia at te e t th ha at t o ot th he er r i in nh hi ib bi it to or rs s a an nd d
c c co o om m mb b bi i in n na a at t ti i io o on n ns s s o o of f f i i in n nh h hi i ib b bi i it t to o or r rs s s wh wh whi i ic c ch h h b b bi i in n nd d d t t to o o a a an n nd d d r r re e ed d du u uc c ce e e t t th h he e e a a ac c ct t ti i iv v vi i it t ty y y o o of f f t t th h he e e t t ta a ar r rg g ge e et t ts s s a a ac c cc c co o or r rd d di i in n ng g g t t to o o t t th h he e e
i i in n nv v ve e en n nt t ti i io o on n n,,, m m ma a ay y y b b be e e i i in n nc c cl l lu u ud d de e ed d d i i in n n a a a c c cu u ul l lt t tu u ur r re e e m m me e ed d di i iu u um m m o o or r r a a a c c cu u ul l lt t tu u ur r re e e m m me e ed d di i iu u um m m s s su u up p pp p pl l le e em m me e en n nt t t i i in n n a a ac c cc c co o or r rd d da a an n nc c ce e e
with the invention.
Inhibitors as described herein may be added to the culture medium to a final concentration that is
appropriate, taking into account the IC50 value of the inhibitor.
For example, SB-202190 may be added to the culture medium at a concentration of between 50
nM and 100 uM, or between 100 nM and 50 uM, or between 1 uM and 50 uM. For example, SB-
202190 may be added to the culture medium at approximately 10 uM.
SB-203580 may be added to the culture medium at a concentration of between 50 nM and 100 uM,
or between 100 nM and 50 uM, or between 1 uM and 50 uM. For example, SB-203580 may be
added to the culture medium at approximately 10 uM.
VX-702 may be added to the culture medium at a concentration of between 50 nM and 100 uM, or
between 100 nM and 50 uM, or between 1 uM and 25 uM. For example, VX-702 may be added to
the culture medium at approximately 5 uM.
VX-745 may be added to the culture medium at a concentration of between 10 nM and 50 uM, or
between 50 nM and 50 uM, or between 250 nM and 10 uM. For example, VX-745 may be added
to the culture medium at approximately 1 uM.
PD-169316 may be added to the culture medium at a concentration of between 100 nM and 200
uM, or between 200 nM and 100 uM, or between 1 uM and 50 uM. For example, PD-169316 may
be added to the culture medium at approximately 20 uM.
RO-4402257 may be added to the culture medium at a concentration of between 10 nM and 50
uM, or between 50 nM and 50 uM, or between 500 nM and 10 uM. For example, RO-4402257
may be added to the culture medium at approximately 1 uM.
BIRB-796 may be added to the culture medium at a concentration of between 10 nM and 50 uM,
or between 50 nM and 50 uM, or between 500 nM and 10 uM. For example, BIRB-796 may be
added to the culture medium at approximately 1 uM.
A83-01 may be added to the culture medium at a concentration of between 10 nM and 10 uM, or
between 20 nM and 5 uM, or between 50 nM and 1 uM. For example, A83-01 may be added to the
culture medium at approximately 500 nM.
SB-431542 may be added to the culture medium at a concentration of between 80 nM and 80 uM,
or between 100 nM and 40 uM, or between 500 nM and 10 uM. For example, SB-431542 may be
added to the culture medium at approximately 1 uM.
SB-505124 may be added to the culture medium at a concentration of between 40 nM and 40 uM,
or between 80 nM and 20 uM, or between 200 nM and 1 uM. For example, SB-505124 may be
added to the culture medium at approximately 500 nM.
SB-525334 may be added to the culture medium at a concentration of between 10 nM and 10 uM,
or between 20 nM and 5 uM, or between 50 nM and 1 uM. For example, SB-525334 may be added
to the culture medium at approximately 100 nM.
LY 36494 may be added to the culture medium at a concentration of between 40 nM and 40 uM,
or between 80 nM and 20 uM, or between 200 nM and 1 uM. For example, LY 36494 may be
added to the culture medium at approximately 500 nM.
Table 1: Exemplary inhibitors as described herein
IC50
Inhibitor Targets (nM) Mol Wt Name Formula
A83-01 ALK5 12 421.52 3-(6-Methyl C25H19N5S
(TGF-βR1) pyridinyl)-N-phenyl
ALK4 45 (4-quinolinyl)-1H-
pyrazole
ALK7 7.5
carbothioamide
ALK5 94 384.39 4-[4-(1,3-benzodioxol- C22H16N4O3
SB-431542
-yl)(2-pyridinyl)-
ALK4
1H-imidazol
ALK7
yl]benzamide
SB-505124 ALK5 47 335.4 2-(5-benzo[1,3]dioxol- C20H21N3O2
-yltert-butyl-
ALK4 129
3Himidazol-
4-yl)methylpyridine
hydrochloride hydrate
SB-525334 ALK5 14.3 343.42 6-[2-(1,1- C21H21N5
Dimethylethyl)(6-
methylpyridinyl)-
1H-imidazol
yl]quinoxaline
SD-208 ALK5 49 352.75 C17H10ClFN6
2-(5-Chloro
fluorophenyl)[(4-
pyridyl)amino]pteridine
LY-36494 TGR-βRI 59 272.31 4-[3-(2-Pyridinyl)-1H- C17H12N4
TGF-βRII 400 pyrazolyl]-quinoline
MLK-7K 1400
LY364947 ALK5 59 272.30 4-[3-(2-pyridinyl)-1H- C H N
17 12 4
pyrazolyl]-quinoline
SJN-2511 ALK5 23 287.32 2-(3-(6- C17H13N5
Methylpyridineyl)-
1H-pyrazolyl)-1,5-
naphthyridine
SB-202190 p38 MAP 38 331.35 4-[4-(4-Fluorophenyl)- C20H14N3OF
kinase 5-(4-pyridinyl)-1H-
imidazolyl]phenol
p38α 50
p38β 100
p38 50 4-[5-(4-Fluorophenyl)- C21H16FN3OS
SB-203580
2-[4-
p38β2 500 377.44
(methylsulfonyl)phenyl
]-1H-imidazol
yl]pyridine
VX-702 p38α 4-20; 6- C19H12F4N4O2
(Kd = [(Aminocarbonyl)(2,6-
3.7) difluorophenyl)amino]-
p38β Kd = 17 2-(2,4-difluorophenyl)-
404.32 3-pyridinecarboxamide
VX-745 p38α 10 436.26 5-(2,6-Dichlorophenyl)- C19H9Cl2F2N3
2-[2,4- OS
difluorophenyl)thio]-
6H-pyrimido[1,6-
b]pyridazinone
p38 89 360.3 4-[5-(4-fluorophenyl)- C20H13FN4O
PD-169316
2-(4-nitrophenyl)-1H-
imidazolyl]-pyridine
RO- p38α 14 Pyrido[2,3-d]pyrimidin-
7(8H)-one,6-(2,4-
4402257
difluorophenoxy)[[3-
hydroxy(2-
hydroxyethyl)propyl]a
mino]methyl-
p38β 480
BIRB-796 p38 4 527.67 1-[2-(4-methylphenyl)- C31H37N5O3
-tert-butyl-pyrazol
yl][4-(2-morpholin-
4-ylethoxy)naphthalen-
1-yl]urea::3-[2-(4-
methylphenyl)tert-
butyl-pyrazolyl]
[4-(2-morpholin
ylethoxy)naphthalen
yl]urea ::3-[3-tert-butyl-
1-(4-methylphenyl)-
1H-pyrazolyl]{4-
[2-(morpholin
yl)ethoxy]naphthalen-
1-yl}urea
SD-208 may be added to the culture medium at a concentration of between 40 nM and 40 uM, or
between 80 nM and 20 uM, or between 200 nM and 1 uM. For example, SD-208 may be added to
the culture medium at approximately 500 nM.
LY364947 may be added to the culture medium at a concentration of between 40 nM and 40 uM,
or between 80 nM and 20 uM, or between 200 nM and 1 uM. For example, LY364947 may be
added to the culture medium at approximately 500 nM.
SJN 2511 may be added to the culture medium at a concentration of between 20 nM and 20 uM, or
between 40 nM and 10 uM, or between 100 nM and 1 uM. For example, SJN 2511 may be added
to the culture medium at approximately 200 nM.
Thus, in some embodiments the inhibitor that directly or indirectly, negatively regulates TGF-beta
or p38 signalling is added to the culture medium at a concentration of between 1nM and 100 μM,
between 10 nM and 100 μM, between 100 nM and 10 μM, or approximately 1 μM, for example,
wherein the total concentration of the one or more inhibitor is between 10 nM and 100 μM,
between 100 nM and 10 μM, or approximately 1 μM.
Additionally to the inhibitor, cell culture media generally contain a number of components which
are necessary to support maintenance and/or expansion of the cultured cells. A cell culture medium
of the invention will therefore normally contain many other components in addition to an inhibitor
as described herein. Suitable combinations of components can readily be formulated by the skilled
person, taking into account the following disclosure. A culture medium according to the invention
will generally be a nutrient solution comprising standard cell culture components, such as amino
acids, vitamins, inorganic salts, a carbon energy source, and a buffer as described in more detail
below. Other standard cell culture components that may be included in the culture include
hormones, such as progesterone, proteins, such as albumin, catalase, insulin and transferrin. These
other standard cell culture components make up the “basal” culture medium.
A culture medium according to the invention may be generated by modification of an existing cell
culture medium. The skilled person will understand from common general knowledge the types of
culture media that might be used for stem cell culture. Potentially suitable cell culture media are
available commercially, and include, but are not limited to, Dulbecco's Modified Eagle Media
(DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow
Minimal Essential Medium (G-MEM), Basal Medium Eagle (BME), DMEM/Ham’s F12,
Advanced DMEM/Ham’s F12, Iscove’s Modified Dulbecco’s Media and Minimal Essential Media
(MEM), Ham's F-10, Ham’s F-12, Medium 199, and RPMI 1640 Media. Thus, in some
embodiments, one of these pre-existing cell culture media is used as the basal culture medium to
which is added the inhibitor that, directly or indirectly, negatively regulates TGF-beta or p38
signalling, and, optionally, to which is added one or more other components as described herein.
In some embodiments, the culture medium of the invention comprises one or more additional
components selected from: a BMP inhibitor, a Wnt agonist, a receptor tyrosine kinase ligand, a
Rock inhibitor, nicotinamide and gastrin. In some embodiments, the culture medium of the
invention comprises any one of Rspondin 1-4 and/or an Rspondin mimic, a TGF-beta inhibitor, a
BMP inhibitor (for example, Noggin) and a Wnt agonist (for example, Wnt(3a)).
In some embodiments, the culture medium of the invention comprises any one of Rspondin 1-4
and/or an Rspondin mimic, a BMP inhibitor (for example, Noggin), a TGF-beta inhibitor, a
receptor tyrosine kinase ligand (for example, EGF), Nicotinamide, a Wnt agonist (for example,
Wnt(3a)), and optionally one or more additional components selected from: a p38 inhibitor,
gastrin, FGF10, HGF and a Rock inhibitor. The optional additional components may be added for
optimisation of the culture medium for culturing cells originating from particular tissues, as
explained in more detail later on.
The culture media of the invention may comprise one or more bone morphogenetic protein (BMP)
inhibitor. BMP ligands signal as dimers by assembling a quadripartite transmembrane
serine/threonine kinase receptor complex consisting of two type I and two type II receptors.
Complex assembly initiates a phosphorylation cascade activating the BMP responsive Smads1/5/8
and resulting in changes in transcriptional activity. Advantageously, the present inventors show
that BMP inhibitors promote expression of Lgr5, and so the presence of a BMP inhibitor in a
culture medium of the invention will likely result in more proliferative organoids than if the BMP
inhibitor is absent (for example, see Example 3). Thus, BMP inhibitors are an advantageous
component of expansion media of the invention. Thus, the use of a BMP inhibitor is advantageous
in the use of an expansion medium when it is desirable to culture the cells for at least 3 months
(e.g. at least 4, 5, 6, 7, 8 or 9 months) without the cells differentiating.
Several classes of natural BMP-binding proteins are known, including Noggin (Peprotech),
Chordin and chordin-like proteins (R&D systems) comprising chordin domains, Follistatin and
follistatin-related protines (R&D systems) comprising a follistatin domain, DAN and DAN-like
proteins (R&D systems) comprising a DAN cystein-knot domain, sclerostin/ SOST (R&D
systems) and apha-2 macroglobulin (R&D systems). A BMP inhibitor is an agent that binds to a
BMP molecule to form a complex wherein the BMP activity is reduced, for example by preventing
or inhibiting the binding of the BMP molecule to a BMP receptor. Alternatively, the inhibitor may
be an agent that binds to a BMP receptor and prevents binding of a BMP ligand to the receptor, for
example, an antibody that binds the receptor. A BMP inhibitor may be a protein or small molecule
and may be naturally occurring, modified, and/or partially or entirely synthetic. A BMP inhibitor
of a culture medium of the invention may be Noggin, DAN, or DAN-like proteins including
Cerberus and Gremlin (R&D systems). These diffusible proteins are able to bind a BMP ligand
with varying degrees of affinity and inhibit their access to signalling receptors. A preferred BMP
inhibitor for use in a culture medium of the invention is Noggin. Noggin may be used at any
suitable concentration. In some embodiments, a basal medium of the culture medium of the
invention may comprise between about 10 ng/ml and about 100 ng/ml of Noggin. For example, a
culture medium may comprise at least 10 ng/ml of Noggin, at least 20 ng/ml of Noggin, at least 50
ng/ml of Noggin, at least 100 ng/ml of Noggin, approximately 100 ng/ml of Noggin or 100 ng/ml
of Noggin. In some embodiments, a culture medium may comprise less than 200 ng/ml of Noggin,
less than 150 ng/ml of Noggin, less than 100 ng/ml of Noggin, less than 75 ng/ml of Noggin, less
than 50 ng/ml of Noggin or less than 30 ng/ml of Noggin. The BMP inhibitor may be added to the
culture medium every second day during culturing, or every day during culturing, or every third
day, every fourth day, every fifth day or as required. BMP inhibitors are particularly advantageous
components of the expansion media, for example for expanding pancreas, small intestine, colon,
liver, prostate stem cells. However, Noggin has been shown to prevent some differentiation (for
example, see example 3). Therefore, in some embodiments a BMP inhibitor is excluded from a
differentiation medium of the invention.
In some embodiments, cells cultured with a BMP inhibitor have upregulated expression of Lgr5
compared to cells cultured without a BMP inhibitor. Therefore, addition of a BMP inhibitor
typically results in more proliferative organoids. This is surprising, because in the literature it is
described that BMP activity is useful for the differentiation of pancreatic cells into both the ductal
(see keratin7 and 19 expression) and endocrine cells. Thus, the skilled person would expect the
inclusion of a BMP inhibitor, such as Noggin, to decrease proliferation and to increase
differentiation. However, the inventors surprisingly found that the use of a BMP inhibitor was
advantageous because it resulted in more proliferative organoids and higher expression of
Lgr5.The culture media of the invention may comprise one or more Wnt agonist. The Wnt
signalling pathway is defined by a series of events that occur when a Wnt protein binds to a cell-
surface receptor of a Frizzled receptor family member. This results in the activation of Dishevelled
family proteins which inhibit a complex of proteins that includes axin, GSK-3, and the protein
APC to degrade intracellular beta-catenin. The resulting enriched nuclear beta-catenin enhances
transcription by TCF/LEF family transcription factors. A Wnt agonist is defined as an agent that
activates TCF/LEF-mediated transcription in a cell. Wnt agonists are therefore selected from true
Wnt agonists that bind and activate a Frizzled receptor family member including any and all of the
Wnt family proteins, an inhibitor of intracellular beta-catenin degradation, and activators of
TCF/LEF. Said Wnt agonist stimulates a Wnt activity in a cell by at least 10%, more preferred at
least 20%, more preferred at least 30%, more preferred at least 50%, more preferred at least 70%,
more preferred at least 90%, more preferred at least 100%, relative to a level of said Wnt activity
in the absence of said molecule. As is known to a skilled person, a Wnt activity can be determined
by measuring the transcriptional activity of Wnt, for example by pTOPFLASH and pFOPFLASH
Tcf luciferase reporter constructs (Korinek et al, 1997 Science 275 1784-1787).
In some embodiments, a Wnt agonist comprises a secreted glycoprotein including Wnt- l/Int-1,
Wnt- 2/Irp (InM -related Protein), Wnt-2b/13, Wnt-3/Int-4, Wnt-3a (R&D sytems), Wnt- 4, Wnt-
5a, Wnt-5b, Wnt-6 (Kirikoshi H et al 2001 Biochem Biophys Res Com 283 798-805), Wnt-7a
(R&D systems), Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a/14, Wnt- 9b/14b/15, Wnt-10a, Wnt-10b/12,
WnM l , and Wnt-16. An overview of human Wnt proteins is provided in "THE WNT FAMILY
OF SECRETED PROTEINS", R&D Systems Catalog, 2004. Further Wnt agonists include the R-
spondin family of secreted proteins, which is implicated in the activation and regulation of Wnt
signaling pathway and which is comprised of 4 members (R-spondin 1 (NU206, Nuvelo, San
Carlos, CA), R-spondin 2 ((R&D systems), R-spondin 3, and R-spondin-4), and Norrin (also called
Nome Disease Protein or NDP) (R&D systems), which is a secreted regulatory protein that
functions like a Wnt protein in that it binds with high affinity to the Frizzled-4 receptor and
induces activation of the Wnt signaling pathway (Kestutis Planutis et al (2007) BMC Cell Biol 8
12). In some embodiments, one or more Wnt agonists for use in the invention is an R-spondin
mimic, for example an agonist of Lgr5 such as an anti-Lgr5 antibody. A small-molecule agonist of
the Wnt signaling pathway, an aminopyrimidine derivative, was recently identified and is also
expressly included as a Wnt agonist (Lm et al (2005) Angew Chem Int Ed Engl 44, 1987-90).
In some embodiments, the Wnt agonist is a GSK-inhibitor. Known GSK-inhibitors comprise
small-interfering RNAs (siRNA, Cell Signaling), lithium (Sigma), kenpaullone (Biomol
International, Leost, M et al (2000) Eur J Biochem 267, 5983-5994), 6-Bromoindirubin
acetoxime (Meyer, L et al (2003) Chem Biol 10, 1255-1266), SB 216763 and SB 415286 (Sigma-
Aldrich), and FRAT-family members and FRAT-derived peptides that prevent interaction of GSK-
3 with axin. An overview is provided by Meijer et al , (2004) Trends in Pharmacological Sciences
, 471-480, which is hereby incorporated by reference. Methods and assays for determining a
level of GSK-3 inhibition are known to a skilled person and comprise, for example, the methods
and assay as described in Liao et al 2004, Endocrinology, 145(6) 2941-2949.
In some embodiments, the Wnt agonist is an inhibitor of RNF43 or ZNRF3. The inventors have
discovered that RNF43 and ZNRF3 reside in the cell membrane and negatively regulate levels of
the Wnt receptor complex in the membrane, probably by ubiquitination of Frizzled. Therefore, the
inventors hypothesise that inhibition of RNF43 or ZNRF3 with antagonistic antibodies, RNAi or
small molecule inhibitors would indirectly stimulate the Wnt pathway. RNF43 and ZNRF3 have a
catalytic ring domain (with ubiquitination activity), which can be targeted in small molecule
inhibitor design. Several anti-RNF43 antibodies and several anti-ZNRF3 antibodies are available
commercially. In some embodiments, such antibodies are suitable Wnt agonists in the context of
the invention.
In some embodiments, said Wnt agonist is selected from the group consisting of Wnt-3a, a GSK-
inhibitor (such as CHIR99021), Wnt 5, Wnt-6a, Norrin, and any other Wnt family protein.
In some embodiments, said Wnt agonist comprises or consists of any one of Rspondin 1, Rspondin
2, Rspondin 3 or Rspondin 4. In a preferred embodiment, said Wnt agonist is selected from one or
more of a Wnt family member, R-spondin 1-4, Norrin, and a GSK-inhibitor. In some
embodiments, said Wnt agonist is a GSK-3 inhibitor, such as CHIR99021 (Stemgent 04-0004). In
some embodiments, CHIR99021 is added to the culture medium to a final concentration of
between 50 nM and 100 uM, for example between 100 nM and 50 uM, between 1 uM and 10 uM,
between 1 uM and 5 uM, or 3 uM. In some embodiments in which a GSK-3 inhibitor is used, the
GSK-3 inhibitor is not BIO (6-bromoindirubin-3’-oxime, Stemgent 04-0003). It was found by the
inventors that the addition of at least one Wnt agonist to the basal culture medium is essential for
proliferation of the epithelial stem cells or isolated crypts.
In a further preferred embodiment, said Wnt agonist comprises or consists of R-spondin 1 or R-
spondin-4. R-spondin 1, R-spondin 2, R-spondin 3 or R-spondin 4 is preferably added to the basal
culture medium at a concentration of at least 50 ng/ml, more preferred at least 100 ng/ml, more
preferred at least 200 ng/ml, more preferred at least 300 ng/ml, more preferred at least 500 ng/ml.
A most preferred concentration of R-spondin 1, R-spondin 2, R-spondin 3 or R-spondin 4 is
approximately 500 ng/ml or 500 ng/ml. In some embodiments, R-spondin 1, R-spondin 2, R-
spondin 3 or R-spondin 4 is added to the culture medium at a concentration of at least 500 ng/ml,
at least 600 ng/ml, at least 700 ng/ml, at least 800 ng/ml, at least 900 ng/ml, at least 1 ug/ml, at
least 1.5 ug/ml or at least 2 ug/ml. In another preferred embodiment, R-spondin 1, R-spondin 2, R-
spondin 3 or R-spondin 4 is added to the culture medium at a concentration of approximately 1
ug/ml or 1 ug/ml. In some embodiments, R-spondin 1, R-spondin 2, R-spondin 3 or R-spondin 4
is added to the basal culture medium at a concentration of less than 1000 ng/ml, for example, less
than 800 ng/ml, less than 600 ng/ml, less than 550 ng/ml, less than 500 ng/ml, less than 400
ng/ml, less than 300 ng/ml or less than 200 ng/ml, or less than 100 ng/ml. In some embodiments,
two or more (e.g. 2, 3 or 4) of Rspondin 1, Rspondin 2, Rspondin 3 and Rspondin 4 (“Rspondin 1-
4”) are added to the medium. Preferably, when two or more of Rspondin 1-4 are added, the total
concentration of Rspondin amounts to the concentrations described above. Where culture media
described herein are said to comprise “Rspondin 1-4”, it is meant that the medium comprises any
one or more of Rspondin 1, Rspondin 2, Rspondin 3 and Rspondin 4.Where culture media
described herein are said to comprise “Rspondin”, it is meant that the medium comprises any one
or more of Rspondin 1, Rspondin 2, Rspondin 3, Rspondin 4 and an Rspondin mimic.
During culturing of stem cells, said Wnt family member is preferably added to the culture medium
every second day, while the culture medium is refreshed preferably every fourth day.
In a preferred embodiment, a Wnt agonist is selected from the group consisting of R-spondin,
Wnt-3a and Wnt-6. More preferably, R-spondin and Wnt-3a are both used as Wnt agonist. This
combination is particularly preferred since this combination surprisingly has a synergistic effect on
organoid formation. Preferred concentrations are approximately 500 ng/ml or 500 ng/ml for R-
spondin and approximately 100 ng/ml or 100 ng/ml for Wnt3a.
The culture media of the invention may comprise one or more receptor tyrosine kinase ligands.
An example of a receptor tyrosine kinase ligand for use in the invention is EGF, which is the
ligand for the receptor tyrosine kinase EGFR. Many receptor tyrosine kinase ligands are also
mitogenic growth factors.
The culture media of the invention may comprise one or more mitogenic growth factor. The one or
more mitogenic growth factor may be selected from a family of growth factors comprising
epidermal growth factor (EGF, Peprotech), Transforming Growth Factor-alpha (TGF-alpha,
Peprotech), basic Fibroblast Growth Factor (bFGF, Peprotech), brain-derived neurotrophic factor
(BDNF, R&D Systems), and Keratinocyte Growth Factor (KGF, Peprotech). EGF is a potent
mitogenic factor for a variety of cultured ectodermal and mesodermal cells and has a profound
effect on the differentiation of specific cells in vivo and in vitro and of some fibroblasts in cell
culture. The EGF precursor exists as a membrane-bound molecule which is proteolytically cleaved
to generate the 53-amino acid peptide hormone that stimulates cells. A preferred mitogenic growth
factor is EGF. EGF is preferably added to the basal culture medium at a concentration of between
and 500 ng/ml or of at least 5 and not higher than 500 ng/ml. A preferred concentration is at least
, 20, 25, 30, 40, 45, or 50 ng/ml and not higher than 500, 450, 400, 350, 300, 250, 200, 150, or
100 ng/ml. A more preferred concentration is at least 50 and not higher than 100 ng/ml. An even
more preferred concentration is about 50 ng/ml or 50 ng/ml. The same concentrations could be
used for a FGF, preferably for FGF10 or FGF7. If more than one FGF is used, for example FGF7
and FGF10, the concentration of a FGF is as defined above and refers to the total concentration of
FGF used. During culturing of stem cells, said mitogenic growth factor is preferably added to the
culture medium every second day, while the culture medium is refreshed preferably every fourth
day. Any member of the FGF family may be used. Preferably, FGF7 and/or FGF10 is used FGF7
is also known as KGF (Keratinocyte Growth Factor). In a further preferred embodiment, a
combination of mitogenic growth factors such as, for example, EGF and KGF, or EGF and BDNF,
is added to the basal culture medium. In a further preferred embodiment, a combination of
mitogenic growth factors such as, for example, EGF and KGF, or EGF and FGF10, is added to the
basal culture medium. The mitogenic growth factor may be added to a culture media at a
concentration of between 5 and 500 nanogram/ml or at least 5 and not more than 500
nanogram/ml, for example at least 10, 20, 25, 30, 40, 45, or 50 ng/ml and not higher than 500, 450,
400, 350, 300, 250, 200, 150, or 100 ng/ml. The mitogenic growth factor may be selected from the
group consisting of EGF, TGF-alpha, KGF, FGF7 and FGF. Preferably, a mitogenic factor is
selected from the groups consisting of EGF, TGF-alpha and KGF or from EGF, TGF-alpha and
FGF7 or from EGF, TGF-alpha and FGF or from EGF and KGF or from EGF and FGF7 or from
EGF and a FGF or from TGF-alpha and KGF or from TGF-alpha and FGF7 or from TGF-alpha
and a FGF. EGF may be replaced by TGF-alpha. In some embodiments, the mitogenic growth
factor is hepatocyte growth factor (HGF). In some embodiments, HGF is added to the culture
medium.
In some embodiments, the receptor tyrosine kinase ligand is a mitogenic growth factor, for
example selected from a family of growth factors consisting of epidermal growth factor (EGF),
Transforming Growth Factor-alpha (TGF-alpha), basic Fibroblast Growth Factor (bFGF), brain-
derived neurotrophic factor (BDNF), Hepatocyte growth factor (HGF) and Keratinocyte Growth
Factor (KGF).
ROCK inhibitors, such as Y-27632 (10 μM; Sigma), can be included in any of the media
described, in particular in the first few days of culture before performing cell sorting experiments,
because it is known to avoid anoikis (a form of programmed cell death which is induced by
anchorage-dependent cells detaching from the surrounding extracellular matrix). Therefore, any of
the media defined herein, may additionally comprise a ROCK inhibitor for the first few days. In
some embodiments, the culture media of the invention additionally comprises a ROCK inhibitor,
such as Y-27632, for example for the first few days of culture before performing cell sorting
experiments.
A further embodiment of a method according to the invention comprises a culture medium
comprising a Rock (Rho-kinase) inhibitor. The addition of a Rock inhibitor was found to prevent
anoikis, especially when culturing single stem cells. Said Rock inhibitor is preferably selected
from R-(+)-trans(l-aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride
monohydrate (Y-27632, Sigma- Aldrich), 5-(l ,4-diazepan- l-ylsulfonyl)isoquinoline (fasudil or
HA1077, Cayman Chemical), and (S)-(+)methyl- l-[(4-methylisoquinolinyl)sulfonyl] -
hexahydro-1H- 1,4-diazepine dihydrochloride (H-1 152, Tocris Bioschience). Said Rho-kinase
inhibitor, for example Y-27632, is preferably added to the culture medium every second day
during the first seven days of culturing said stem cells. A Rock inhibitor is preferably included in
the medium in the first few days e.g. for the first 1, 2, 3, 4, 5, 6 or 7 days of culture after single cell
seeding or after a split. Any suitable concentration of the Rock inhibitor may be used, for example,
1-200 uM, 1-100 uM, 5-50 uM or approximately 10uM. A preferred concentration for Y27632 is
10uM. Therefore, in some embodiments, the invention provides a method for culturing stem cells
and/or a method for obtaining an organoid wherein a Rock inhibitor is added to the culture
medium for the first 1, 2, 3, 4, 5, 6 or 7 days, optionally every second day. In some embodiments,
the Rock inhibitor is not added to the culture medium after the first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
Addition of a Rock inhibitor is particularly important when culturing single stem cells (as
mentioned above), i.e. when the starting material for an organoid is a single stem cell. Therefore,
in some embodiments the invention provides a method for obtaining an organoid, wherein the
method comprises culturing stem cells, optionally single stem cells, wherein a Rock inhibitor is
added to the culture medium for the first 1, 2, 3, 4, 5, 6 or 7 days, optionally every second day, and
optionally not adding the Rock inhibitor to the culture medium after the first 2, 3, 4, 5, 6, 7, 8, 9 or
days.
The Rock inhibitor is less important, and sometimes not necessary, when culturing multiple cells,
for example when the starting material for an organoid is a tissue fragment. Therefore, in some
embodiments, the invention provides a method for obtaining an organoid, wherein the method
comprises culturing stem cells, optionally a tissue fragment, wherein the Rock inhibitor is not
added to the culture medium either at all or after the first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
After the cells are split into multiple cultures, a Rock inhibitor may be added to the culture
medium in the same way, meaning for the first 1, 2, 3, 4, 5, 6 or 7 days, optionally every second
day, after the split, particularly when the split involves taking single stem cells from a first culture
and placing these into a second culture. If the split involves taking multiple stem cells from the
first culture and placing these into a second culture then addition of a Rock inhibitor is less
important, and sometimes not necessary. Therefore, in some embodiments, wherein the method for
obtaining organoids or for culturing stem cells involves a split, optionally where a single cell is
involved in the split, a Rock inhibitor is added to the new culture medium for the first 1, 2, 3, 4, 5,
6 or 7 days, optionally every second day, after the split. In some embodiments, wherein the method
for obtaining organoids or for culturing stem cells involves a split, optionally where multiple cells
are involved in the split, is not added to the culture medium either at all or after the first 2, 3, 4, 5,
6, 7, 8, 9 or 10 days.
In yet a further embodiment, a method according to the invention comprises a culture medium
further comprising a Notch agonist. Notch signaling has been shown to play an important role in
cell-fate determination, as well as in cell survival and proliferation. Notch receptor proteins can
interact with a number of surface-bound or secreted ligands, including but not limited to Delta 1,
Jagged 1 and 2, and Delta-like 1, Delta-like 3, Delta-like 4. Upon ligand binding, Notch receptors
are activated by serial cleavage events involving members of the ADAM protease family, as well
as an intramembranous cleavage regulated by the gamma secretase presenilin. The result is a
translocation of the intracellular domain of Notch to the nucleus where it transcriptionally activates
downstream genes. A preferred Notch agonist is selected from Jagged 1 and Delta 1, or an active
fragment or derivative thereof. A most preferred Notch agonist is DSL peptide (Dontu et al., 2004.
Breast Cancer Res 6. R605-R615) with the sequence CDDYYYGFGCNKFCRPR. Said DSL
peptide is preferably used at a concentration between 10μM and 100nM or at least 10μM and not
higher than 100nM. The addition of a Notch agonist, especially during the first week of culturing,
increases the culture efficiency by a factor of 2-3. Said Notch agonist is preferably added to the
culture medium every second day during the first seven days of culturing said stem cells.
Therefore, in some embodiments, the invention provides a method for culturing stem cells and/or a
method for obtaining an organoid wherein a Notch agonist is added to the culture medium for the
first 1, 2, 3, 4, 5, 6 or 7 days, optionally every second day. In some embodiments, the Notch
agonist is not added to the culture medium after the first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
A Notch agonist is defined as a molecule that stimulates a Notch activity in a cell by at least 10%,
more preferred at least 20%, more preferred at least 30%, more preferred at least 50%, more
preferred at least 70%, more preferred at least 90%, more preferred at least 100%, relative to a
level of a Notch activity in the absence of said molecule. As is known to a skilled person, a Notch
activity can be determined by measuring the transcriptional activity of Notch, for example by a
4xwtCBFl-luciferase reporter construct as described (Hsieh et al, 1996 Mol Cell. Biol. 16, 952-
959).
In a further embodiment, the cell culture medium is supplemented with a gamma-secretase
inhibitor, such as DAPT or DBZ. Gamma-secretase inhibitors can influence cell fate decisions
during differentiation. For example, in some embodiments, gamma-secretase inhibitors can
influence cell fate towards secretory cells, such as goblet cells. . Any suitable concentration of the
gamma-secretase inhibitor may be used, for example, between 1 nM and 10 uM, 1 nM and 1 uM,
between 1 and 100 nM, or preferably between 1 and 20nM. For example, a gamma-secretase
inhibitor may be added to the culture medium to a final concentration of approximately 1 nM.
In a further embodiment, the cell culture medium is supplemented with gastrin (or a suitable
alternative such as Leu15-gastrin). Gastrin (or a suitable alternative) may be added to the culture
medium to a final concentration of between 1 nM and 10 uM, 1 nM and 1 uM, between 5 and 100
nM, or preferably between 10 and 50nM. For example, Leu15-gastrin may be added to the culture
medium to a final concentration of approximately 10 nM. Gastrin is not necessary for some
culture media of the invention. Therefore, in some embodiments the culture medium of the
invention does not comprise gastrin. In particular, gastrin is not required for culturing intestinal
stem cells or for obtaining intestinal (crypt-villus or colon crypt) organoids. However, even where
gastrin is not required, it may still be added to the culture medium without negative effects.
In a further embodiment, the culture medium of the invention is supplemented with nicotinamide.
Addition of nicotinamide has been found to improve culture efficiency and lifespan of human
colon organoids. Nicotinamide may be added to the culture medium to a final concentration of
between 1 and 100 mM, between 5 and 50 mM, or preferably between 5 and 20mM. For example,
nicotinamide may be added to the culture medium to a final concentration of approximately 10
In a preferred embodiment of the invention, the culture medium is supplemented with
nicotinamide and gastrin (or a suitable alternative, such as Leu15-gastrin), wherein nicotinamide
and gastrin are added to the culture medium at any of the concentrations described above.
In some embodiments, the culture medium is supplemented with an activator of the prostaglandin
signalling pathway (see Figure 24, Antagonism of the prostaglandin D receptors DP and CRTH2
as an approach to treat allergic diseases. Roy Pettipher, Trevor T. Hansel & Richard Armer Nature
Reviews Drug Discovery 6, 313-325 (April 2007)). For example, the culture medium is
supplemented with any one or more of the compounds selected from the list comprising:
Phospholipids, Arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2),
prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For example, in
some embodiments, the culture medium is supplemented with PGE2 and/or AA. In some
embodiments, PGE2 is added to the medium to a final concentration of at least 10 nM, for example
at least 20nM, at least 30nM, at least 40nM, at least 45nM, between 10 nM and 500 nM, between
nM, and 400 nM, between 10 nM and 300 nM, between 10 nM and 200 nM, between 10 nM
and 100 nM, between 20 nM and 50 nM. In a preferred embodiment, PGE2 is added to the
medium to a final concentration of 50 nM. In some embodiments, AA is added to the medium to a
final concentration of at least 1 ug/ml, at least 5 ug/ml , at least 8 ug/ml, at least 9 ug/ml, at least 10
ug/ml, for example between 1 ug/ml and 1000 ug/ml, between 1 ug/ ml and 500 ug/ml, between 1
ug/ml and 100 ug/ml, between 1 ug/ml and 50 ug/ml, or between 5 ug/ml and 20 ug/ml. In a
preferred embodiment, AA is added to the medium to a final concentration of 10 ug/ml. AA and
PGE2 are interchangeable in the context of the culture media of the invention. Therefore, where a
culture medium described herein is said to include PGE2, it may alternatively include AA (at an
appropriate concentration) instead of PGE2. Conversely, where a culture medium described herein
is said to include AA, it may alternatively include PGE2 (at an appropriate concentration) instead
of AA. Furthermore, the skilled person would understand that where PGE2 and/or AA are included
in a culture medium of the invention, the culture medium could instead comprise any one or more
of the compounds selected from the following list in replacement or in addition to PGE2 and/or
AA: Phospholipids, prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2
(PGH2), and prostaglandin D2 (PGD2).
In a futher embodiment, the culture medium of the invention is supplemented with RANK ligand
(also referred to herein as RANKL). RANK ligand can be useful for directing differentiation
towards particular cell fates. For example, when RANK ligand is included in the culture medium
for small intestinal cells, preferably in the medium for differentiating small intestinal cells, it
results in a greater proportion of the cells being differentiated into M cells. Therefore, in some
embodiments, the invention provides a culture medium comprising RANKL. In particular, the
invention provides a culture medium for culturing, preferably for differentiating small intestinal
cells, wherein the culture medium comprises RANKL. Any suitable concentration of the RANKL
may be used, for example, between 10ng/ml and 1000ng/ml, between 10 and 500 ng/ml, or
between 50 and 100ng/ml. For example, RANKL may be added to the culture medium to a final
concentration of approximately 100 ng/ml.
A culture medium comprising EGF, Noggin and R-spondin is referred to herein as the “ENR
medium”. A culture medium comprising the ENR medium and a Wnt agonist such as Wnt-3a is
referred to herein as the “WENR medium”. In a preferred embodiment of the invention, the culture
medium comprises a WENR medium. In a most preferred embodiment of the invention, the culture
medium comprises a WENR medium supplemented with gastrin and/or nicotinamide (i.e.,
WENRg or WENR+nicotinamide or WENRg+nicotinamide).
The pH of the medium may be in the range from about 7.0 to 7.8, in the range from about 7.2 to
7.6, or about 7.4. The pH may be maintained using a buffer. A suitable buffer can readily be
selected by the skilled person. Buffers that may be used include carbonate buffers (e.g. NaHCO ),
and phosphates (e.g. NaH PO ). These buffers are generally used at about 50 to about 500 mg/l.
Other buffers such as N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesul-phonic acid] (HEPES) and 3-
[N-morpholino]-propanesulfonic acid (MOPS) may also be used, normally at around 1000 to
around 10,000 mg/l. A culture medium may comprise a pH indicator, such as phenol red, to enable
the pH status of the medium to be easily monitored (e.g. at about 5 to about 50 mg/litre).
A culture medium for use in the invention may comprise one or more amino acids. The skilled
person understands the appropriate types and amounts of amino acids for use in stem cell culture
media. Amino acids which may be present include L-alanine, L-arginine, L-asparagine, L-aspartic
acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-
leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,
L-tyrosine, L-valine and combinations thereof. Some culture media will contain all of these amino
acids. Generally, each amino acid when present is present at about 0.001 to about 1 g/L of medium
(usually at about 0.01 to about 0.15 g/L), except for L-glutamine which is present at about 0.05 to
about 1 g/L (usually about 0.1 to about 0.75 g/L). The amino acids may be of synthetic origin.
A culture medium for use in the invention may comprise one or more vitamins. The skilled person
understands the appropriate types and amounts of vitamins for use in stem cell culture media.
Vitamins which may be present include thiamine (vitamin B1), riboflavin (vitamin B2), niacin
(vitamin B3), D-calcium pantothenate (vitamin B5), pyridoxal/pyridoxamine/pyridoxine (vitamin
B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol
(vitamin D2), DL-alpha tocopherol (vitamin E), biotin (vitamin H) and menadione (vitamin K).
A culture medium for use in the invention may comprise one or more inorganic salts. The skilled
person understands the appropriate types and amounts of inorganic salts for use in stem cell culture
media. Inorganic salts are typically included in culture media to aid maintenance of the osmotic
balance of the cells and to help regulate membrane potential. Inorganic salts which may be present
include salts of calcium, copper, iron, magnesium, potassium, sodium, zinc. The salts are normally
used in the form of chlorides, phosphates, sulphates, nitrates and bicarbonates. Specific salts that
may be used include CaCl , CuSO -5H O, Fe(NO )-9H O, FeSO -7H O, MgCl, MgSO , KCl,
2 4 2 3 2 4 2 4
NaHCO , NaCl, Na HPO , Na HPO -H O and ZnSO -7H O.
3 2 4 2 4 2 4 2
The osmolarity of the medium may be in the range from about 200 to about 400 mOsm/kg, in the
range from about 290 to about 350 mOsm/kg, or in the range from about 280 to about 310
mOsm/kg. The osmolarity of the medium may be less than about 300 mOsm/kg (e.g. about 280
mOsm/kg).
A culture medium for use in the invention may comprise a carbon energy source, in the form of
one or more sugars. The skilled person understands the appropriate types and amounts of sugars to
use in stem cell culture media. Sugars which may be present include glucose, galactose, maltose
and fructose. The sugar is preferably glucose, particularly D-glucose (dextrose). A carbon energy
source will normally be present at between about 1 and about 10 g/L.
A culture medium of the invention may contain serum. Serum obtained from any appropriate
source may be used, including fetal bovine serum (FBS), goat serum or human serum. Preferably,
human serum is used. Serum may be used at between about 1% and about 30% by volume of the
medium, according to conventional techniques.
In other embodiments, a culture medium of the invention may contain a serum replacement.
Various different serum replacement formulations are commercially available and are known to
the skilled person. Where a serum replacement is used, it may be used at between about 1% and
about 30% by volume of the medium, according to conventional techniques.
In other embodiments, a culture medium of the invention may be serum-free and/or serum
replacement-free. A serum-free medium is one that contains no animal serum of any type. Serum-
free media may be preferred to avoid possible xeno-contamination of the stem cells. A serum
replacement-free medium is one that has not been supplemented with any commercial serum
replacement formulation.
In a preferred embodiment, the cell culture medium is supplemented with a purified, natural, semi-
synthetic and/or synthetic growth factor and does not comprise an undefined component, such as
fetal bovine serum or fetal calf serum. For example, supplements such as B27 (Invitrogen), N-
Acetylcysteine (Sigma) and N2 (Invitrogen) stimulate proliferation of some cells. In some
embodiments, the cell culture medium is supplemented with one or more of these supplements, for
example one, any two or all three of these supplements.
In other embodiments, the cell culture medium is supplemented with Exendin-4. Exendin-4, a 39
amino acid peptide, activates GLP-1 (glucagon-like peptide-1) receptors to increase intracellular
cAMP in pancreatic acinar cells and has no effect on VIP (vasoactive intestinal peptide) receptors.
A culture medium for use in the invention may comprise one or more trace elements, such as ions
of barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium,
titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium,
cadmium, zinc and/or aluminium.
The medium may comprise a reducing agent, such as beta-mercaptoethanol at a concentration of
about 0.1 mM.
A culture medium of the invention may comprise one or more additional agents, such as nutrients
or growth factors previously reported to improve stem cell culture, such as
cholesterol/transferrin/albumin/insulin/progesterone, putrescine, selenite/other factors.
A culture medium of the invention may be diffused into an extracellular matrix (ECM). In a
preferred method of the invention, isolated tissue fragments or isolated epithelial stem cells are
attached to an ECM. ECM is composed of a variety of polysaccharides, water, elastin, and
glycoproteins, wherein the glycoproteins comprise collagen, entactin (nidogen), fibronectin, and
laminin. ECM is secreted by connective tissue cells. Different types of ECM are known,
comprising different compositions including different types of glycoproteins and/or different
combination of glycoproteins. Said ECM can be provided by culturing ECM-producing cells, such
as for example fibroblast cells, in a receptacle, prior to the removal of these cells and the addition
of isolated tissue fragments or isolated epithelial stem cells. Examples of extracellular matrix-
producing cells are chondrocytes, producing mainly collagen and proteoglycans, fibroblast cells,
producing mainly type IV collagen, laminin, interstitial procollagens, and fibronectin, and colonic
myofibroblasts producing mainly collagens (type I, III, and V), chondroitin sulfate proteoglycan,
hyaluronic acid, fibronectin, and tenascin-C. Alternatively, said ECM is commercially provided.
Examples of commercially available extracellular matrices are extracellular matrix proteins
(Invitrogen) and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse
sarcoma cells (e.g. Matrigel (BD Biosciences)). A synthetic extracellular matrix material, such
as ProNectin (Sigma Z378666) may be used. Mixtures of extracellular matrix materials may be
used, if desired. The use of an ECM for culturing stem cells enhanced long-term survival of the
stem cells and the continued presence of undifferentiated stem cells. In the absence of an ECM,
stem cell cultures could not be cultured for longer periods and no continued presence of
undifferentiated stem cells was observed. In addition, the presence of an ECM allowed culturing of
three-dimensional tissue organoids, which could not be cultured in the absence of an ECM. The
extracellular matrix material will normally be a drop on the bottom of the dish in which cells are
suspended. Typically, when the matrix solidifies at 37ºC, the medium is added and diffuses into
the ECM. The cells in the medium stick to the ECM by interaction with its surface structure, for
example interaction with integrins. A fibronectin solution of about 1mg/ml (stock solution) used at
approximately 1 μg/cm may be used to coat a cell culture vessel, or between about 1 μg/cm to
2 2 2
about 250 μg/cm , or at about 1 μg/cm to about 150 μg/cm . In some embodiments, a cell culture
vessel is coated with fibronectin at between 8 μg/cm and 125 μg/cm .
An example of an ECM for use in a method of the invention comprises at least one glycoprotein,
such as laminin.
A preferred ECM for use in a method of the invention comprises at least two distinct
glycoproteins, such as two different types of collagen or a collagen and laminin. The ECM can be
a synthetic hydrogel extracellular matrix or a naturally occurring ECM. A further preferred ECM is
provided by Matrigel (BD Biosciences), which comprises laminin, entactin, and collagen IV. In
some embodiments the extracellular matrix is a laminin-containing extracellular matrix such as
Matrigel (BD Biosciences).
In some embodiments, the single stem cell, population of cells, or tissue fragment is embedded in
matrigel, which is optionally growth factor reduced and/or phenol red-free.
In some embodiments, the culture medium is placed on top of the ECM. The culture medium can
then be removed and replenished as and when required. In some embodiments, the culture medium
is replenished every 1, 2, 3, 4, 5, 6 or 7 days. If components are “added” or “removed” from the
media, then this can in some embodiments mean that the media itself is removed from the ECM
and then a new media containing the “added” component or with the “removed” component
excluded is placed on the ECM.
In some embodiments the culture medium of the invention is in contact with an extracellular
matrix or a 3D matrix that mimics the extracellular matrix by its interaction with the cellular
membrane proteins, such as integrins.
In some embodiments, the basal culture medium comprises or consists of Advanced DMEM/F12
supplemented with penicillin/streptomycin, 10mM HEPES, Glutamax, 1× N2, 1× B27 (all from
Invitrogen) and 1 mM N-acetylcysteine (Sigma)).
Examples of culture media of the invention
In one embodiment, the cell culture medium comprises a TGF-beta inhibitor that binds to and
reduces the activity of ALK5 and a p38 inhibitor that binds to and reduces the activity of p38. For
example, in one embodiment the cell culture media comprises A83-01 and/or SB202190,
preferably A83-01+ SB202190. The use of A83-01+SB202190 together in a culture medium of
the invention has surprisingly be found to synergistically increase the number of passages of
human colon organoids. In one embodiment, the cell culture media comprises WENR+A83-
01+SB202190. In one embodiment, the cell culture media comprise WENR+A83-
01+SB202190+nicotinamide. In one embodiment, the cell culture media comprises
WENRg+nicotinamide +A83-01+SB202190 (where “g” is gastrin). In one embodiment, the cell
culture medium comprises WENR+A83-01+Nicotinamide+ FGF10. In one embodiment, the cell
culture medium comprises WENRg+A83-01+Nicotinamide+ FGF10. In one embodiment, the cell
culture medium comprises WENRg+A83-01+Nicotinamide+FGF10+SB202190. In one
embodiment, the cell culture media is used for obtaining colon organoids. A colon organoid
obtainable by culturing epithelial cells using a cell culture media as described in this embodiment
is also provided.
For example, in one embodiment the cell culture media comprises WENRg+A83-01+FGF10,
wherein the Wnt agonist is R-Spondin but no other Wnt agonist is present and no nicotinamide is
present. For example, in some embodiments, the cell culture media comprises EGF (e.g. 50
ng/ml), R-Spondin (e.g. 10% or 1 ug/ml), Noggin (e.g. 100ng/ml), FGF10 (e.g. 100ng/ml), A8301
(e.g. 500 nM) and Gastrin (e.g. 10uM) and optionally SB202190. These components may be
added to a basal medium, such as DMEM/F12 media. In some embodiments, the basal medium is
further supplemented with any one or more (for example, 1, 2, 3, 4 or 5) or all, of the components
selected from the list comprising: P/S, Glutamax, 10nmM Hepes, B27, N2 and N-Acetylcysteine.
The use of such a cell culture media has been found to be useful for obtaining pancreatic
organoids. A pancreatic organoid obtained by culturing epithelial cells using a cell culture media
as described in this embodiment is also provided. In some embodiments, gastrin or nicotinamide or
gastrin and nicotinamide are excluded from the culture medium.
Tissue-specific culture media of the invention
Particularly preferred culture media are described in the Examples herein. The culture medium of
the invention can be adapted for use with different tissues, for example as described below.
Intestinal culture media
In some embodiments, the culture medium for small intestinal crypts, such as murine small
intestinal crypts, comprises or consists of a basal medium, for example as described above,
additionally comprising: EGF, such as murine EGF; a BMP inhibitor, such as murine Noggin; and
Rspondin, such as human Rspondin-1 or 4. In some embodiments, this culture medium further
comprises a TGF-beta inhibitor (such as A83-01) and/or a p38 inhibitor (such as SB202190). In
some embodiments, the culture medium for colonic crypts, such as murine colonic crypts,
comprises or consists of a basal medium, for example as described above, additionally comprising:
a Wnt agonist, such as recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF, such as
murine EGF; a BMP inhibitor, such as murine Noggin; and Rspondin, such as human Rspondin-1
or 4. In some embodiments, this culture medium further comprises a TGF-beta inhibitor (such as
A83-01) and/or a p38 inhibitor (such as SB202190).
In some embodiments, the culture medium for human intestinal stem cells, human small intestinal
crypts or human colonic crypts (also known as the HISC culture medium), comprises or consists of
a basal medium, for example as described above, additionally comprising: a Wnt agonist, such as
recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP inhibitor, such as
Noggin; Rspondin, such as human Rspondin-1; a TGF-beta inhibitor, such as A83-01; a p38
inhibitor, such as SB202190; gastrin; and nicotinamide. In some embodiments, the p38 inhibitor
and/or gastrin can be excluded from the HISC culture medium.
In some embodiments the invention provides a culture medium for culturing intestinal cells,
comprising or consisting of a basal medium, Wnt-3a, EGF, Noggin, any one of Rspondin 1-4, a
TGF-beta inhibitor, nicotinamide, and preferably a p38 inhibitor.
In some embodiments, the culture medium for expanding small intestine or colon stem cells, for
example human small intestine or colon cells, comprises or consists of a basal medium (for
example comprising Advanced DMEM/F12, B27 (50x), n-Acetylcysteine (1 mM) and
glutamin/glutamax), Wnt3A (optionally conditioned medium), any one of Rspondin 1-4
(preferably 1 ug/ml), Noggin (preferably 50-100 ng/ml), nicotinamide (preferably 10 mM), EGF
(preferably 10-50 ng/ml), gastrin (preferably 10 nM), a TGF-beta inhibitor, for example A83-01
(preferably 500 nM). In a further embodiment, this culture medium additionally comprises a p38
inhibitor, for example SB202190 (preferably 100 nM). In a further embodiment, this culture
medium additionally comprises a Rock inhibitor, for example LY2157299.
In some embodiments, the invention provides a culture medium for differentiating intestinal cells,
comprising or consisting of a basal medium, EGF, Noggin, a TGF-beta inhibitor and a p38
inhibitor.
In some embodiments, the culture medium for differentiating small intestine or colon stem cells,
for example human small intestine or colon cells, comprises or consists of a basal medium (for
example comprising Advanced DMEM/F12, B27 (50x), n-Acetylcysteine (1 mM) and
glutamin/glutamax), Noggin (preferably 50-100 ng/ml), EGF (preferably 10-50 ng/ml), gastrin
(preferably 10 nM), a TGF-beta inhibitor, for example A83-01 (preferably 500 nM) and a p38
inhibitor, for example SB202190 (preferably 100 nM). In some embodiments, gastrin can be
excluded from this differentiation medium. In some embodiments, a gamma-secretase inhibitor
may be added to the differentiation medium (preferably at a concentration of 1 uM). Gamma-
secretase inhibitors can influence cell fate decisions during differentiation e.g. towards secretory
cells, such as goblet cells. In some embodiments, a RANKL may be added to the differentiation
medium (for example at a concentration of 100 ng/ml). RANKL can influence cell fate decisions
during differentiation e.g. towards M-cells.
Cancer culture media
In some embodiments, the culture medium for colon cancer cells, comprises or consists of a basal
medium, for example as described above, additionally comprising: a Wnt agonist, such as
recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP inhibitor, such as
Noggin; Rspondin, such as human Rspondin-1; a TGF-beta inhibitor, such as A83-01; a p38
inhibitor, such as SB202190; gastrin; and nicotinamide.
In one embodiment, the culture medium for colon carcinoma, for example human colon
carcinoma, comprises a basal medium (for example comprising Advanced DMEM/F12, B27 (50x),
n-Acetylcysteine (1 mM), primocin and/or P/S (antibiotics) (500x) and hepes), Rspondin
(optionally conditioned medium) (preferably 1 ug/ml), Noggin (preferably 100 ng/ml),
Nicotinamide (preferably 10 mM), EGF (preferably 50 ng/ml), gastrin (preferably 50 nM), a TGF-
beta inhibitor, for example A83-01 (preferably 500 nM), a p38 inhibitor, such as SB202190
(preferably 10 uM), optionally PGE2 (preferably 10 nM) and/or a Rock inhibitor (preferably 10
uM).
In some embodiments, colon cancer cells can also be grown in the HISC culture medium. In some
embodiments, colon cancer cells can be cultured in the HISC culture medium, wherein one or
more or all of the following are excluded from the medium: EGF, Noggin, Rspondin, TGF-beta
inhibitor and p38 inhibitor. Cancer cells may have mutations that consitutively activate or
deactivate certain growth pathways. For example, many colon cancers result in constitutive
activation of the Wnt pathway. In such cases, a culture medium would not require a Wnt agonist.
Other mutations would allow other factors to be left out of the medium as described above. Other
epithelial cancers (carcinomas) can also be grown in culture media of the invention. In a preferred
embodiment, a cancer organoid obtained from cancer stem cells is grown in a culture medium that
is suitable for growth of the corresponding normal tissue organoid obtained from normal stem
cells, optionally with certain factors excluded from the medium. For example, a stomach cancer
organoid obtained by culturing stomach cancer stem cells may be grown in the same culture
conditions as a normal gastric organoid obtained by culturing gastric stem cells, optionally with
certain factors excluded from the medium. In another example, a pancreatic cancer organoid
obtained by culturing pancreatic cancer stem cells may be grown in the same culture conditions as
a normal pancreatic organoid obtained by culturing pancreatic stem cells, optionally with certain
factors excluded from the medium. In another example, a prostate cancer organoid obtained by
culturing prostatic cancer stem cells may be grown in the same culture conditions as a normal
prostate organoid obtained by culturing prostatic stem cells, optionally with certain factors
excluded from the medium. In another example, a liver cancer organoid obtained by culturing liver
cancer stem cells may be grown in the same culture conditions as a normal liver organoid obtained
by culturing liver stem cells, optionally with certain factors excluded from the medium. In many
situations it may be preferable (or at least more convenient) to grow cancer organoids in the
normal tissue medium (without any factors excluded). The normal tissue medium should allow
cancers with all genetic backgrounds to grow, without excluding any particular cancer mutations.
Therefore, in some embodiments, the invention provides a culture medium for culturing cancer
cells, for example cancer stem cells, such as adenocarcinoma or carcinoma cells from a tissue type
of interest, wherein the culture medium comprises or consists of the components of the culture
medium used for culturing the cells from the corresponding non-cancerous tissue type of interest,
optionally wherein one or more of the following are excluded from the medium that is used to
culture the non-cancerous cells of the tissue type of interest: Wnt-3a, EGF, Noggin, Rspondin,
TGF-beta inhibitor, p38 inhibitor, nicotinamide, gastrin, FGF10 and HGF.
Adenoma culture medium
In some embodiments, the culture medium for intestinal adenomas, such as murine intestinal
adenomas comprises a basal medium, for example as described above, additionally comprising
EGF, such as murine EGF.
Gastric culture media
In some embodiments, the invention provides a culture medium for culturing gastric cells,
comprising or consisting of a basal medium, Wnt-3a, EGF, Noggin, any one of Rspondin 1-4, a
TGF-beta inhibitor, gastrin, nicotinamide, FGF-10, and preferably a p38 inhibitor.
In some embodiments, the culture medium for gastric stem cells, for example human gastric stem
cells comprises or consists of a basal medium (for example comprising Advanced DMEM/F12,
B27 (50x), n-Acetylcysteine (1 mM), primocin and/or P/S (antibiotics) (500x) and
glutamin/glutamax), any one of Rspondin 1-4 (optionally conditioned medium) (preferably
1ug/ml), Noggin (optionally conditioned medium) (preferably 100ng/ml), Wnt3A (optionally
conditioned medium), nicotinamide (preferably 5 mM), EGF (preferably 50 ng/ml), FGF10
(preferably 200 ng/ml), gastrin (preferably 1 nM), a TGF-beta inhibitor, for example A83-01
(preferably 2 uM). The culture medium for gastric stem cells optionally further comprises a p38
inhibitor, for example SB202190 (preferably 10 nM). The culture medium for gastric stem cells
optionally further comprises PGE2 (preferably 500 nM). The culture medium for gastric stem cells
optionally further comprises a Rock inhibitor (preferably 10 uM).
In some embodiments, the culture medium for gastric stem cells, for example murine gastric cells
comprises or consists of a basal medium (for example comprising Advanced DMEM/F12, B27
(50x), n-Acetylcysteine (1 mM), primocin and/or P/S (antibiotics) (500x) and glutamin/glutamax),
any one of Rspondin 1-4 (optionally conditioned medium) (preferably 1ug/ml), Noggin (optionally
conditioned medium) (preferably 100ng/ml), Wnt3A (optionally conditioned medium), EGF
(preferably 50 ng/ml), FGF10 (preferably 200 ng/ml), gastrin (preferably 1 nM) and a Rock
inhibitor (preferably 10 uM). In some embodiments, this culture medium further comprises a TGF-
beta inhibitor (such as A83-01) and/or a p38 inhibitor (such as SB202190).
Prostate culture media
In some embodiments, the culture medium for expanding prostate stem cells comprises
testosterone, optionally dihydrotestosterone (also referred to herein as DHT). Testosterone is a
steroid hormone from the androgen group. In humans, a large percentage of testosterone undergoes
5α-reduction to form the more potent androgen, dihydrotestosterone. Testosterone,
dihydrotestosterone or a testosterone mimic (for example, a molecule that mimics the activity of
testosterone binding to an androgen receptor) can be added to a culture medium of the invention.
Therefore, where the term testosterone is used, it can always be replaced by dihydrotestosterone or
a testosterone mimic. The inventors have shown that addition of testosterone to a culture medium
for prostate stem cells, results in increased differentiation but also in continued expansion of the
stem cell population (for example, see Figures 41 - 45). This is highly surprising because the
literature teaches that testosterone plays an important role in the differentiation of cells by acting to
suppress proliferation and maintain terminal differentiation (Mirochnik et al. PLoS One, 7(3),
e31052, 2012; Niu et al. Oncogene 29, 3593-3604, 2010). The skilled person would have expected
that addition of testosterone to a culture medium for prostate would result in completely
differentiated organoids with no further expansion potential. This would be similar to what is
observed when the colon, pancreas and liver organoids are differentiated in a differentiation
medium. However, by contrast, the present inventors have found that although testosterone
increases differentiation, it also allows stem cell expansion to continue. Therefore, organoids
grown in a culture medium comprising testosterone surprisingly comprise stem cells and
differentiated cells i.e. luminal cells and basal cells.
In some embodiments, the culture medium for obtaining a prostate organoid comprises a basal
medium and testosterone, optionally dihydrotestosterone and anyone of Rspondin 1-4 or an
Rspondin mimic. In some embodiments, the culture medium further comprises a BMP inhibitor,
for example Noggin. In some embodiments, the culture medium further comprises a tyrosine
receptor kinase ligand, optionally wherein the tyrosine receptor kinase ligand is a mitogenic
growth factor, such as EGF, FGF, KGF or HGF. In some embodiments, the culture medium for
obtaining a prostate organoid comprises EGF, Noggin, any one or Rspondin 1-4 and testosterone.
In a preferred embodiment, the culture medium for prostate cells comprises a TGF-beta inhibitor.
In some embodiments, the culture medium for prostate cells comprises EGF, Noggin, any one of
Rspondin 1-4, a TGF-beta inhibitor and testosterone. In some embodiments, the culture medium
for prostate cells further comprises a p38 inhibitor. In some embodiments, a culture medium for
obtaining a prostate organoid does not comprise an inhibitor of the invention, for example a TGF-
beta inhibitor and/or a p38 inhibitor. In some embodiments the culture medium for prostate stem
cells does not comprise testosterone. In some embodiments, the culture medium comprises a basal
medium, EGF, Noggin and any one of Rspondin 1-4 and optionally a TGF-beta inhibitor, and does
not comprise testosterone.
In some embodiments, the invention provides a culture medium for culturing prostate cells,
comprising or consisting of a basal medium, EGF, any one of Rspondin 1-4, Noggin, nicotinamide
a TGF-beta inhibitor, and preferably Wnt-3a and FGF-10. In some embodiments, the culture
medium for culturing prostate cells further comprises testosterone, for example
(dihydro)testosterone. In some embodiments the culture medium further comprises a p38
inhibitor.In some embodiments, the culture medium for prostate cells, for example mouse, human,
normal or carcinoma, comprises a basal medium (for example comprising Advanced DMEM/F12,
B27 (50x), n-Acetylcysteine (1 mM) and glutamin/glutamax), any one of Rspondin 1-4 (optionally
conditioned medium) (preferably 1 ug/ml), Noggin (optionally conditioned medium) (preferably
100 ng/ml), nicotinamide (preferably 10mM), EGF (preferably 50 ng/ml), FGF10 (preferably 100
ng/ml), a TGF-beta inhibitor, for example A83-01 (preferably 500 nM), (Dihydro)testosterone
(preferably 1 nM) 10 nM and optionally Wnt-3a. In some embodiments, this culture medium
further comprises a Rock inhibitor (preferably 10 uM). In some embodiments, the culture medium
for prostate cells further comprises a p38 inhibitor, for example SB202190. In some embodiments,
wherein mouse prostate cells are cultured, the TGF-beta inhibitor can be excluded from the culture
medium. In other embodiments, nicotinamide, FGF10 and/or the Rock inhibitor can be excluded
from the culture medium.
Pancreatic culture media
In some embodiments, the invention provides a culture medium for expanding pancreas cells,
comprising or consisting of a basal medium, any one of Rspondin 1-4, Noggin, EGF, FGF10,
gastrin, a TGF-beta inhibitor, and preferably exendin 4 and Wnt-3a.
In some embodiments, the culture medium for expanding pancreatic stem cells, for example
human pancreatic stem cells comprises or consists of a basal medium (for example comprising
Advanced DMEM/F12, B27 (50x), n-Acetylcysteine (1 mM) and glutamin/glutamax), any one of
Rspondin 1-4 (optionally conditioned medium) (preferably 1ug/ml), Noggin (optionally
conditioned medium) (preferably 100ng/ml), nicotinamide (preferably 10 mM), EGF (preferably
50 ng/ml), FGF10 (preferably 100 ng/ml), gastrin (preferably 100 nM), and a TGF-beta inhibitor,
for example A83-01 (preferably 2 uM). In a further embodiment, this culture medium additionally
comprises Wnt-3a. In a further embodiment, this culture medium additionally comprises a p38
inhibitor, for example SB202190 (preferably 100 nM). In a further embodiment, this culture
medium additionally comprises a Rock inhibitor, for example LY2157299 (preferably 10 uM). In a
further embodiment, this culture medium additionally comprises Exendin 4 (preferably 50 ng/ml).
In some embodiments, the culture medium for expanding pancreatic stem cells, for example mouse
pancreatic stem cells comprises or consists of a basal medium (for example comprising Advanced
DMEM/F12, B27 (50x), n-Acetylcysteine (1 mM), primocin and/or P/S (antibiotics), Hepes and
glutamin/glutamax), any one of Rspondin 1-4 (optionally conditioned medium) (preferably
1ug/ml), Noggin (optionally conditioned medium) (preferably 100ng/ml), nicotinamide (preferably
mM), EGF (preferably 50 ng/ml), FGF10 (preferably 100 ng/ml), gastrin (preferably 100 nM),
and a TGF-beta inhibitor, for example A83-01 (preferably 2 uM). In a further embodiment, this
culture medium additionally comprises a Rock inhibitor, for example LY2157299 (preferably 10
uM). In some embodiments, the culture medium for pancreatic cells further comprises a p38
inhibitor, for example SB202190.
In some embodiments, the invention provides a culture medium for differentiating pancreas cells
comprising or consisting of a basal medium, Noggin, EGF, FGF10, gastrin, a TGF-beta inhibitor,
gamma-secretase inhibitor and preferably exendin 4.
In some embodiments, the culture medium for differentiating pancreatic stem cells, for example
human pancreatic stem cells comprises or consists of a basal medium (for example comprising
Advanced DMEM/F12, B27 (50x), n-Acetylcysteine (1 mM) and glutamin/glutamax), Noggin
(preferably 100 ng/ml), EGF (preferably 50 ng/ml), FGF10 (preferably 10 nM), gastrin (preferably
100 nM), a TGF-beta inhibitor, for example A83-01 (preferably 50nM) and gamma-secretase
inhibitor (DAPT/DBZ) (preferably 10 uM). In a further embodiment, this culture medium
additionally comprises Exendin 4 (preferably 50 ng/ml). In some embodiments, the culture
medium for pancreatic cells further comprises a p38 inhibitor, for example SB202190.
In some embodiments, the culture medium for differentiating pancreatic stem cells, for example
mouse pancreatic stem cells comprises or consists of a basal medium (for example comprising
Advanced DMEM/F12, B27 (50x), n-Acetylcysteine (1 mM) and glutamin/glutamax), EGF
(preferably 50 ng/ml) and gamma-secretase inhibitor (for example DAPT/DBZ) (preferably 10
uM).
Barrett’s Esophagus culture medium
In some embodiments, the culture medium for Barrett’s Esophagus, comprises or consists of a
basal medium, for example as described above, additionally comprising: a Wnt agonist, such as
recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP inhibitor, such as
Noggin; Rspondin, such as human Rspondin-1; a TGF-beta inhibitor, such as A83-01; a p38
inhibitor, such as SB202190; gastrin; nicotinamide; and an FGF, such as human FGF10 (i.e.
HISC+FGF). In some embodiments, gastrin is excluded from this culture medium.
In some embodiments, the invention provides a method for obtaining a Barrett’s Esophagus
organoid, wherein the method comprises culturing isolated epithelium from Barrett’s Esophagus
for 1, 2, 3, 4, 5, 6, 7 or more days in HISC culture medium, optionally additionally comprising
FGF10; and withdrawing nicotinamide and SB202190 after the first 1, 2, 3, 4 or more days. In
some embodiments the culture medium additionally comprises a Notich inhibitor, such as DBZ. In
some embodiments, a Barrett’s Esophagus organoid cultured in the presence of a Notch inhibitor
comprises almost no or no proliferating cells, and comprises more goblet cells relative to an
organoid cultured in the absence of a Notch inhibitor (see figure 5).
Liver culture media
In some embodiments, liver cells can be grown in a first “expansion” culture medium (also
referred to herein as EM), preferably followed by culturing the cells in a second “differentiation”
culture medium (also referred to herein as DM). However, in some embodiments, the step of
differentiating in DM media is not carried out, for example in some methods, cells are transplanted
and allowed to differentiate in vivo. Similarly, there are expansion culture media and
differentiation culture media for other tissues, such as the pancreas, small intestine and colon (see
above).
In one embodiment, the liver expansion medium comprises EGF, a Wnt agonist, FGF, and
Nicotinamide. Preferably, the Wnt agonist is R-spondin 1-4 (for example any one or more of
Rspondin 1, 2, 3, and 4) and so the expansion medium is referred to as “ERFNic”. A particularly
preferred expansion medium additionally comprises HGF and is referred to as “ERFHNic”.
In some embodiments, the liver expansion medium is supplemented with a TGF beta inhibitor. In
some embodiments, TGF beta is present at at least 5 nM, for example, at least 50nM, at least
100nM, at least 300nM, at least 450nM, at least 475nM, for example 5nM-500mM, 10nM-
100mM, 50nM-700uM, 50nM-10uM, 100nM-1000nM, 350-650nM or more preferably 500nM.
The presence of a TGF beta inhibitor in the expansion media is particularly preferred for human
cell embodiments.
In some embodiments, the invention provides a culture medium for expanding liver cells,
comprising or consisting of a basal medium, any one of Rspondin 1-4, Noggin, nicotinamide,
EGF, FGF10, HGF, gastrin, a TGF-beta inhibitor and PGE2, and preferably Wnt-3a.
In some embodiments, the liver expansion medium further comprises a p38 inhibitor.
In some embodiments, the liver expansion medium is supplemented with an activator of the
prostaglandin signalling pathway (also called a prostaglandin pathway activator) (see Figure 24).
For example, the liver expansion medium may be supplemented with any one or more of the
compounds selected from the list comprising: Phospholipids, Arachidonic acid (AA),
prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2
(PGH2), prostaglandin D2 (PGD2). For example, in some embodiments, the liver expansion
medium is supplemented with PGE2 and/or AA. In some embodiments, PGE2 is added to the liver
expansion medium to a final concentration of at least 10 nM, at least 30nM, at least 40nM, at least
45 nM, at least 50nM, for example between 10 nM and 500 nM, between 10 nM and 400 nM,
between 10 nM and 300 nM, between 10 nM and 200 nM, between 10 nM and 100 nM, between
20 nM and 50 nM. In a preferred embodiment, PGE2 is added to the liver expansion medium to a
final concentration of 50 nM. In some embodiments, AA is added to the liver expansion medium
to a final concentration of at least 1 ug/ml, for example at least 3 ug/ml, at least 5 ug/ml, at least 8
ug/ml, at least 9 ug/ml, at least 10 ug/ml, between 1 ug/ml and 1000 ug/ml, between 1 ug/ ml and
500 ug/ml, between 1 ug/ml and 100 ug/ml, between 1 ug/ml and 50 ug/ml, or between 5 ug/ml
and 10 ug/ml. In a preferred embodiment, AA is added to the medium to a final concentration of
ug/ml.
In a preferred embodiment, the liver expansion medium is supplemented with both a TGF-beta
inhibitor and an activator of the prostaglandin signalling pathway (for example, PGE2 and/or AA)
and optionally a p38 inhibitor.
In preferred embodiments, the liver expansion medium additionally comprises gastrin.
In one embodiment, the liver differentiation medium comprises EGF, a TGF-beta inhibitor, FGF
(for example, FGF10, FGF2 or any other suitable FGF family member) and a Notch inhibitor. In
one embodiment, the TGF-beta inhibitor is A83-01 and/or the Notch inhibitor is DAPT. This
differentiation medium is referred to herein as “EAFD” and is a preferred differentiation medium
of the invention. FGF may optionally be replaced by HGF or alternatively both FGF and HGF may
be present or absent in the differentiation medium. In some embodiments, EGF might be replaced
by HGF or another receptor tyrosine kinase ligand. Dexamethasone may also be added, for
example at a concentration of between 10nM to 10uM. The liver differentiation medium may
optionally include a prostaglandin pathway activator, such as PGE2 or AA. However, this
component may also be excluded from the differentiation medium. In some embodiments,
oncostatin M may also be added, for example at a concentration range between 1ng/ml to 1mg/ml,
to help differentiation to hepatocyte fate.
In some embodiments, the invention provides a culture medium for differentiating liver cells
comprising or consisting of a basal medium, Noggin, EGF, gastrin, a TGF-beta inhibitor, a
gamma-secretase inhibitor such as DAPT or DBZ, and preferably Wnt-3a.
In some embodiments, the liver cells may initially be cultured in an expansion medium that
additionally contains Wnt and Noggin, for example an “ENRW” medium containing EGF,
Noggin, R-spondin and Wnt (for example, Wnt-3A), optionally a prostaglandin pathway activator,
such as PGE2 or AA, optionally a TGF beta inhibitor and optionally FGF, HGF, Nicotinamide. In
a preferred embodiment, the liver expansion medium is supplemented with one of or more
preferably both of a TGF-beta inhibitor and an activator of the prostaglandin signalling pathway
(for example, PGE2 and/or AA).
In some embodiments, the expansion media for liver comprises EGF, Noggin, Gastrin, FGF,
Nicotinamide, a TGF-beta inhibitor such as A83-01, HGF, RSpondin 1-4 (e.g. any one or more of
Rspondin 1, 2, 3 and 4) and PGE2.
In a preferred embodiment, the liver cells may initially be cultured in an expansion media that
contains EGF, noggin, gastrin, FGF10, nicotinamide, A8301, HGF and any one of Rspondin 1-4
supplemented with PGE2 and/or AA. Rspondin 1-4 may be provided in the form of Rspo
conditioned media. For example, the expansion media may contain EGF (100ng/ml, Invitrogen);
noggin (25ng/ml, peprotech); gastrin (10nM, sigma); FGF10 (e.g. 100ng/ml, peprotech);
nicotinamide (10mM, sigma); A8301 (500nM, Tocris); HGF (50ng/ml, peprotech); Rspo
conditioned media (10%, e.g. 1ug/ml) supplemented with PGE2 (50nM) and/or AA (10 ug/ml).
The expansion medium may also contain a Rock inhibitor.
When expanding mouse liver cells, one or more of the following components may be excluded
from the culture medium described above: TGF-beta inhibitor (e.g. A83-01) and PGE2.
The inventors have found that this medium is optimal for stimulating initial expansion of cells for
the first few days. Therefore, this first expansion medium is sometimes referred to herein as EM1.
In some embodiments, the Wnt and Noggin are removed after approximately 1 day, 2 days, 3 days,
4 days, 5 days, 6 days, 7 days or more, for example 2 weeks, 1 month, 5 weeks, 8 weeks, 2 months
3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, 14 months or more. In some embodiments, the cells
may then be expanded in an expansion medium of the invention, as described above that does not
contain Wnt or Noggin. This second expansion medium is sometimes referred to herein as EM2. In
some embodiments, the cells are cultured in EM2 for approximately 1 day, 2 days, 3 days, 4 days,
days, 6 days, 7 days or a longer time period, such as 3, 4, 5, 10, 20 or more weeks. The culture
medium may then be changed to an optimised differentiation medium, as described above, that
contains a TGF-beta inhibitor and a Notch inhibitor. Typically, the differentiation medium does
not contain a Wnt agonist, R-spondin or Nicotinamide. In some embodiments, the differentiation
medium does not contain a prostaglandin pathway activator, such as PGE2 or AA. This encourages
the differentiation of the cells towards mature hepatocytes and cholangyocytes. These cells are
suitable for transplantation into humans or animals.
Expansion Medium (EM2) for liver:
In one aspect of the present invention there is provided a cell culture medium which comprises or
consists of a basal medium for animal or human cells to which is added: Epidermal Growth Factor,
an FGF able to bind to FGFR2 or FGFR4, preferably FGF10 as a mitogenic growth factor,
Nicotinamide, and preferably, a Wnt agonist, preferably R-spondin 1-4. This medium is referred to
as EM2. This “EM2” medium is preferred for expanding liver cells.
In some embodiments, EM2 comprises a prostaglandin pathway activator such as PGE2 and/or
AA.
In some embodiments, EM2 comprises a TGF-beta inhibitor such as A83-01.
Preferably, the Wnt agonist is R-spondin 1-4. A medium comprising EGF, R-spondin 1-4, FGF
and Nicotinamide is referred to herein as ERFNic.
In some embodiments, the EM2 medium does not comprise noggin, and more preferably does not
comprise a BMP-inhibitor. In some embodiments, the EM2 medium does not comprise Wnt, for
example Wnt-3a.
In some embodiments, HGF is present in addition to FGF. A preferred medium comprising HGF
in addition to FGF is ERFHNic (EGF + R-spondin (preferably R-spondin1-4) + FGF (preferably
FGF10) + HGF + Nicotinamide + a prostaglandin pathway inhibitor such as PGE2 and/or AA and
a TGF-beta inhibitor. The inventors have found that the ERFHNic medium containing the TGF-
beta inhibitor and the prostaglandin pathway activator is the optimal medium for long-term
expansion of cells. In the absence of HGF, cells did not remain viable in culture for longer than
three months. Further, in the absence of HGF, after 10 passages, cells showed a growth
disadvantage compared to cells cultured in the presence of HGF as evidenced by a lower
proliferation ratio. In particular, after 15 passages, the cells were not growing organoids at the
same speed ratio in the absence of HGF as in the presence of HGF. Thus, HGF was found to be
essential for maintaining a good proliferation rate during long-term culture. Thus the invention
provides the use of an ERFHNic medium of the invention for culturing cells for at least 2 weeks, at
least 1 month, at least 2 months, more preferably at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 24, at least 25, at least 30 or more
months, for example 3 or more years. In practice, some embodiments of the invention comprise
the use of EM2 for around 20-50 passages of the cells. For example, the cells may be split 4-8
times once a week for 7-10 weeks in a row. Preferably the cells will expand at a rate of about 4-5
fold per week or more than two population doublings a week. The invention further provides the
use of an ERFHNic medium of the invention for culturing cells for at least 8 passages, for
example, at least 9, at least 10, at least 11, at least 12, at least 15, at least 20, at least 25, at least 30,
at least 40, at least 50, at least 60 passages or for between 15-35 passages, for example
approximately 20-30 passages. In some embodiments, a TGF-beta inhibitor such as A83-01 is
additionally present in the EM2 medium. This is particularly useful when human cells or
organoids are being cultured. In some embodiments, the A83-01 is present at a concentration of
between 400-600 nM, for example 450-550 nM, 470-530 nM or approximately 500 nM. In
embodiments in which a TGF-beta inhibitor is present in EM2, a Notch inhibitor is preferably not
present. In some embodiments, EM2 additionally comprises a p38 inhibitor.
Expansion Medium (EM1) for liver:
In one aspect, the invention provides a cell culture medium comprising or consisting of a basal
medium for animal or human cells to which is added EGF, a BMP inhibitor, R-spondin Wnt.
Preferably, the BMP inhibitor is Noggin and the EM1 medium is termed “ENRW” (EGF, Noggin,
R-spondin and Wnt (e.g. Wnt3A)). This medium is referred to as EM1. In some embodiments,
EM1 additionally comprises a prostaglandin pathway activator such as PGE2 and/or AA. In some
embodiments, EM1 comprises a TGF-beta inhibitor such as A83-01. More preferably, the EM1
additionally comprises a prostaglandin pathway activator and a TGF-beta inhibitor. The inventors
have found that a medium containing Wnt and Noggin is ideal for stimulating initial expansion of
cells. Thus, in some embodiments, the EM1 medium is used for just 1 passage or 1 week but it is
also envisaged that EM1 medium can be used for around a year because it is not harmful for the
cells. Thus, in some embodiments, an EM1 medium is used for culturing cells from day 0 to day
, for example from days 0-7, days 0- 6, days 0-5, days 0-4, days 0-3, days 0-2, days 0-1, wherein
day 0 is the day that the cells are isolated from their tissue of origin and day 1 is the subsequent
day or is used for 1 or more weeks for example 2, 3, 4 or more weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12 or more months. In some embodiments, the EM1 medium is used only for the first day or
first two days of culture. In some embodiments, EM1 medium is used for 1 or more passages, for
example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more passages, for example, 20-30 passages,
-35 passages, 32-40 passages or more. In some embodiments, the EM1 medium is used
subsequent to a freezing step or any other transportation step involving a medium or temperature
change that does not combine with optimal growth. This “EM1” medium is preferred for
expanding liver cells.
The EM1 medium is supplemented with one or more of the compounds selected from the group
consisting of FGF, HGF, Nicotinamide, gastrin, B27, N-acetylcystein and N2. In the case of
starting the cultures from a frozen stock or from a single cell, the EM1 media is preferably
supplemented with a ROCK inhibitor. Y27632 is the preferred ROCK inhibitor for use in the
invention.
Thus, in one embodiment there is provided a cell culture medium which comprises or consists of a
basal medium for animal or human cells to which is added: Epidermal Growth Factor, an FGF (for
example, an FGF able to bind to FGFR2 or FGFR4), preferably FGF10 and HGF as mitogenic
growth factors,
a prostaglandin pathway activator, such as PGE2 and/or AA;
a TGF-beta inhibitor;
gastrin, Nicotinamide, B27, N2 and N-Acetylcysteine, and preferably;
a BMP inhibitor, preferably Noggin; and
a Wnt agonist, preferably R-spondin 1 and/or Wnt-3a.
B27 (Invitrogen), N-Acetylcysteine (Sigma) and N2 (Invitrogen), Gastrin (Sigma) and
Nicotinamide (Sigma) are also added to the medium defined above and are believed to control
proliferation of the cells and assist with DNA stability. In the context of the invention,
Nicotinamide is also referred to herein as “Nic”.
‘N2 Supplement’ is available from Invitrogen, Carlsbad, CA; www.invitrogen.com; catalog no.
17502-048; and from PAA Laboratories GmbH, Pasching, Austria; www.paa.com; catalog no.
F005-004; Bottenstein & Sato, PNAS, 76(1):514-517, 1979. N2 Supplement is supplied by PAA
Laboratories GmbH as a 100x liquid concentrate, containing 500μg/ml human transferrin,
500μg/ml bovine insulin, 0.63μg/ml progesterone, 1611μg/ml putrescine, and 0.52μg/ml sodium
selenite. N2 Supplement may be added to a culture medium as a concentrate or diluted before
addition to a culture medium. It may be used at a 1x final concentration or at other final
concentrations. Use of N2 Supplement is a convenient way to incorporate transferrin, insulin,
progesterone, putrescine and sodium selenite into a culture medium of the invention. In some
embodiments in which the medium comprises B27, it does not also comprise N2. The
embodiments of the present invention can therefore be adapted to exclude N2 when B27 is present,
if desired.
‘B27 Supplement’ (available from Invitrogen, Carlsbad, CA; www.invitrogen.com; currently
catalog no. 17504-044; and from PAA Laboratories GmbH, Pasching, Austria; www.paa.com;
catalog no. F01-002; Brewer et al., J Neurosci Res., 35(5):567-76, 1993) may be used to formulate
a culture medium that comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone,
putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol
(vitamin E), albumin, insulin and transferrin. B27 Supplement is supplied by PAA Laboratories
GmbH as a liquid 50x concentrate, containing amongst other ingredients biotin, cholesterol,
linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-
iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin. Of these
ingredients at least linolenic acid, retinol, retinyl acetate and tri-iodothyronine (T3) are nuclear
hormone receptor agonists. B27 Supplement may be added to a culture medium as a concentrate or
diluted before addition to a culture medium. It may be used at a 1x final concentration or at other
final concentrations. Use of B27 Supplement is a convenient way to incorporate biotin, cholesterol,
linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-
iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin into a
culture medium of the invention.
For example, a cell culture medium may comprise or consist of a basal medium to which is added:
EGF and R-spondin 1 supplemented with FGF10, HGF and Nicotinamide; for example, EGF (50
ng/ml) and R-spondin 1 (1ug/ml) supplemented with FGF10 (100ng/ml), HGF (25-50ng/ml),
Nicotinamide (1-10mM), a prostaglandin pathway activator, such as PGE2 (50 nM) and/or AA (10
ug/ml) and a TGF-beta inhibitor such as A83-01 (500nM). In some embodiments the medium
additionally comprises a p38 inhibitor. The inventors have found that this medium may be used for
long-term expansion of cells. Thus, this cell culture medium is preferred for use as an EM2 of the
invention. The basal medium is preferably supplemented with B27, N2 and 200ng/ml N-
Acetylcysteine. In some embodiments, the basal medium is Advanced-DMEM/F12. However,
any other suitable basal medium may be used.
Another example of a cell culture medium, and method of using this medium comprises or
consists of Advanced-DMEM/F12 preferably supplemented with B27, N2, 200ng/ml N-
Acetylcysteine, 50ng/ml EGF, 1 µg/ml R-spondin1, 10 nM gastrin, 100 ng/ml FGF10, 10mM
Nicotinamide, 50ng/ml HGF, 50% Wnt conditioned media, a prostaglandin pathway activator,
such as PGE2 (50 nM) and/or AA (10 ug/ml) and a TGF-beta inhibitor such as A83-01 (500nM)
and, preferably 10-100ng/ml Noggin. Wnt conditioned media comprises Advanced DMEM, P/S,
B27, N2 and also FCS. 293T cells transfected with Wnt3A expression plasmid produce Wnt. The
whole medium is taken after a few days (i.e. with secreted Wnt) and used as the Wnt source.
The invention therefore provides a cell culture medium, comprising or consisting of a basal
medium for animal or human cells to which is added:
Epidermal Growth Factor, an FGF able to bind to FGFR2 or FGFR4, preferably FGF10 and
HGF as mitogenic growth factors,
a prostaglandin pathway activator, such as PGE2 and/or AA,
a TGF-beta inhibitor;
gastrin, Nicotinamide, B27, N2 and N-Acetylcystein, and preferably
a BMP inhibitor more preferably Noggin and
a Wnt agonist, more preferably R-spondin 1 and/or Wnt-3a.
The invention thus encompasses a first preferred culture medium comprising or consisting of a
basal medium for animal or human cells to which is added:
Epidermal Growth Factor, FGF10 and HGF as mitogenic growth factors,
a prostaglandin pathway activator, such as PGE2 and/or AA,
a TGF-beta inhibitor;
gastrin, Nicotinamide, B27, N2 and N-Acetylcysteine,
a BMP inhibitor more preferably Noggin and
a Wnt agonist, more preferably R-spondin 1 and Wnt-3a.
In some embodiments, a p38 inhibitor is added to the expansion medium.
This medium may be used as an EM1 cell culture medium of the invention to stimulate initial
expansion of cells. In some embodiments, the medium used as an EM1 cell culture medium
comprises all the components of an EM2 culture medium of the invention and additionally
comprises Wnt-3a and Noggin.
In embodiments in which the basal medium is supplemented with N-Acetylcysteine, B27 and N2,
the following are preferably added to the culture media: EGF, R-spondin1, gastrin, FGF10,
Nicotinamide and HGF and Wnt-conditioned media and a prostaglandin pathway activator, such as
PGE2 and/or AA. Preferably, the basal medium is supplemented with N-Acetylcysteine, EGF, R-
spondin1, gastrin, FGF10, Nicotinamide and HGF and Wnt-conditioned media and a prostaglandin
pathway activator, such as PGE2 and/or AA in accordance with the quantities described
hereinabove. Preferably, a TGF-beta inhibitor is also present at the quantities described herein.
For example, in some embodiments the basal medium may be supplemented with 150ng/ml to 250
ng/ml N-Acetylcysteine; preferably, the basal medium is supplemented with, about or exactly
200ng/ml N-Acetylcysteine. For example, in some embodiments the basal medium may be
supplemented with 40ng/ml to 60ng/ml EGF; preferably, the basal medium is supplemented with
about or exactly 50ng/ml EGF. For example, in some embodiments the basal medium may be
supplemented with 0.5 µg/ml to 1.5 µg/ml R-spondin1; preferably, the basal medium is
supplemented with about or exactly 1 µg/ml R-spondin1. For example, in some embodiments the
basal medium may be supplemented with 5nM to 15nM gastrin; preferably, the basal medium is
supplemented with about or exactly 10nM gastrin. For example, in some embodiments the basal
medium may be supplemented with 25-200ng/ml FGF10, for example
70 ng/ml to 130 ng/ml FGF10; preferably, the basal medium is supplemented with about or exactly
100 ng/ml FGF10. For example, in some embodiments the basal medium may be supplemented
with 5mM to 15mM Nicotinamide; preferably, the basal medium is supplemented with about or
exactly 10mM Nicotinamide. For example, in some embodiments the basal medium may be
supplemented with 25ng/ml to 100 ng/ml HGF, for example 35ng/ml to 65ng/ml HGF; preferably,
the basal medium is supplemented with about or exactly and 50ng/ml HGF. For example, in some
embodiments the basal medium may be supplemented with 35% to 65% Wnt-conditioned media;
preferably, the basal medium is supplemented with about or exactly 50% Wnt-conditioned media.
For example, in some embodiments, the liver expansion medium is supplemented with an activator
of the prostaglandin signalling pathway (see Figure 24). For example, the liver expansion medium
may be supplemented with any one or more of the compounds selected from the list comprising:
Phospholipids, Arachidonic acid (AA), prostaglandin E2 (PGE2), prostaglandin G2 (PGG2),
prostaglandin F2 (PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For example, in
some embodiments, the liver expansion medium is supplemented with PGE2 and/or AA. In some
embodiments, PGE2 is added to the liver expansion medium to a final concentration of at least 10
nM, for example between 10 nM and 500 nM, between 10 nM, and 400 nM, between 10 nM and
300 nM, between 10 nM and 200 nM, between 10 nM and 100 nM, between 20 nM and 50 nM. In
a preferred embodiment, PGE2 is added to the liver expansion medium to a final concentration of
50 nM. In some embodiments, AA is added to the liver expansion medium to a final concentration
of at least 1 ug/ml, for example between 1 ug/ml and 1000 ug/ml, between 1 ug/ ml and 500 ug/ml,
between 1 ug/ml and 100 ug/ml, between 1 ug/ml and 50 ug/ml, or between 5 ug/ml and 10 ug/ml.
In a preferred embodiment, AA is added to the medium to a final concentration of 10 ug/ml.
In some embodiments one or both of gastrin and N2 are not present in the cell culture medium.
Preferably, the basal medium is advanced-DMEM/F12.
This first culture medium (for example, EM1, EM2 or both EM1 and EM2) is preferably used
during the first two weeks of the culture method of the invention. However, it may be used for a
shorter time period, such as for 1, 2, 3, 5, 7, or 10 days, or a longer time period, such as 3, 4, 5, 10,
20 or more weeks, 5 months or more, for example, 6, 7, 8, 9, 10, 11, 12 months or more.
Differentiation Medium (DM) for liver:
In another aspect, there is provided a second cell culture medium which comprises or consists of a
basal medium for animal or human cells to which is added: EGF, a TGF-beta inhibitor, a Notch
inhibitor and a prostaglandin pathway activator, such as PGE2 and/or AA. The inventors have
found that this medium is useful for differentiating cells. The medium used for differentiating the
cells may be referred to herein as DM.
Preferably, the second cell culture medium also comprises FGF and/or HGF.
In one embodiment, the second culture medium comprises or consists of a basal medium for
animal or human cells to which is added:
Epidermal Growth Factor, FGF10 and HGF as mitogenic growth factors;
a Notch inhibitor;
a TGF-beta inhibitor; and
a prostaglandin pathway activator, such as PGE2 and/or AA.
In one embodiment, the TGF-beta inhibitor is A83-01 and/or the Notch inhibitor is DAPT. In
another embodiment, the DM cell culture medium additionally comprises Dexamethasone. In
another embodiment, the DM cell culture medium additionally comprises Oncostatin M. In another
embodiment, the DM cell culture medium additionally comprises gastrin.
A preferred second cell culture medium, and method of using this medium, is described in the
examples, and comprises or consists of a basal medium to which is added: 50ng/ml EGF, 100
ng/ml FGF10, 50 nM A8301 and 10 uM DAPT. Advanced-DMEM/F12 may be used as the basal
medium as may any other suitable basal medium.
In some embodiments, the differentiation medium for liver cells, for example for human liver
cells, comprises or consists of a basal medium (for example comprising Advanced DMEM/F12,
B27 (50x), n-Acetylcystein (1 mM) glutamin/glutamax), Noggin (preferably 100 ng/ml), EGF
(preferably 50 ng/ml), gastrin (preferably 10nM), TGF-beta inhibitor, such as A83-01 (preferably
50 nM) and a gamma-secretase inhibitor (for example DAPT/DBZ) (preferably 10 uM).
In some embodiments, the differentiation medium for liver cells, for example for mouse liver cells,
comprises or consists of a basal medium (for example comprising Advanced DMEM/F12, B27
(50x), n-Acetylcystein (preferably 1mM) glutamin/glutamax), EGF (preferably 50 ng/ml), FGF10
(preferably 100 ng/ml) gastrin (preferably 10nM), TGF-beta inhibitor, such as A83-01 (preferably
50 nM) and a gamma-secretase inhibitor (for example DAPT/DBZ) (preferably 10 uM).
In some embodiments, the second cell culture medium does not comprise R-spondin or Wnt. In
some embodiments, the second cell culture medium does not comprise a Wnt agonist. In some
embodiments, the second cell culture medium does not comprise Nicotinamide. In some
embodiments, the second cell culture medium does not comprise a BMP inhibitor. In some
embodiments, the second cell culture medium does not comprise a prostaglandin pathway
activator, such as PGE2 and/or AA.
The inventors have discovered that R-spondin1 and Nicotinamide both inhibit the expression of
the mature hepatocyte marker CYP3A11 and yet promote the expression of the hepatoblast marker
albumin. Therefore, to increase differentiation of the cells to more mature liver fates, the inventors
removed R-spondin and Nicotinamide from the cell culture. The inventors have also discovered
that the expression of specific biliary transcription factors is highly upregulated in expansion
cultures containing R-spondin1, indicating that the culture gene expression was unbalanced
towards a more biliary cell fate. Notch and TGF-beta signaling pathways have been implicated in
biliary cell fate in vivo. In fact, deletion of Rbpj (essential to achieve active Notch signalling)
results in abnormal tubulogenesis (Zong Y. Development 2009) and the addition of TGF-beta to
liver explants facilitates the biliary differentiation in vitro (Clotman F. Genes and Development
2005). Since both Notch and TGF-beta signalling pathways were highly upregulated in the liver
cultures (Figure 22) the inventors reasoned that inhibition of biliary duct cell-fate might trigger the
differentiation of the cells towards a more hepatocytic phenotype. It was found that addition of a
TGF-beta inhibitor (such as A8301) and a Notch inhibitor (such as DAPT) to a differentiation
medium that preferably does not contain R-spondin or Wnt, enhances the expression of mature
hepatocyte markers and increases the number of hepatocyte-like cells (for example, see example
General culture media
A cell culture medium according to the invention allows the survival and/or proliferation and/or
differentiation of epithelial stem cells or isolated crypts on an extracellular matrix. In some
embodiments, a cell culture medium according to the invention allows the survival and/or
proliferation and/or differentiation of an organoid of the invention, such as a crypt-villus organoid,
a colon organoid, a pancreatic organoid, a gastric organoid, a Barret’s Esophagus organoid, an
adenocarcinoma organoid or a colon carcinoma organoid on an extracellular matrix. In some
embodiments, a cell culture medium according to the invention allows the survival and/or
proliferation and/or differentiation of an organoid of the invention, such as a small intestinal
(crypt-villus) organoid, a colon organoid, a pancreatic organoid, a gastric organoid, a Barret’s
Esophagus organoid, an adenocarcinoma organoid, a carcinoma organoid, a colon carcinoma
organoid, a prostate organoid or a prostate carcinoma organoid on an extracellular matrix.
Preferably, in embodiments in which a TGF-beta inhibitor and/or p38 inhibitor is present the cell
culture medium allows the survival and/or proliferation, preferably the survival and proliferation
of a population of cells or organoid of the invention. Preferably, embodiments in which a TGF-
beta inhibitor and/or p38 inhibitor is initially present in a cell culture medium but is then removed
from the medium (e.g. by failing to add it when the medium is refreshed), allow the survival and/or
differentiation, preferably the survival and differentiation of a population of cells or organoid of
the invention.
In some embodiments, a p38 inhibitor is added to any of the media described herein.
The term cell culture medium is synonymous with medium, culture medium or cell medium.
Uses of culture media of the invention
The invention also provides the use of a culture medium of the invention for expanding and/or
differentiating a stem cell, population of stem cells, tissue fragment or organoid.
In some embodiments, the stem cell, population of stem cells, tissue fragment or organoid is
selected from the group consisting of one or more intestinal stem cells, small intestinal crypts,
colonic crypts, gastric stem cells, liver stem cells, pancreas stem cells and prostate stem cells.
In some embodiments, the stem cell, population of stem cells, tissue fragment or organoid is
obtainable from a normal tissue.
In some embodiments, the stem cell, population of stem cells, tissue fragment or organoid is
obtainable from a diseased tissue, for example from an adenoma, a carcinoma, an adenocarcinoma,
an intestine of a patient having cystic fibrosis or an intestine of a patient having inflammatory
bowel disease.
Stem cells cultured according to the invention
Stem cells are found in many organs of adult humans and mice. Although there may be great
variation in the exact characteristics of adult stem cells in individual tissues, adult stem cells share
at least the following characteristics: they retain an undifferentiated phenotype; their offspring can
differentiate towards all lineages present in the pertinent tissue; they retain self-maintenance
capabilities throughout life; and they are able to regenerate the pertinent tissue after injury. Stem
cells reside in a specialised location, the stem cell niche, which supplies the appropriate cell-cell
contacts and signals for maintenance of said stem cell population. The stem cells described herein
preferably express Lgr5.
In one embodiment, the invention provides a population of cells or one or more organoids
comprising said stem cells that have been generated or obtained by culturing stem cells or tissue
fragments according to the invention, which have been cultured for at least 3 months, preferably at
least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least
12 months or more.
A ‘population’ of cells is any number of cells greater than 1, but is preferably at least 1x10 cells,
4 5 6 7 8
at least 1x10 cells, at least 1x10 cells, at least 1x10 cells, at least 1x10 cells, at least 1x10 cells,
or at least 1x10 cells.
The stem cells cultured according to the invention may be human stem cells. The stem cells
cultured according to the invention may be epithelial stem cells.
In some embodiments, the stem cells cultured according to the invention are not embryonic stem
cells. In some embodiments the stem cells cultured according to the invention are not human
embryonic stem cells. Preferably, the stem cells are adult stem cells.
In a preferred embodiment, the stem cells may be human epithelial stem cells. Human epithelial
stem cells include stem cells of human epithelial tissue origin. These include, but are not limited to
pancreatic, small intestinal, large intestinal, corneal, olfactory, respiratory tissues, gastric tissues,
liver and skin, mammary and/or prostatic tissues, for example, a tissue selected from the group
consisting of pancreatic, small intestinal, large intestinal, corneal, olfactory, and respiratory
tissues. Epithelial stem cells are able to form the distinct cell types of which the epithelium is
composed. Some epithelia, such as skin or intestine, show rapid cell turnover, indicating that the
residing stem cells must be continuously proliferating. Other epithelia, such as the liver or
pancreas, show a very slow turnover under normal conditions.
Intestinal stem cells
The self-renewing epithelium of the small intestine is ordered into crypts and villi (Gregoreff and
Clevers, 2005 Genes Dev 19, 877-90). Each cell along the crypt-villus axis is polarized, whereby
cells on the top of the intestinal villi, or in the upper positions of colonic crypts, are the most
differentiated and are continuously lost into the lumen by apoptosis. Continuous proliferation of
stem cells residing in the base of the crypts, and massive proliferation of progenitor cells residing
in the middle of the crypts, ensures proper replacement of the shed cells. There is a resulting
epithelial turnover time of 5 days in the mouse. Self-renewing stem cells have long been known to
reside near the crypt bottom and to produce the rapidly proliferating transit amplifying (TA) cells
capable of differentiating towards all lineages. The estimated number of stem cells is between 4
and 6 per crypt (Bjerknes and Cheng, 1999 Gastroenterology 1 16, 7- 14). Three differentiated cell
types, enterocytes, goblet cells and enteroendocrine cells, form from TA cells and continue their
migration in coherent bands along the crypt-villus axis. Each villus receives cells from multiple
different crypts. The fourth major differentiated cell-type, the Paneth cell, resides at the crypt
bottom.
The colon resembles the small intestine but with a flat surface epithelium rather than villi. Colon
crypts are organized like small intestinal crypts. Paneth cells are not present in colon crypts;
instead there are so-called “Deep Crypt Secretory” cells. The flat surface of the epithelium
contains the differentiated cells (colonocytes and secretory cells). Differentiated goblet cells occur
throughout the crypt, also intermingled with transit amplifying cells.
Isolation of tissue fragments and stem cells
Crypts can be isolated from the small and large intestine, including the duodenum, jejunum, ileum
and colon, and the pyloric and corpus region of the stomach by protocols that are known to the
skilled person. For example, crypts can be isolated by incubation of isolated tissue with chelating
agents that release cells from their calcium- and magnesium-dependent interactions with the
basement membrane and stromal cell types. After washing the tissue, the epithelial cell layer is
scraped from the submucosa with a glass slide and minced. This is followed by incubation in
trypsin or, more preferred, EDTA and/or EGTA and separation of undigested tissue fragments and
single cells from crypts using, for example, filtration and/or centrifugations steps. Other
proteolytic enzymes, such as collagenase and/or dispase I, can be used instead of trypsin. Similar
methods are used to isolate fragments of the pancreas and stomach. Similar methods may be used
to isolated fragments of other tissues described herein. The culture media of the invention are
suitable for culturing such tissue fragments (see Example 1).
A culture medium according to the invention allows the establishment of long-term culture
conditions under which single crypts undergo multiple crypt fission events, while simultaneously
generating villus-like epithelial domains in which all differentiated cell types are present. Cultured
crypts undergo dramatic morphological changes after taking them into culture. The upper opening
of freshly isolated crypts becomes sealed and this region gradually balloons out and becomes filled
with apoptotic cells, much like apoptotic cells are pinched off at the villus tip. The crypt region
undergoes continuous budding events which create additional crypts, a process reminiscent of
crypt fission. In a preferred embodiment, the organoids comprise crypt-like extensions which
comprise all differentiated epithelial cell types, including proliferative cells, Paneth cells,
enterocytes and goblet cells. No myofibroblasts or other non-epithelial cells were identified in the
organoids at any stage.
Expansion of the budding crypt structures creates organoids, comprising crypt-like structures
surrounding a central lumen lined by a villus-like epithelium and filled with apoptotic cell bodies.
The crypt-villus organoids comprise a central lumen lined by a villus-like epithelium. The lumen is
opened at consecutive time intervals to release the content into the medium.
A similar crypt-villus organoid structure is formed when single epithelial stem cells are cultured.
After about one week, structures are formed that strongly resemble the crypt-villus organoid
structures that are obtained with intact crypts.
Methods to isolate stem cells are known and suitable methods for use with this invention can be
selected by the skilled person depending on the stem cell type that is used. For example, isolation
of epithelial stem cells may be performed using compounds that bind to Lgr5 and/or Lgr6, which
are unique cell surface markers on epithelial stem cells. Examples of such compounds are anti-
Lgr5 and anti-Lgr6 antibodies.
In some embodiments, single Lgr5+ epithelial stem cells, for example from the colon, small
intestine, or pancreas, may be used to form organoids, such as colonic, crypt-villus or pancreatic
organoids respectively.
In a further example, single Lgr5+ epithelial stem cells from the liver, prostate or stomach may be
used to obtain organoids, such as liver, prostate or gastric organoids respectively.
In an alternative embodiment, tissue fragments, such as cultured crypts from the intestinal tract,
comprising Lgr5+ stem cells may be used to obtain organoids using methods and culture media
described herein.
In some embodiments the single Lgr5+ epithelial stem cell or tissue fragment may be a cancer
stem cell or cancer tissue fragment, for example from a carcinoma or adenocarcinoma. In some
embodiments the single Lgr5+ epithelial stem cell may be a stem cell or tissue fragment from a
neoplastic pathology or diseased tissue, for example Barrett’s esophagus, cystic fibrosis or
adenoma. Organoids obtained from cancerous, neoplastic or diseased starting material have
characterisitics resembling the in vivo starting material and therefore are useful as a research tool
for drug screening, target validation, target discovery, toxicology and toxicology screens,
personalized medicine, regenerative medicine and ex vivo cell/organ models, for example disease
models.In one embodiment, the invention provides organoids generated or obtained by culturing
human stem cells or tissue fragments according to a method of the invention. In one embodiment,
the invention provides crypt-villus organoids or gastric organoids or pancreatic organoids or colon
organoids or Barrett’s Esophagus organoids or adenocarcinoma organoids or colon carcinoma
organoids generated or obtained by culturing human stem cells or tissue fragments according to a
method of the invention. In one embodiment, the invention provides prostate organoids generated
or obtained by culturing human stem cells or tissue fragments according to a method of the
invention. Such a population of organoids, for example, crypt-villus, gastric or pancreatic
organoids, generated or obtained by culturing human stem cells or tissue fragments according to a
method of the invention, may each comprise more than 10, preferably more than 20, more
preferably more than 40 organoids. Said collection of organoids preferably comprises at least 10%
viable cells, more preferred at least 20% viable cells, more preferred at least 50% viable cells,
more preferred at least 60% viable cells, more preferred at least 70% viable cells, more preferred at
least 80% viable cells, more preferred at least 90% viable cells. Viability of cells may be assessed
using Hoechst staining or Propidium Iodide staining in FACS.
The inventors have shown that the culture media and methods of the invention may be used for
culture of cancer cell lines, including colorectal cancer and adenocarcinoma (see Example 1). As
explained in Example 1, the culture technology is widely applicable as a research tool for
infectious, inflammatory and neoplastic pathologies. Accordingly, the stem cells described herein
may be cancer stem cells. In some embodiments, cancer stem cells can form adenoma or colon
cancer organoids. In some embodiments, these organoids comprise cells which are Ki67+ (Thermo
Scientific* Cellomics, Millipore).
Similarly, the inventors have shown that the culture media and methods of the invention may be
used for culturing stem cells with other diseased genotypes and/or phenotypes. For example,
intestinal stem cells taken from patients with cystic fibrosis can be expanded using the culture
media and methods of the invention. These stem cells maintain the cystic fibrosis genotype and
phenotype. Therefore, in some embodiments of the invention, the stem cells are taken from a
patient with a disease, for example cystic fibrosis, inflammatory bowel disease (such as Crohn’s
disease), carcinoma, adenoma, adenocarcinoma, colon cancer, diabetes (such as type I or type II),
Barrett’s esophagus Gaucher’s disease, alphaantitrypsin deficiency, Lesch-Nyhan syndrome,
anaemia, Schwachman-Bodian-Diamond syndrome, polycythaemia vera, primary myelofibrosis,
glycogen storage disease, familial hypercholestrolaemia, Crigler-Najjar syndrome, hereditary
tyrosinanaemia, Pompe disease, progressive familial cholestasis, Hreler syndrome, SCID or leaky
SCID, Omenn syndrome, Cartilage-hair hypoplasia, Herpes simplex encephalitis, Scleroderma,
Osteogenesis imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy, Dyskeratosis
congenitor, etc. In some embodiments of the invention, disease organoids can be obtained by
culturing stem cells taken from a human or animal with a disease. Disease organoids still have
characteristics of the tissue from which they were obtained. Therefore, a cystic fibrosis small
intestinal organoid grown from a small intestinal crypt falls within the definition of a small
intestinal organoid. Similarly, a colon carcinoma organoid falls within the definition of a colon
organoid.
There is some confusion in the literature as to the definition of a cancer stem cell. Here, we follow
the consensus reached at a recent AACR workshop (Clarke et al., 2006. Cancer Res. 66:9339-44),
which states that the cancer stem cell "is a cell within a tumor that possesses the capacity to self-
renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. Cancer
stem cells can thus only be defined experimentally by their ability to recapitulate the generation of
a continuously growing tumor". Alternative terms in the literature include tumor-initiating cell and
tumorigenic cell. Assays for cancer stem cell activity need to address the potential of self-renewal
and of tumor propagation. The gold-standard assay currently is serial xeno-transplantation into
immunodeficient mice. In addition, cancer stem cells described herein normally express Lgr5.
However, in some embodiments, cancer initiating/propagating/stem cells that do not express Lgr5
can also be cultured by the culture media and methods of the invention.
Genomic and phenotypic integrity of stem cells and organoids comprising said stem cells
Clinical and research applications for stem cells and their differentiated progeny require
reproducible stem cell culture methods that provide populations of cells of suitable quality.
Generally, in vitro expansion of stem cells aims to provide a population of cells which resemble
their in vivo counterparts as closely as possible. This property is herein referred to as the “genomic
and phenotypic integrity” of the cells. Organoids obtained by culturing diseased cells, such as
cancer cells or cystic fibrosis cells, also resemble their in vivo counterparts i.e. they maintain their
disease genotype and/or phenotype and therefore, also maintain their “genomic and phenotypic
integrity” in that sense i.e. they maintain the genetic or phenotypic instability characteristic of the
disease that is remincent of the in vivo situation. Therefore, in some embodiments, the invention
provides “normal” organoids obtained from healthy tissue. In other embodiments, the invention
provides “disease” organoids, such as cancer organoids (for example, colon carcinoma organoids
or adenocarcinoma organoids) or cystic fibrosis small intestinal organoids obtained from diseased
tissue.
For the first time, the inventors have discovered that it is possible to expand human epithelial stem
cells in culture, with minimal loss of genomic and phenotypic integrity, for at least 3 months,
preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9
months, or at least 12 months or more (see Example 1). Under the improved culture conditions of
the invention, human intestinal organoids displayed budding organoid structures, rather than the
cystic structures seen under previous culture conditions. Metaphase spreads of organoids more
than 3 months old consistently revealed 46 chromosomes in each of the 20 cells taken from three
different donors. Furthermore, microarray analysis revealed that the stem cells in culture possessed
similar molecular signatures to intestinal crypt cells including intestinal stem cell genes.
Therefore, in some embodiments the invention provides organoids that have been grown for at
least 3 months, preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months,
at least 9 months, or at least 12 months or more with minimal loss of genomic and phenotypic
integrity.
In some embodiments, the invention provides human intestinal organoids comprising budding
structures. In some embodiments of the invention, human intestinal organoids do not comprise
cystic structures. In some embodiments of the invention, human intestinal organoids comprise
more budding structures than cystic structures.The inventors also demonstrated that the human
intestinal organoids generated by media and methods of the present invention, mimicked in vivo
cell fate decisions in response to external factors. For example, it has previously been shown that
Notch inhibition in intestinal stem cells, terminates intestinal epithelial proliferation and induces
goblet cell hyperplasia in vivo. The inventors were able to show that the intestinal organoids of the
invention, when treated with a Notch inhibitor, ceased proliferation and most cells converted into
goblet cells within 3 days.
These results show the dramatic improvement in the genomic and phenotypic integrity of the stem
cells and organoids produced by the methods and media of the present invention compared to
previous methods and media.
The genomic integrity of stem cells described herein can be confirmed by karyotype analysis.
Stem cells and their progeny can be karyotyped using known methods as described in Sato, T et
al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.
Nature 459, 262-265, 2009.
A “normal karyotype” is one where all chromosomes are present (i.e. euploidy) with no noticeable
alterations. Accordingly, in preferred embodiments more than 50%; more than 70%; more than
80%; more than 90%; more than 95%; or more than 99% of the stem cells and differentiated cells
in an expanded population exhibit normal karyotypes after 1, 2, 3, 4, 5, 6, 9, 12 or more months.
The term “expanded population” encompasses organoids.
A “normal phenotype” refers to cells which display, to a first approximation, the same visual
characteristics, gene expression and behaviour as the average in vivo counterpart cell. In preferred
embodiments more than 50%; more than 70%; more than 80%; more than 90%; more than 95%; or
more than 99% of the stem cells in an expanded population cultured according to the invention
exhibit normal phenotypes after 1, 2, 3, 4, 5, 6, 9, 12 or more months.
For example, visually a normal phenotype may be judged by the number of dead cells outside the
organoid, the amount of ‘budding’ of the organoid compared to cystic growth (budding structures
are preferred), and the overall integrity of the single layer of epithelial cells (e.g. columnar
squamous phenotype). In addition the cell types present may help to judge whether an organoid is
visually “normal”.
Preferred properties of the stem cells and organoids are outlined below.
Stem cell markers
When mouse genes are referred to herein, a human organoid of the invention may have a similar
gene profile but wherein the human gene counterparts are substituted for the mouse genes. Thus,
also described herein is a human organoid having a gene expression profile as described herein,
but in respect of the corresponding human genes. The human counterparts of the mouse genes
listed herein will be readily available to the skilled person.
In one embodiment, described herein is a population of adult stem cells characterised by natural
expression of Lgr5. In a preferred embodiment, described herein is a population of adult stem
cells characterised by natural expression of at least Lgr5 and one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23) of stem cell markers from the group
consisting of: LGR4, epcam, Cd24a, Cdca7, Axin, CK19, Nestin, Somatostatin, CXCR4 ,
CD133 , DCAMKL-1, CD44, Sord, Sox9, CD44, Prss23, Sp5, Hnf1α, Hnf4a, Sox9, KRT7 and
KRT19, Tnfrsf19. The stem cell markers may be tissue specific. For example, pancreatic stem
cells or organoids may be characterised by natural expression of one or more (for example 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 1314, or 15 for example, 1, 2, 3 or 4) of: CK19, Nestin, Somatostatin,
insulin, glucagon, CXCR4 , Ngn3, Pdx1, NeuroD, Nkx2.2, Nkx6.1, Pax6, Mafa, Hnf1b, optionally
Tnfrsf19 at a significant level; gastric organoids may be characterised by natural expression of one
or more (for example 1, 2, 3 or 4) of: CD133 , DCAMKL-1, CD44, optionally Tnfrsf19 at a
significant level; and crypt-villus organoids may be characterised by expression of one or more or
all (for example 1 or 2) of: Sord and/or Prss23, at a significant level or all genes of table/figure 14,
for example, at a significant level.
The term “significant level” as used herein in the context of marker expression is used
synonymously with the term “detectable level”, as described below.
Small intestinal and gastric organoid cell populations also express markers of progenitor
populations common to the small intestine and stomach, such as one or both of Cd44 and Sox9
(Barker & Huch et al Cell stem cell 2010). These are highly expressed in the stem cells described
herein. Cells according to this embodiment may also up-regulate Wnt target genes, including for
example, one, two or all of MMP7, Sp5 Tnfrs19 and axin2. This provides strong evidence of the
requirement for an active and robust canonical Wnt signalling activity to maintain the self
renewing capacity of these cultures.
The inventors have observed that expression of the ‘stem cell’ genes is present in the early
organoids at a level significantly higher then the differentiated cells that become the offspring of
these stem cells. For example, the genes LGR5, LGR4, Epcam, CD44, Tnfrsf19, Sox9, Cd24a,
Sp5, Prom1/CD133, Cdca7, are preferably expressed in the organoids of the invention but are
preferably significantly downregulated upon differentiation of the pancreas, liver, small intestine
and colon organoids. In addition, the genes RNF43 and ZNRF3 are preferably expressed in the
organoids of the invention.
By “natural expression” is meant that the cells have not been manipulated recombinantly in any
way, i.e., the cells have not been artificially induced to express these markers or to modulate these
markers’ expression by introduction of exogenous genetic material, such as introduction of
heterologous (non-natural) or stronger promoters or other regulatory sequences operably linked to
either the endogenous genes or exogenously-introduced forms of the genes. Natural expression is
from genomic DNA within the cells, including introns between the exon coding sequences where
these exist. Natural expression is not from cDNA. Natural expression can if necessary be proven
by any one of various methods, such as sequencing out from within the reading frame of the gene
to check that no extraneous heterogenous sequence is present. “Adult” means post-embryonic.
With respect to the stem cells described herein, the term “adult stem cell” means that the stem cell
is isolated from a tissue or organ of an animal at a stage of growth later than the embryonic stage.
This stem cell population can also be characterised by a lack of natural expression of certain
markers at any significant level, many of which are associated with cellular differentiation.
Specifically, the cells of the isolated adult stem cell population do not naturally express one or
more of Cd11b, CD13, CD14, AFP, Pdx1, any CYP member (e.g. CYP3A11, CYP 11A1) at a
significant level. As defined herein, these markers are said be to be negative markers.
Detecting markers and isolating cells
The term “expressed” is used to describe the presence of a marker within a cell. In order to be
considered as being expressed, a marker must be present at a detectable level. By “detectable
level” is meant that the marker can be detected using one of the standard laboratory methodologies
such as PCR, blotting or FACS analysis. A gene is considered to be expressed by a cell of the
population if expression can be reasonably detected after 30 PCR cycles, which corresponds to an
expression level in the cell of at least about 100 copies per cell. The terms “express” and
“expression” have corresponding meanings. At an expression level below this threshold, a marker
is considered not to be expressed. The comparison between the expression level of a marker in a
cell as described herein, and the expression level of the same marker in another cell, such as for
example an embryonic stem cell, may preferably be conducted by comparing the two cell types
that have been isolated from the same species. Preferably this species is a mammal, and more
preferably this species is human. Such comparison may conveniently be conducted using a reverse
transcriptase polymerase chain reaction (RT-PCR) experiment.
In some embodiments, a population of cells or an organoid is considered to express a marker if at
least about 5%, (for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or
100%) of the cells in the cell population as described herein or organoid according to the invention
show expression of the marker.
In some embodiments, the cells express a cell marker at a significant level if they comprise
2 5 2 4 3 4
between 1 x 10 to 1 x 10 , for example 5 x 10 to 1 x 10 or 1 x 10 to 1 x 10 fold more copies of
the mRNA encoding the cell marker relative to the number of mRNA copies of the housekeeping
gene GADPH.
In some embodiments, the expression of a gene in an organoid of the invention or cell as described
herein when cultured in expansion medium is several fold (e.g. at least 1.5 fold, 2 fold, 3 fold, 4
fold, 5 fold) higher than when the organoid or cell is cultured in differentiation medium or in the
fully differentiated adult tissue. In some embodiments, a cell as described herein or organoid of
the invention when cultured under differentiation conditions, exhibits an increase in expression of
genes that are known as differentiation genes compared to a cell as described herein or organoid of
the invention when cultured under expansion conditions and also may show a decrease in the level
of expression of at least one or more stem cell/progenitor genes compared to a cell as described
herein or organoid of the invention when cultured in expansion medium.
Any one of a number of physical methods of separation known in the art may be used to select the
cells as described herein and distinguish these from other cell types. Such physical methods may
involve FACS and various immuno-affinity methods based upon makers specifically expressed by
the cells as described herein. As described above, Lgr5, CD44 and Sox9 are three of the cell
markers expressed at high levels in the stem cells as described herein. Therefore, by way of
illustration only, the stem cells as described herein may be isolated by a number of physical
methods of separation, which rely on the presence of these.
In one embodiment, the cells as described herein may be isolated by FACS utilizing an antibody,
for example, against one of these markers. Fluorescent activated cell sorting (FACS) can be used
to detect markers characteristic of a particular cell type or lineage. As will be apparent to one
skilled in the art, this may be achieved through a fluorescent labeled antibody, or through a
fluorescent labeled secondary antibody with binding specificity for the primary antibody.
Examples of suitable fluorescent labels includes, but is not limited to, FITC, Alexa Fluor® 488,
GFP, CFSE, CFDA-SE, DyLight 488, PE, PerCP, PE-Alexa Fluor® 700, PE-Cy5 (TRI-
COLOR®), PE-Cy5.5, PI , PE-Alexa Fluor® 750, and PE-Cy7. This list is provided by way of
example only, and is not intended to be limiting.
It will be apparent to a person skilled in the art that FACS analysis using an anti-Lgr5 antibody
will provide a purified stem cell population. However, in some embodiments, it may be preferable
to purify the cell population further by performing a further round of FACS analysis using one or
more of the other identifiable markers.
Immunohistochemistry may also be used to understand the distribution and localisation of
biomarkers and differentially expressed proteins in different parts of a cell population or organoid.
Visualising an antibody-antigen interaction can be accomplished in a number of ways that are well
known in the art, such as those that are described in described in Barker et al, Identification of
stem cells in small intestine and colon by marker gene Lgr5.Nature, 2007 Oct 25;449(7165):1003-
In another embodiment, the cells as described herein may be isolated by immuno-affinity
purification, which is a separation method well known in the art. By way of illustration only, the
cells as described herein may be isolated by immuno-affinity purification directed towards c-kit.
As will be apparent to one skilled in the art, this method relies upon the immobilisation of
antibodies on a purification column. The cell sample is then loaded onto the column, allowing the
appropriate cells to be bound by the antibodies, and therefore bound to the column. Following a
washing step, the cells are eluted from the column using a competitor which binds preferentially to
the immobilised anti-c-kit antibody, and permits the cells to be released from the column.It will be
apparent to a person skilled in the art that immuno-affinity purification using an immobilised
antibody will provide a purified cell population. However, in some embodiments, it may be
preferable to purify the cell population further by performing a further round of immuno-affinity
purification using one or more of the other identifiable markers, and use an aliquot of the isolated
clones to ascertain the expression of other relevant intracellular markers.
It will be apparent to a person skilled in the art that LGR5 or stem cell purification can be preceded
by any number of purification steps,such as purification of the epithelium with methods known in
the art, for example EDTA purification or Epcam FACS sorting of the epithelium.
It will be apparent to a person skilled in the art that the sequential purification steps are not
necessarily required to involve the same physical method of separation. Therefore, it will be clear
that, for example, the cells may be purified through a FACS step using an anti-Lgr5 antibody,
followed by an immuno-affinity purification step using a SSEA-1 affinity column. In certain
embodiments, the cells may be cultured after isolation for at least about 15, at least about 20 days,
at least about 25 days, or at least about 30 days. In certain aspects, the cells are expanded in
culture longer to improve the homogeneity of the cell phenotype in the cell population.
Mircroarray analysis, cluster analysis and comparative gene expression profiling can be used to
compare population phenotype with phenotype of the original parent cells or of the appropriate in
vivo counterparts (Sato T et al., Paneth cells constitute the niche for Lgr5 stem cells in intestinal
crypts. Nature 469 415-418).
Lineage tracing of Lgr5+ stem cells shows preservation of crypt-villus characteristics in organoids.
In another embodiment, high content analysis may be used to assess phenotypic integrity of stem
cells as described herein. For example, a number of high content screening kits and platforms
exist, such as point scanning 4 color ImageXpress ULTRA (Molecular Devices, Union City,
USA), the spinning disk (nipkow disk) Pathway 855 and 435 from BD Biosciences (formerly Atto
Biosciences, Rockville, Maryland), Opera (PerkinElmer Inc., Waltham, MA) and the slit scanning
IN Cell 3000 (GE/Amersham Biosciences, Cardiff, UK), Arrayscan VTI (Cellomics (Cellomics)),
IN Cell Analyzer 2000 (GE Healthcare Piscataway, New Jersey, USA), Acumen eX3 (TTP
LabTech Ltd (Acumen eX3)), Scanalyzer (Scanalyzer LemnaTec, Aachen Germany) and
ImageXpress MICRO (Molecular Devices, Union City, USA), IN Cell 1000 (GE/Amersham
Biosciences Piscataway, New Jersey, USA), the Pathway HT (Becton Dickinson Biosciences) and
the ImageXpress MICRO (Molecular Devices, Union City, USA), Scan^R (Olympus Soft Imaging
Solutions, Germany).
Plating density
In some embodiments as described herein, single-cell suspensions or small clusters of cells (2-50
cells/cluster) will normally be seeded, rather than large clusters of cells, as is known in the art. As
they divide, such cells will be seeded onto a support at a density that promotes cell proliferation.
Typically, when single cells are isolated the plating density of at least 1-500 cells/well is used, the
surface of the well being 0.32 cm . When clusters are seeded the plating density is preferably 250-
2500 cells/cm . For replating, a density of between about 2500 cells/ cm and about 5,000 cells/
cm may be used. During replating, single-cell suspensions or small cluster of cells will normally
be seeded, rather than large clusters of cells, as in known in the art.
Further differentiation
In some embodiments as described herein, certain components of the expansion medium can be
withdrawn to change the cell fate of the cultured cells towards differentiation. Any components of
the culture medium that are responsible for maintaining an undifferentiated state and/or activating
stem cell or progenitor genetic programs may be withdrawn from the culture medium.
In some embodiments as described herein, withdrawal of the inhibitors as described herein can
enable cells of the organoid to differentiate to mature cells, such as mature goblet and
enteroendocrine cells in crypt-villus organoids. Thus in some embodiments, described herein is a
method for further differentiating the organoids using a second culture medium which does not
comprise an inhibitor as described herein. For example, see Example 1.
For example, in some embodiments, the inhibitor of TGF-beta and/or the inhibitor of p38 are
withdrawn from the cell culture medium to allow the cells to differentiate. By “withdrawn” or
“withdrawal” of a component from the cell culture medium is meant that when the cells are
replated and the medium is changed, the component is not added to the fresh medium.
In some embodiments, Wnt is present in the expansion medium but not in the differentiation
medium. For example, some embodiments comprise withdrawal of Wnt for differentiation of
colon organoids to mature enterocytes. Wnt may also be withdrawn to enable differentiation of
crypt-villus organoids.
In some embodiments, Rspondin is present in the expansion medium but not in the differentiation
medium. For example, some embodiments comprise withdrawal of Rspondin for differentiation of
colon organoids to mature enterocytes. Rspondin may also be withdrawn to enable differentiation
of crypt-villus organoids. In some embodiments Rspondin and Wnt may withdrawn to enable
differentiation of crypt-villus organoids.
In some embodiments, nicotinamide is present in the expansion medium but not in the
differentiation medium. Thus, in some embodiments, nicotinamide and SB202190 (or another p38
inhibitor) are withdrawn from the cell culture medium to enable differentiation of the cells, for
example, into crypt-villus organoids or colon organoids.
Thus, a method of obtaining differentiated cells or organoids may comprise culturing epithelial
cells in a culture method of the invention which comprises a TGF-beta and/or p38 inhibitor to
enable the cells to survive and/or proliferate (i.e. expansion medium) and then continuing to
culture the cell and replenish the media, wherein the replenished media does not comprise a TGF-
beta inhibitor and/or p38 inhibitor (i.e. differentiation medium).
In some embodiments, the differentiation medium comprises additional components. For example
in some embodiments the differentiation medium comprises a gamma secretase inhibitor, for
example DAPT or DBZ. In some embodiments the differentiation medium comprises RANK
ligand (also referred to herein as RANKL). As mentioned above, the addition of a gamma
secretase inhibitor can direct the differentiation of intestinal organoids cells, such as small
intestinal organoid cells, towards secretory cells, such as goblet cells. The addition of RANKL to
the culture medium can direct differentiation intestinal organoid cells such as small intestinal
organoid cells, towards M cells.
In some embodiments the invention provides a culture medium for differentiating stem cells from
a tissue of interest, wherein the culture medium comprises or consists of the components of the
culture medium used for expanding the stem cells from the tissue type of interest but wherein one
or more of the following are excluded from the medium for differentiating stem cells: Wnt,
Rspondin, BMP inhibitor, TGF-beta inhibitor, receptor tyrosine kinase ligand, p38 inhibitor and
nicotinamide.
Furthermore, the invention provides a method for expanding a single stem cell or a population of
stem cells, preferably to generate an organoid, wherein the method comprises culturing the single
stem cell or population of stem cells in a culture medium according to the invention, wherein the
method comprises:
culturing the stem cell, population of stem cells or tissue fragments in a first expansion
medium;
continuing to culture the stem cell, population of stem cells or tissue fragments and
replenishing the medium with a differentiation medium, wherein the differentiation
medium does not comprise one or more of, preferably all of the factors selected from: a
TGF-beta inhibitor, a p38 inhibitor, nicotinamide and Wnt.
In general, where a component is described as being “removed” from a medium, it is meant that
that component is not added when the medium is replenished i.e. the component is excluded from
the replenished medium. When the medium is “replenished” this can mean that the medium is
physically removed from the extracellular matrix and then replaced with fresh medium.
For the colon, liver and pancreas, very few differentiated cells are present in the expansion
medium. Only once the expansion medium is replaced with a differentiation medium, do the cells
begin to differentiate. At this stage, the organoids also start to lose their stem cells. Differentiated
organoids may be appropriate for certain uses, such as (but not limited to) transplantation, drug
screening of metabolic diseases, toxicology (for example using liver organoids comprising
hepatocytes) and for studying antibacterial functions of the small intestine. Expanding organoids
may generally be more appropriate for other uses, such as (but no limited to) regenerative
medicine and drug screening, for example for cancer or cystic fibrosis. Expanding organoids
generally have more growth potential (and thus greater longevity) than differentiated organoids. In
some embodiments, the colon, liver and pancreatic organoids are not further differentiated.
The small intestine and prostate organoids differ from the colon, liver and pancreatic organoids, in
that they maintain an expanding stem cell population whilst also differentiating at the same time.
They do not need to be cultured in a separate differentiation medium in order for differentiated cell
types to be present. They can be considered to have the properties of both an expanding and a
differentiated organoid. However, to achieve full differentiation of small intestinal organoids, they
can be cultured in a separate differentiation medium that preferably does not comprise Wnt3a and
that preferably comprises a gamma secretase inhibitor and/or a RANK ligand (also referred to
herein as RANKL). By “full” differentiation it is meant that all differentiated cell types are present
including goblet cells, neuroendocrine cells, tuft cells, M-cells, enterocytes and paneth cells. Some
of these differentiated cell types, for example paneth cells, are also present (sometimes in smaller
quantities) in the expanding organoids.
Organoids
The cells described above grow into organoids. Accordingly, an organoid obtainable by a method
of the invention is a further aspect of the invention. Also provided is an organoid as described
herein. The organoid is preferably a human organoid. To the best of our knowledge, this is the
first time that human organoids have been obtained that are functional and alive after such an
extended period of time (i.e at least 3 months, preferably at least 4 months, at least 5 months, at
least 6 months, at least 7 months, at least 9 months, or at least 12 months or more of culture; see
examples included herein). Functionality is preferably characterized by the presence of tissue-
specific markers and/or by the structure of said organoid as defined herein. Since the final amount
of organoids obtained correlates with the duration of culture, the skilled person will understand
that the invention is a pioneer invention and potentially opens new possibilities in for example
regenerative medicine. Thus, there is provided an organoid as described herein that is functional
and alive after at least 3 months (e.g. at least 4, 5, 6, 7, 8 or more months) of culture. For example,
there is provided an organoid as described herein that retains at least one or more (e.g. 1, 2 or 3) of
its structure, marker expression and function after at least 3 months (e.g. at least 4, 5, 6, 7, 8 or
more months) of culture.
For example, an organoid according to the present invention may comprise a population of cells of
3 4 5 6 7
at least 1x10 cells, at least 1 x 10 cells, at least 1x10 cells, at least 1x10 cells, at least 1x10
cells or more. Each organoid comprises between approximately 1x10 cells and 5x10 cells. The
inventors have shown that it is possible to grow organoids from single Lgr5+ stem cells into
organoids comprising a population of cells as described above or comprising a population of cells
of approximately 10 cells. For example, it has now been shown for mouse that it is possible to
start growth of an organoid from single stem cells. Thus, the invention provides a method for
generating an organoid from a single stem cell. In some embodiments, the organoid comprises
approximately 10 cells. In some embodiments, 10-20, or 20-30 or 30-40 or 40-50 organoids may
be grown together in one well of a 24 well plate.
In some embodiments, the invention provides an organoid or population of cells, which is capable
of surviving in culture for at least 3 months, for example at least 4 months, at least 5 months, at
least 6 months, at least 7 months, at least 9 months, or at least 12 months or more, when cultured in
a culture medium of the invention.
In some embodiments, the invention provides an organoid or population of cells, wherein the
organoid or population of cells expands at a rate of at least 3 fold, at least 4 fold, at least 5 fold, at
least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold or at least 10 fold per week.
Preferably the population of cells or organoids will expand at a rate of about 4-5 fold per week or
more than two population doublings a week. Therefore, in some embodiments, the population of
cells or organoids will expand at a rate of at least 3 fold, at least 4 fold, at least 5 fold, at least 6
fold, at least 7 fold, at least 8 fold, at least 9 fold or at least 10 fold per week.
Organoids of the invention may be obtained using cells isolated from any suitable source.
Generally, the cells used to generate an organoid will be isolated from the same tissue type as the
organoid which is generated. The organoids are preferably mammalian, for example, murine,
bovine, porcine or human. Most preferably, the organoids are human.
In some embodiments, the invention provides an organoid or population of cells, wherein the
organoid or population of cells is a normal (healthy) organoid or population of cells or a disease
organoid or population of cells, for example obtained by culturing stem cells taken from a human
or animal with a disease.
In some embodiments, the invention provides an organoid or population of cells which is frozen
o o o o o o
C, below -10 C, below -20 C, below -40 C, below -60 C, or below -80 C,
and stored at below -5
o o o
below -100 C, below -150 C or at approximately -180 C. The organoid or population of cells of
the invention may be stored in liquid nitrogen. Therefore, in some embodiments, the invention
provides an organoid or population of cells which is stored in liquid nitrogen.
In some embodiments, the invention provides an organoid of the invention, wherein the organoid
is a small intestine organoid, a colon organoid, a gastric organoid, a pancreatic organoid, a liver
organoid or a prostatic organoid.
Organoid structure and morphology
Organoids of the invention, obtainable by expansion of stem cells, provide a population of cells
which resemble their in vivo counterparts.
Image analysis may be used to assess characteristics of cells in culture such as cell morphology;
cell structures; evidence for apoptosis or cell lysis; and organoid composition and structure. Many
types of imaging analysis are well known in the art, such as electron microscopy, confocal
microscopy, stereomicroscopy, fluorescence microscopy. Histological analysis can reveal basic
architecture and cell types.
Illustrative examples of organoids generated according to the invention are given in the
accompanying figures. It can be seen that organoids according to the invention may possess a layer
of cells with at least one bud and a central lumen. The organoids in the outside of the matrigel tend
to be larger than the organoids in the center of the matrigel, perhaps because they have better
access to the necessary growth factors. Structurally, organoids according to the invention are often
elongated in shape. They may include one or more budding structure – a single cell epithelial layer
with similarities to ducts or islets. Under confocal microscopy, the structures may stain positive for
keratin. They may include cells with polarised nuclei and small cytoplasm. The organoids may
have a section which is formed of multiple layers; such cells often tend to have their nuclei more
central to the cells, i.e. not polarized. The cells in the multilayer section may organise themselves
to include a gap, or lumen between the cells.
In some embodiments the organoids of the invention comprise or consist of epithelial cells. In
some embodiments, the organoids comprise or consist of a single layer of epithelial cells. In some
embodiments non-epithelial cells are absent from the organoids. In some embodiments, the
organoids of the invention comprise all the differentiated cell types that exist in their
corresponding in vivo tissue counterpart.
In some embodiments human intestinal organoids displayed budding organoid structures, rather
than the cystic structures seen under previous culture conditions. Metaphase spreads of organoids
more than 3 months old consistently revealed 46 chromosomes in each of the 20 cells taken from
three different donors.
In some embodiments the organoids of the invention comprise a single monolayer of cells. In some
embodiments the organoids of the invention have a section which is formed of multiple layers.
Multiple layers of cells are also referred to herein as regions of “stratified” cells. By “stratified” it
is meant that there are multiple (more than one) layers of cells. In some embodiments the
organoids of the invention comprise single monolayers that are folded (or invaginated) to form two
or more layers. It can sometimes be difficult to distinguish between folded (or invaginated)
monolayers and regions of stratified cells. In some embodiments an organoid comprises both
regions of stratified cells and regions of folded monolayers. In some embodiments the organoids of
the invention have a section which is formed of multiple layers and a section comprising a single
monolayer of cells. Morphologically, the cells appear like their corresponding in vivo tissue
counterpart.
Therefore, in some embodiments the invention provides an organoid, preferably obtainable using
the culture media and methods of the invention, which is a three-dimensional organoid comprising
epithelial cells surrounding a central lumen, wherein optionally the epithelial cells exist in distinct
dividing domains and differentiating domains. In some embodiments the organoid of the invention
is a three-dimensional organoid comprising epithelial cells arranged in regions of monolayers,
optionally folded monolayers and regions of stratified cells. In some embodiments, non-epithelial
cells are absent from said organoid. In some embodiments, all differentiated cell types of the
normal in vivo tissue are present in said organoid.
Crypt-villus organoids
In small intestinal crypt-villus organoids the structural arrangement of the organoids is very similar
to the structure of in vivo crypt-villi: the Lgr5+ stem cell and their niche cells (Paneth cells) are
next to each other at the base of the crypt, followed by the transit amplifying cells, just above the
base of the crypt and leading into the sides of the villi and finally the differentiated cells, such as
enterocytes that make up the rest of the villi and become more and more differentiated towards the
top of the villi. It can be seen that organoids according to the invention may possess a layer of cells
with at least one bud and a central lumen. The organoids in the outside of the matrigel tend to be
larger than the organoids in the center of the matrigel, perhaps because they have better access to
the necessary growth factors. Structurally, organoids according to the invention are often elongated
in shape. Under confocal microscopy, the structures may stain positive for keratin. They may
include cells with polarised nuclei and small cytoplasm. The crypt-villus organoids are generally
single-layered.
In some embodiments, for example for a mouse crypt-villus organoid, a crypt-villus organoid is a
three-dimensional organoid, comprising crypt-like domains surrounding a central lumen lined by
villus-like epithelial domains, which are epithelial domains comprising differentiated cell types. In
some embodiments, non-epithelial cells are absent from said organoid.
In some embodiments, for example for a human crypt-villus organoid, a crypt-villus organoid is a
three-dimensional organoid, comprising crypt-like domains surrounding a central lumen. In some
embodiments, dividing cells are confined to the budding structures. No or few differentiated cells
are present. Under differentiation conditions the differentiated cells of the intestine are formed. In
some embodiments, non-epithelial cells are absent from said organoid. In some embodiments,
when the organoid is expanding, for example when it is in an expansion culture medium according
to the invention, the organoid has few or no differentiated cells.
In some embodiments, a small intestinal organoid of the invention cultured in a culture medium of
the invention comprising RANKL, comprises M-cells. In some embodiments of the invention, a
small intestinal organoid of the invention cultured in a culture medium of the invention comprising
a gamma-secretase inhibitor, comprises goblet cells. In some embodiments, a small intestinal
organoid cultured in a differentiation medium (for example wherein the differentiation medium
comprises a basal medium, Noggin, EGF, a TGF-beta inhibitor and a p38 inhibitor, a gamma-
secretase inhibitor and a RANKL) comprises all differentiated cell types including, for example,
goblet cells, neuroendocrine cells, tuft cells, M-cells, enterocytes and paneth cells. Some of these
differentiated cell types, for example paneth cells, are also present (sometimes in smaller
quantities) in the expanding organoids.
Human intestinal organoids display budding organoid structures, rather than the cystic structures
seen under previous culture conditions. The upper opening of freshly isolated crypts becomes
sealed and this region gradually balloons out and becomes filled with apoptotic cells, much like
apoptotic cells are pinched off at the villus tip. Thus, in some embodiments, the crypt-villus
organoids have a crypt-like structure surrounding a central lumen lined by a villus-like epithelium
and filled with apoptotic cell bodies. In some embodiments, the lumen is opened at consecutive
time intervals to release the content into the medium.
In some embodiments, the crypt region undergoes continuous budding events which create
additional crypts, a process reminiscent of crypt fission.
The inventors also demonstrated that the human intestinal organoids generated by media and
methods of the present invention, mimicked in vivo cell fate decisions in response to external
factors. For example, it has previously been shown that Notch inhibition in intestinal stem cells,
terminates intestinal epithelial proliferation and induces goblet cell hyperplasia in vivo. Thus in
some embodiments, when a crypt-villus organoid of the invention is treated with a Notch inhibitor,
proliferation ceases and most cells (for example more than 50%, more than 60%, more than 70%,
more than 80%, more than 90%, more than 95%, more than 98%) convert into goblet cells within 3
days.
Metaphase spreads of organoids more than 3 months old consistently revealed 46 chromosomes in
each of the 20 cells taken from three different donors. Furthermore, microarray analysis revealed
that the stem cells in culture possessed similar molecular signatures to intestinal crypt cells
including intestinal stem cell genes.
Colon organoids
Colon organoids exhibit a similar cell composition to crypt-villus organoids. Thus, the comments
for crypt-villus organoids above apply to colon organoids mutatis mutandis. For example, see
figures 1 and 2.
Typically, the difference between the colon and small intestinal organoids is that the crypts are
shallower in the colon making it look a little like a “football” rather than a sphere with protrusions.
Both small intestinal and colon organoids have domains that contain stem cells and transit
amplifying (TA) cells, and other domains containing differentiating and/or differentiated cells. For
the small intestinal organoids the differentiated domains are sometimes referred to as “villus-like”.
The differentiated domains of the colon organoids are typically similar in cell composition to the
“villus-like” domains of the small intestine but the colon itself does not have villi.
The amount of Wnt present can influence the size of the budding structures (i.e. the depth of the
crypts) in the organoids. More Wnt reduces budding. The colon produces more Wnt than the small
intestine and so requires less additional Wnt in the culture medium and typically has shallower
crypts than the small intestine. The same difference is seen in the organoids.
In some aspects, colon organoids are provided by the invention. The inventors have found that
mouse colon organoids can be obtained by culturing colon crypts in an ENR + Wnt3A (WENR)
cell culture media. Thus, in some embodiments, the invention provides a colon organoid obtained
by culturing colon crypts in WENR media.
The inventors have also surprisingly found that human colon organoids can be maintained using a
culture medium comprising WENR plus gastrin plus nicotinamide. In some embodiments, a
human colon organoid of the invention is obtainable by using a media comprising WENR plus
gastrin plus nicotinamide and also comprising an inhibitor of TGF beta. For example, in some
embodiments, the following cell culture media may be used to obtain a human colon organoid:
WENR+gastrin+nicotinamide+A8301+SB202190. In other embodiments, the following cell
culture medium may be used to obtain a human colon organoid: WENR+Nicotinamide+A83-01
In some embodiments, a mouse colon organoid has a maximal diameter of approximately 200-
700um, for example 250-600 um, 300-500 um, 320-450 um, 340-400 um, 300-380 um, for
example approximately 360um. In some embodiments, a colon organoid has a minimal diameter
of approximately 100-400um, for example 150-350 um, 170-300 um, 190-280 um, 195-250 um,
for example, approximately 235um. In a further embodiment, the organoids can have a diameter
of up to 1mm. In some embodiments, a human colon organoid has a maximal diameter of
approximately 300-800um, for example 350-700 um, 400-600 um, 450-550 um, 475-540 um, 500-
530 um, for example approximately 500um. In some embodiments, a colon organoid has a
minimal diameter of approximately 200-500um, for example 250-450 um, 300-415 um, 350-400
um, 325-380 um, for example, approximately 375um. In a further embodiment, the organoids can
have a diameter of up to 1mm. In some embodiments, a colon organoid of the invention comprises
budding structures. These may be visible by using EdU stain to visualize proliferating cells.
Human colon organoids retain their characteristic budding structure under the Human Intestinal
Stem Cell Culture (“HISC”) condition (WENRg+nicotinamide+TGF-beta inhibitor (e.g. A83-
01)+p38 inhibitor (e.g. SB202190)).In some embodiments, a colon organoid is a three-dimensional
organoid, comprising budding structures which are proliferating and contain stem cells. These
stem cell domains surround a central lumen. Dividing cells are generally confined to the budding
structures. In some embodiments, no or few differentiated cells are present. Under differentiation
conditions the differentiated cells of the intestine are formed, for example mature enterocytes. In
some embodiments, non-epithelial cells are absent from said organoid.
Pancreatic organoids
Pancreatic organoids of the invention preferably exhibit budding. In some embodiments, the
pancreatic organoids are from 100-1000 micrometers in diameter, for example, 200-900
micrometers, 300-1000 micrometers, 400-700 micrometers. The pancreatic organoids are
preferably single layered. There are only the very beginnings of islet or ductal structures. Budding
structure are indicative of a healthy proliferation status and stem cell maintenance.
In some embodiments, for example when pancreatic organoids are grown in a culture medium of
the invention (and in the absence of TGF-beta inhibitors), pancreatic organoids are mainly cystic
structures with few budding structures or duct-like domains. The cystic structures comprise mainly
monolayers but some regions of stratified cells may be present. The cells express stem cell and
progenitor (ductal) markers. No differentiated cells, such as beta-cells, are present in the organoids.
The cyst is mainly formed by a monolayer, but stratified parts exist. Cell types resemble stem cells
/ progenitor (duct cell gene expression). There are no differentiated cells (β-cells).
In other embodiments, for example when pancreatic organoids are grown in the presence of a
TGF-beta inhibitor, such as A83-01, for example in a culture medium of the invention, the
pancreatic organoids comprise more budding structures/ductal-like domains (this means cells are
duct-like cells more than the structure is like a duct), as shown by Krt19 staining (for example, see
figure 31). Monolayers of polarized cells can be identified, but also areas with stratified cells.
Adenocarcinoma and colon cancer organoids
Adenocarcinoma and colon cancer organoids generally form cystic structures instead of budding
structures. This is reminiscent of the absence of good cell niche support. Adenoma crypts cultured
with EFG+Noggin show approximately 16x expansion in the first 10 days. Adeno(carcino)ma and
colon cancer organoids may provide useful research tools and drug screening models.
Carcinoma, adenoma and adenocarcinoma organoids are largely cystic (for example, see figures 4
and 9). However, in some embodiments, they may also comprise structures that resemble their
normal tissue organoid counterparts.
Barrett’s Esophagus (BE) organoids
A BE organoid of the invention comprises budding structures (for example, see figure 5).
Morphologically, the cells in the organoids of the invention appear like their corresponding in vivo
tissue counterpart.
Barrett’s Esophagus is a disease marked by the presence of columnar epithelium in the lower
esophagus, replacing the normal squamous cell epithelium as a result of metaplasia. The
histological hallmark of Barrett’s esophagus is the presence of intestinal goblet cells in the
esophagus. Exploiting the similarity between Barrett’s Esophagus and the intestinal epithelium, the
inventors showed that the culture medium and methods of the invention could be used to maintain
Barrett’s Esophagus epithelium for up to 1 month. The inventors also demonstrated, for the first
time, that addition of FGF10 to the culture medium of the invention enabled the Barrett’s
Esophagus organoids to form budding structures and significantly prolonged the culture duration
to more than three months. Thus, a Barrett’s Esophagus organoid is an example of an organoid of
the invention. In some embodiments, a Barrett’s Esophagus organoid has a cystic structure. In
some embodiments, a Barrett’s Esophagus organoid of the invention comprises Paneth cells. In
some embodiments, a Barrett’s Esophagus organoid of the invention expresses lysozyme.
The inventors, therefore, also describe a culture medium according to the invention, comprising
FGF10, for the culture of Barrett’s Esophagus epithelium.
In some embodiments of the invention, Barrett’s Esophagus organoids may be grown using a
culture medium according to invention also comprising FGF10. In some embodiments, these
Barrett’s Esophagus organoids express Ki67 and have a minimal number, preferably less than
%, less than 5% or less than 1% PAS-positive cells and Mucin-positive cells. In some
embodiments, the Barrett’s Esophagus organoids comprise lysozyme-positive Paneth cells.
Stomach (gastric) organoids (for example, see figure 46)
Mouse gastric organoids grown in a culture medium of the invention are three-dimensional
organoids, comprising or consisting of a single layer epithelia, that comprises a gastric gland base
like domains (formed by stem and progenitor cells) surrounding a central lumen lined by epithelial
domains comprising differentiated cell types, and optionally wherein non-epithelial cells are absent
from said organoid.
Human gastric organoids grown in a culture medium of the invention comprise cystic structures.
The cystic structure is a monolayer of polarized cells. These human gastric organoids grown in the
presence of a TGF-beta inhibitor resemble mouse gastric organoids much more closely than
human organoids grown in the absence of TGF-beta inhibitor.
Prostatic organoids (see figures 41 to 43)
Under culture conditions comprising EGF, Noggin, Rspondin, murine prostatic organoids form
three dimensional cystic structures with a lumen. In time the layers fold inward forming 3-4 layers
of (stratified) epithelial cells. The outer layer is mostly composed of CK5+ basal epithelial cells
whereas the inner layers are mostly composed of CK8+ luminal epithelial cells. No stem cell
compartment has been identified; all domains contain dividing cells. Therefore, in some
embodiments, a prostate organoid grown in the absence of testosterone comprises stratified layers
of dividing epithelial cells. In a further embodiment, the prostate organoid comprises an outer layer
of cells comprising CK5+ basal epithelial cells and inner layers comprising CK8+ luminal
epithelial cells. In some embodiments, a prostate organoid grown in the absence of testosterone
does not contain any stem cells.
Addition of testosterone to the prostate culture medium
The inventors have shown that the addition of (DiHydro) testosterone to the culture conditions for
the prostatic organoids, results in the majority of cells differentiating into CK8+ luminal cells
which form a single layer of epithelium that folds onto itself into two layers. Prostate organoids
grown in the presence of testosterone consist of mostly luminal cells with or without a second
layer of basal cells. The structure resembles the in vivo structure. Both differentiated and dividing
cells are present, as well as stem cells and progenitors. Therefore, in some embodiments, for
example when cultured in a medium comprising testosterone, a prostate organoid is a three-
dimensional organoid comprising cystic structures and a lumen. In some embodiments the prostate
organoid comprises CK8+ luminal cells which form a monolayer of epithelium. In some
embodiments the monolayer is folded into two or more layers. In other embodiments, the organoid
may comprises regions of stratified cells. In some embodiments, the prostate organoid comprises
differentiated cells while maintaining the dividing stem cell population.In some embodiments, the
shape of the organoid is determined by the origin of the cellular or tissue starting material (i.e. the
position in the prostate before isolation). The prostate consists of different lobes or regions which
display the different epithelial structures described above (stratified and folded), After in vitro
culturing the organoids appear to some extent to maintain the different macroscopic structure
(stratified or folded) of the part of the prostate from which it originated.
Liver organoids
Structurally, mouse liver organoids according to the invention are often elongated in shape. They
may include one or more budding structure – a single cell epithelial layer which has a structure not
unlike a bile duct. Under confocal microscopy, the structures may stain positive for keratin. They
may include cells with polarised nuclei and small cytoplasm. The organoids may have a section
which is formed of multiple layers; such cells often tend to have their nuclei more central to the
cells, i.e. not polarized. The cells in the multilayer section may organise themselves to include a
gap, or lumen between the cells. Human liver organoids of the invention, in some embodiments
have a generally cystic structure.
In some embodiments, a liver organoid is a three-dimensional organoid, with a cystic structure (for
example, see figure 30). Under expansion conditions the organoid may consist of stem cells and
progenitor cells where two domains are defined: (1) A duct-like domain, formed by a single-layer
cubical epithelia (positive for the ductal marker Krt19) with cells lining a central lumen; and (2) a
pseudo-stratified epithelial domain where krt19 positive cells and scattered albumin positive cells
are detected. This architecture (areas with single layer epithelia together with areas of
pseudostratified epithelia) resembles the embryonic liver bud. Under expansion conditions fully
differentiated cells are not present, although expression of hepatocyte/hepatoblast-specific markers
can in some embodiments be detected. Differentiation conditions result in the formation of a cystic
organoid where the duct-like domain (single layer epithelia) is lost and the entire structure
becomes a pseudo-stratified epithelia containing >50% polarized hepatocytes.
A liver organoid, preferably comprises a hepatocyte and a cholangiocyte cell (although
hepatocytes are especially seen following differentiation in DM and are not required for
expansion), more preferably wherein at least one of the following markers could be detected: at
least one hepatocyte marker such as albumin, transthyretrin, B-1 integrin and Glutamine synthetase
and/or at least one of CYP3A11, FAH, tbx3, TAT and Gck and/or at least one cholangiocyte
maker such as Keratin 7 and 19. The skilled person knows how to detect each of these markers
(i.e. RT-PCR and/or immunofluorescence). Preferably the expression of each of these markers is
assessed as carried out in the experimental part. Each of these markers is usually expressed after at
least two weeks, three weeks or one month of culture using a method of the invention. Microarray
analysis of the organoids in both culture conditions showed that liver organoids resemble adult
liver tissue.
Preferably all cells in a liver organoid express hepatocyte surface markers. For example, in some
embodiments, at least 50% (for example 50-60%), at least 60%, at least 70%, at least 80%, at least
90%, at least 99% or 100% of cells in a liver organoid express hepatocyte markers. In some
embodiments, approximately 35% of the cells in a liver organoid express a hepatocyte surface
marker, for example, 25-45%, 30-40%, 33-37%, 35% or less, or 15-35% of cells. In some
embodiments, the expansion phase would have less hepatocytes, for example less than 20%, less
than 10%, less than 5% of the cells, less than 2%, less than 1%, preferably 0% of the cells.
Preferably, cells and organoids generated according to the invention also possess hepatocyte
functions, such as expressing or staining positive for the mature hepatic markers albumin, B-1
integrin,, CK-8, CK-18, transthyretin (TTR), glucose 6P, Met, Glutamine synthase (Glul),
transferrin, Fahd1, Fahd2a, K7, K19 and cytochrome P450 isoforms 3A13 (CYP3A13), 51
(CYP51) 2D10 (CYP2D10), 2j6 (CYP2j6), 39A1 (CYP39A1), 4A10 (CYP4A10), 4F13
(CYP4F13) 4F16 (CYP4F16), CYP4B1and 20A1(CYP20A1). Also, embryonic liver gene AFP is
in some embodiments not detected in neither of both culture conditions, as in adult liver. In some
embodiments, the expression of alpha fetal protein is just above the background gene expression.
Also, the well-known liver transcription factors as HNF1a, HNF1b and HNF4a are highly
expressed in both conditions.
Since liver and pancreas are closely related organs, we investigated whether our liver cultures also
expressed pancreas-specific genes. The pancreas is functionally divided into endocrine and
exocrine pancreas. The endocrine pancreas is mainly characterized for expressing insulin,
glucagon and somatostatin. The expression of these hormones is tightly regulated by a set of
endocrine pancreas-specific transcription factors, the most important being Pdx1 and NeuroD. The
exocrine pancreas is formed by acinar and ductal compartments responsible of producing the
digestive enzymes amylase, pancreatic lipase and chymotrypsin, among others. The expression of
these genes is also regulated by specific exocrine pancreatic genes as Ptf1.
The pancreas specific genes Ptf1a, pancreatic amylase (Amy2a4), pancreatic lipase (Pnlip), insulin
(ins1 and ins2), glucagon (Gcg), chymotrypsin (cela1), Pdx1 and NeuroD were absent in the liver
cultures here described.
In some embodiments, one or more or all of the following genes are expressed in the liver
organoids at a similar level to the corresponding gene in adult liver hepatocytes: Aqp1, Bmp2,
Apo3, Apol7a, Sord, C3, Ppara, Pparg, tbx3, lgf1, ll17rb, ll1b, Tgfbi, Apoa1, Apoa4, Apob,
Cyp26b1, Cyp27a1, Cyp2b13, Cyp2b9, Cyp2c37, Cyp2f2, Cyp2g1, Cyp2j13, Cyp3a11, Cyp4a10
and Cypf14. For example, see Figure 27A.
In some embodiments, one or more of the following genes is expressed in the liver organoids at a
similarly shut down level compared to the corresponding gene in adult liver hepatocytes: Ccl2,
Osmr, Icam1 and Cxcl2.
In some embodiments, one or both of the following genes is differentially expressed in both a liver
organoid and newborn liver: mKi67 and cdkn3, meaning that the expression of these genes is
higher in the organoids than in the differentiated organoids or whole organ.
In some embodiments, one, two or all of the following genes are expressed at a similar level in a
liver organoid and a newborn liver: cyp2j6, olfm4 and Lefty 1. For example, see Figure 27B.
In some embodiments, a liver organoid of the invention has a ductal phenotype when cultured in
expansion medium of the invention (e.g. EM1 or EM2).
In some embodiments, a liver organoid of the invention expresses adult liver markers when
cultured in a differentiation medium of the invention.
In one embodiment, a liver organoid of the invention has a gene expression profile as shown in
Figure 27C.
In a particularly preferred embodiment, a mouse liver cell population or organoid of the invention
has the gene expression profile as shown in Figure 28. For example, in one preferred embodiment,
a mouse liver cell population or organoid of the invention:
a) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11), preferably all of the
following stem cell markers: lgr5, lgr4, epcam, Cd44, Tnfrsf19, Sox9, Sp5, Cd24a, Prom1, Cdca7
and Elf3; and/or
b) does not express the following stem cell marker: lgr6; and/or
c) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19), preferably all of the following hepatocyte or cholangiocyte markers when grown in expansion
medium of the invention: Hnf1a, Hnf1b, Hnf4a, Hhex, Onecut1, Onecut2, Prox1, Cdh1, Foxa2,
Gata6, Foxm1, Cebpa, Cebpb, Cebpd, Cebpg, Glul, Krt7, Krt19 and Met; and/or
d) does not express at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17 ) of the following genes when grown in expansion medium of the invention: afp, Ins1, Ins2,
Gcg, Ptf1a, Cela1, Cela2a, Cela3b, Neurod1, Neurod2, Neurog1, Neurog2, Neurog3, Amy2a4,
Igf1r, Igf2 and Cd34; and/or
e) expresses at least one (e.g. 1, 2 or 3) of the following reprogramming genes: Klf4,
Myc and Pou5f1 and/or
f) does not express the following reprogramming gene: Sox2.
wherein the expression of the genes is preferably detected by measuring expression at the
mRNA level, for example, using a microarray.
More preferably a mouse liver cell population or organoid of the invention has all of features a) to
f) above.
In some embodiments, the gene expression profile described above for a mouse liver cell
population or liver organoid of the invention is for a mouse cell population or organoid cultured in
liver expansion medium of the invention.
In some embodiments, there is provided a human liver cell population or organoid of the invention
that has the gene expression signature shown in Figure 29. For example, a human liver cell
population or organoid cultured in EM1 of the invention preferably expresses the genes indicated
in Figure 29 as being expressed in EM1 cell culture medium. For example, a human liver cell
population or organoid cultured in EM2 of the invention preferably expresses the genes indicated
in Figure 29 as being expressed in EM2 cell culture medium. For example, a human liver cell
population or organoid cultured in DM of the invention preferably expresses the genes indicated in
Figure 29 as being expressed in DM cell culture medium.
For example, in one preferred embodiment, a human liver cell population or organoid of the
invention:
a) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9), preferably all of the following
stem cell signature genes: LGR4, TACSTD1/Epcam, CD44, SOX9, SP5, CD24, PROM1, CDCA7
and ELF3; and/or
b) expresses at least one (e.g. 1, 2, 3, 4), preferably all of the following
reprogramming genes: KLF4, MYC, POU5F1 and SOX2; and/or
c) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19), preferably all of the following hepatocyte/cholangiocyte specific genes: HNF1A, HNF1B,
HNF4A, HHEX, ONECUT1, ONECUT2, PROX1, CDH1, FOXA2, GATA6, FOXM1, CEBPA,
CEBPB, CEBPD, CEBPG, GLUL, KRT7, KRT19 and MET; and/or
d) does not express at least one (e.g. 1, 2, 3, 4, 5, 6), preferably all of the following
hepatocyte/cholangiocyte specific genes: NEUROG2, IGF1R and CD34, AFP, GCG and PTF1A,
for example, it does not express NEUROG2, IGF1R and CD34; and/or
e) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18), preferably all of the following hepatocyte specific genes: TTR, ALB, FAH, TAT, CYP3A7,
APOA1, HMGCS1, PPARG, CYP2B6, CYP2C18, CYP2C9, CYP2J2, CYP3A4, CYP3A5,
CYP3A7, CYP4F8, CYP4V2 and SCARB1;
wherein the expression of the genes is preferably detected by measuring expression at the
mRNA level, for example, using a microarray.
More preferably a human liver cell population or organoid of the invention has all of features a) to
e) above.
In some embodiments, the genes in a human liver cell population or organoid of the invention are
upregulated or downregulated relative to expression of a reference RNA as shown in Figure 29.
Preferably, the reference RNA is Universal Human Reference RNA (Stratagene, Catalog
#740000). In some embodiments, a gene is upregulated or downregulated relative to the reference
RNA if it is also shown in Figure 29 as being upregulated or downregulated relative to the
reference RNA but the extent of upregulation or downregulation need not be the same. In other
embodiments, the extent of upregulation or downregulation is +/-35%, +/-30%, +/-25%, +/-20%,
+/-20%, +/-15%, +/-10%, +/-5%, +/-3%, or more preferably +/-1.5-fold, +/fold, +/fold, +/-
-fold or approximately the same as shown in Figure 29. In other embodiments, the absolute level
of expression of the genes in a human organoid of the invention is +/-35%, +/-30%, +/-25%, +/-
%, +/-15%, +/-10%, +/-5%, +/-3%, or +/-1.5-fold, +/fold, +/fold, +/- 5-fold or
approximately the same as shown in Figure 29.
The human liver cell population or organoids of the invention also preferably express Lgr5 and/or
Tnfrsf19, preferably both. In some embodiments, the human liver cell population or organoids,
when cultured in expansion medium of the invention express Lgr5 and/or Tnfrsf19, preferably
both. Preferably, expression of Lgr5 and/or Tnfrsfr19 is detected by RT PCR. In some
embodiments, Lgr5 and/or Tnfrsf19 are present at much lower levels of expression in organoids or
cells when cultured in the differentiation medium compared to their level of expression organoids
or cells when cultured in the expansion medium (for example at least 2-fold, at least 3-fold, at least
4-fold, at least 5-fold, at least 10-fold, at least 15-fold lower).
Liver cells and organoids according to the present invention may preferably be capable of
secreting albumin, for example, at a rate of between approximately 1μg per hour per 10 cells and
10μg per hour per 10 cells, preferably between 2μg and 6μg per hour per 10 cells.
Furthermore, such liver cells and organoids may secrete urea. For example, in a 35mm dish of
cells, the activity of urea synthesis may be between 1μg and 50μg in 48 hours, preferably between
5μg and 30μg.
Liver cells and organoids according to the invention may show visible glycogen stores, for
example, when stained. The capacity for cells and organoids according to the invention to
synthesize glycogen actively can be tested by switching the culture media from low-glucose
differentiation media to high-glucose DMEM supplemented with 10% FBS and 0.2μM
dexamethasone for two days.
Liver cells and organoids according to the invention may possess inducible cytochrome P450
activity (e.g. CYP1A). Such activity may be tested, for example, using an ethoxyresorufin-O-
deethylase (EROD) assay (Cancer Res, 2001, 61: 8164-8170). For example, cells or organoids
may be exposed to a P450 substrate such as 3-methylcholanthrene and the levels of EROD activity
compared to control cells.
Morphologically, the liver organoid cells appear hepatocyte-like.
A preferred liver organoid comprises or consists of a cystic structure with on the outside a layer of
cells with buds and a central lumen as depicted in Figure 30. This liver organoid may have one or
more (e.g. 2, 3, or all 4) of the following characteristics: (a) having a cell density of >5×10
3 5 3
cells/cm , preferably >10×10 cells/cm ; (b) having a thickness equivalent to 2-30 layers of cells,
preferably a thickness equivalent to 2-15 layers of cells; (c) the cells mutually contact in three
dimensions, (d) demonstrate a function inherent to healthy liver tissue, (e) have an elongated
shape, with 2 defined domains, i.e. a single layered epithelial domain where highly polarized cells
are detected and keratin markers are expressed (this domain resembles the bile duct domain) and
the other domain constitutes the main body of the organoid and is formed by a multilayered
epithelia with non-polarized cells wherein albumin expression may be detected. It is clear to the
skilled person that such a liver organoid is preferably not a liver fragment and/or does not
comprise a blood vessel, and/or does not comprise a liver lobule or a bile duct.
Within the context of the invention, a liver fragment is a part of an adult liver, preferably a human
adult liver. Preferably a liver organoid as identified herein is therefore not a liver fragment. A liver
organoid is preferably obtained using a cell from an adult liver, preferably an epithelial stem cell
from an adult liver, more preferably an epithelial stem cell from an adult liver expressing Lgr5. A
liver organoid may also be obtained from any cell which upon damage or culturing expresses Lgr5
and is therefore an Lgr5-expressing cycling stem cell.
In some embodiments, a liver organoid comprises cells that express Lgr5. For example, in some
embodiments, at least 2%, more preferably at least 5%, at least 10%, at least 20%, at least 30%, at
least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the
cells in the liver organoid express Lgr5. Similarly, the invention provides a cell or a population of
cells which express Lgr5, wherein said cells are obtained from a liver organoid of the invention.
The progeny of such cells is also encompassed by the invention.
In an embodiment, a liver organoid is a liver organoid which is still being cultured using a method
of the invention and is therefore in contact with an extracellular matrix. Preferably, a liver
organoid is embedded in a non-mesenchymal or mesenchymal extracellular matrix. Within the
context of the invention, “in contact” means a physical or mechanical or chemical contact, which
means that for separating said liver organoid from said extracellular matrix a force needs to be
used.
In a preferred embodiment, a liver organoid could be cultured during at least 2, 3, 4, 5, 6, 7, 8, 9,
weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 months or longer. In some embodiments, the liver organoid
is expanded or maintained in culture for at least 3 months, preferably at least 4 months, at least 5
months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months or more.
Preferably, a liver organoid cultured using expansion media of the invention comprising a TGF
beta inhibitor may be cultured for at least 4 weeks, more preferably at least 5 weeks at 5 fold
expansion a week or two or more population doublings per week (e.g.. for at least 10 doublings, at
least 20 doublings, more preferably at least 25 doublings, for example, at least 30 doublings).
Preferably, a liver organoid cultured using expansion media of the invention comprising a
prostaglandin pathway activator in addition to a TGF beta inhibitor may be cultured for at least 7
weeks, more preferably at least 8 weeks at 2 or more doublings (e.g. 2-3 doublings) per week (i.e.
at least 15 doublings, at least 25 doublings, at least 30 doublings, at least 32 doublings, at least 35
doublings, e.g. 32-40 doublings or at least 40 doublings, for example, at least 50 doublings). Thus,
preferably, a liver organoid of the invention, for example a human liver organoid, is obtained using
expansion media of the invention.
In another preferred embodiment, a liver organoid originates from a single cell, preferably
expressing Lgr5, more preferably wherein the single cell comprises a nucleic acid construct
comprising a nucleic acid molecule of interest.
Organoid composition and gene expression
The crypt-villus, colon crypt and pancreatic organoids typically comprise stem cells and/or
progenitor cells and, therefore, these organoids share certain patterns of gene expression. In some
embodiments, one or more (for example, 1, 2, 3, 4, 5, 6 or 7) or all of the following markers can be
detected: LGR5, LGR4, epcam, Cd44, Sox9, Cd24a, and CD133/Prom1 and optionally Tnfrsf19.
In another embodiment, the expression of one or two or all of the following progenitor genes can
be detected: Pdx1, Nkx2.2, and Nkx6.1. After differentiation, gene expression patterns of the
crypt-villus, colon crypt and pancreatic organoids are expected to diverge as the differentiated
organoids express tissue-specific adult markers, such as insulin in the pancreas for example.
Crypt villus organoids
In some embodiments of the invention, the organoids comprise crypt-villus like extensions which
comprise all differentiated epithelial cell types, including proliferative cells, Paneth cells,
enterocytes and goblet cells. In some embodiments, the crypt-villus organoids of the invention do
not contain myofibroblasts or other non-epithelial cells. A crypt-villus organoid of the invention
preferably comprises enterocytes, including absorptive enterocytes, goblet cells, enteroendocrine
cells, and Paneth cells in a crypt-villus-like structure. Preferably at least one (for example, 2, 3, 4,
or 6) of the following markers could be detected: SMOC2, CDCA7, OLFM4, ASCL2, AXIN2
and/or Lgr5 Tnfrsf19, CD24a, Sox9, CD44, Prom1 (see Figure 2e and Figure 14). In some
embodiments, the markers RNF43 and ZNRF3 can be detected. In some embodiments, one or
more (for example 1, 2, 3, 4 or 5) or all of SMOC2, CDCA7, OLFM4, ASCL2, AXIN2 and/or
Lgr5 are at least 2-fold, 3-fold, or 4-fold upregulated in crypts, whereas markers that are at least 2-
fold, 3-fold, or 4-fold downregulated in crypts include at least one or more (for example 1, 2, 3 or
4) or all of ABCG1, ENPP3, CSTE, MUC17 and/or APOA1. In this context “upregulation” is
relative to the villus of the intestine or to the top section of the colon crypt. Microarray analysis,
comparing the gene expression of differentiated organoid cells to stem cells, revealed that the
small intestinal crypt-villus and colonic organoids possess comparable molecular signatures of
intestinal crypts including the expression of intestinal stem cell genes. Thus, the invention also
provides a colonic organoid having the molecular signature described above for crypt-villus
organoids. Organoids cultured in-vitro clearly exhibit a similar expression profile to freshly
isolated small intestinal crypts and express known stem cell markers.
In some embodiments, the mRNA encoding one or more genes (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) listed in Figure 14 (for example all of the
genes shaded in Figure 14) as being upregulated in crypt-villus organoids or colon organoids
respectively is upregulated in a crypt-villus organoid or colon organoid of the invention compared
to a freshly isolated small intestinal villi, as determined by microarray. In some embodiments, the
mRNA encoding one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24 or 25) genes listed in Figure 14 (for example all of the genes shaded in Figure 14) as
being downregulated in crypt-villus organoids or colon organoids respectively is downregulated in
a crypt-villus organoid or colon organoid of the invention compared to a freshly isolated small
intestinal villi, as determined by microarray. In some embodiments, the fold upregulation or
downregulation is as indicated in Figure 14 + /– 25%, for example, +/ – 20%, +/ – 15%, + /– 10%,
+/-5%, +/-3% or approximately as quoted in Figure 14. For example, a crypt-villus organoid of
the invention may have ADORA2B upregulated 9.54 fold +/- 25% compared to freshly isolated
small investinal villi. The same applies, mutatis mutandis, to the other genes listed in Figure 14.
In some embodiments, the crypt villus organoids show natural expression of Lgr5. In some
embodiments, the crypt villus organoids show natural expression of at least Lgr5 and one or more
(for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) or all of stem cell markers from the
group consisting of: CK19, Nestin, Somatostatin, CXCR4 , CD133 , DCAMKL-1, CD44, Sord,
Sox9, CD44, Prss23, Sp5, Hnf1α, Hnf4a, Sox9, KRT7 and KRT19. In addition or alternatively,
crypt-villus organoids may be characterised by expression of one or more or all (for example 1 or
2) of: Sord and/or Prss23. In addition or alternatively, crypt-villus organoids may be characterised
by expression of CD44 and/or Sox9. In another embodiment, the crypt-villus organoids show
expression of one or more (for example 1, 2, 3, 4, 5, 6, 7, 8, 9) or all of the markers from the group
consisting of: lgr5, lgr4, epcam (tacstd1), Cd44, Tnfrsf19, Sox9, Sp5, Cd24a, Prom1, and Cdca7.
In some embodiments, a crypt villus organoid comprises Paneth cells expressing lysozyme.
Colon organoids
In some embodiments, a colon organoid contains enteroendocrine cells (e.g. as detectable using
chromagranin A stain), goblet cells (as detectable using mucin 2 stain). In some embodiments,
less than 10% of the cells in the colon organoid are enteroendocrine cells (e.g. 0.01-5%, 0.1-3%).
In some embodiments, less than 30% of the cells in the colon organoid are goblet cells (e.g. 1-
%, 1-15%, 5-10%). In some embodiments, the distribution of the enteroendocrine cells and/or
the goblet cells is as shown in the Figure 1d.
In some embodiments, a colon organoid contains mature enterocytes (e.g. as visualised by alkaline
phosphatise staining). In some embodiments, less than 10% of the cells in the colon organoid are
mature enterocytes (e.g. less than 5%, less than 3%, 0.01-5%, 0.1-3%, 0.1-5%).
In preferred embodiments, a colon organoid does not comprise Paneth cells because there are no
Paneth cells in a naturally occurring in vivo colon.
In some embodiments, the colon organoids show natural expression of Lgr5.
In some embodiments, a colon organoid expresses one or more (e.g. 1, 2, 3 or 4) of Villin1, Alpi,
ChgA and Muc2. In some embodiments, the relative amount of Villin1 mRNA expressed by a
colon organoid of the invention compared to a freshly isolated colon crypt is at least 3% (e.g. at
least 5%, at least 8%, at least 10%), for example between 5-15%. In some embodiments, the
relative amount of Alpi mRNA expressed by a colon organoid of the invention compared to a
freshly isolated colon crypt is at least 0.5% (e.g. at least 1%, at least 2%), for example, between
0.5-5%. In some embodiments, the relative amount of ChgA mRNA expressed by a colon
organoid of the invention compared to a freshly isolated colon crypt is at least 15% (e.g. at least
20%, at least 22%), for example, between 15-30%. In some embodiments, the relative amount of
Muc2 mRNA expressed by a colon organoid of the invention compared to a freshly isolated colon
crypt is at least 20% (e.g. at least 25%, at least 30%, at least 35%), for example, between 25-37%.
In some embodiments, a human colon organoid of the invention expresses known stem cell
markers.
Pancreatic organoids
The pancreas contains three classes of cell types: the ductal cells, the acinar cells, and the
endocrine cells. The endocrine cells produce the hormones glucagon, insulin somatostatin and
pancreatic polypeptide (PP), which are secreted into the blood stream and help the body regulate
sugar metabolism. The acinar cells are part of the exocrine system, which manufactures digestive
enzymes, and ductal cells from the pancreatic ducts, which connect the acinar cells to digestive
organs. During development, Islets of Langerhans are thought to descend from progenitor
endocrine cells which emerge from the pancreatic duct and after differentiation aggregate to form
Islets of Langerhans. Islets of Langerhans comprise α cells, β cells, δ cells, and PP cells.
Pancreatic organoid cells may have an expression pattern that resembles ductal cell markers, such
as one or more (e.g. 1, 2 or all) of K7, K19 and Hnf1b and/or one or more general stem cell
markers such as Sox9 and/or Onecut1. This is likely to be part of their stem cell signature.
Generally, fewer differentiation markers are seen. In some embodiments in which a cell is isolated
from a pancreatic duct in order to generate a pancreatic organoid of the invention, the cell type
that gives rise to a pancreatic organoid of the invention is not a ductal cell (meaning the epithelial
cells positive for keratin 7 and keratin 19 that form the ductal tube), but it is a cell attached to the
pancreatic duct, meaning a cell that is located in the next layer of cells after the duct in contact
with the pancreatic tissue (i.e. not facing the lumen of the duct.) Thus, in embodiments in which
the cell type that gives rise to a pancreatic organoid is not a ductal cell, the pancreatic organoid
will not express K7 or K19. However, such a pancreatic organoid will still preferably express one
or more general stem cell progenitor markers such as Sox9.
A pancreatic organoid of the invention preferably comprises α cells, β cells, δ cells, and PP cells.
In a further preferred embodiment, a pancreatic organoid comprises beta-cells. For example, a
pancreatic organoid may comprise more than 1%, more than 5%, more than 10%, more than 15%,
or more than 20% beta-cells. Expression of insulin may be used as a marker for beta cells. In an
alternative embodiment, the pancreatic organoid comprises progenitor cell types, optionally with a
ductal origin, that can give rise to differentiated cell-types upon transplantation into a human or
animal. In a preferred embodiment, the progenitor cell types can give rise to insulin-secreting beta-
cells upon transplantation into a human or animal. The inventors have shown that human
pancreatic organoids, grown according to the media and methods of the invention, can be
transplanted into mice and stimulate insulin-secreting cells within one month (see example 4). It
can be easily understood that this could lead to revolutionary treatments for patients with diabetes
and insulin-deficiencies.
In some embodiments, a pancreatic organoid of the invention may comprise ductal cells, acinar
cells and endocrine cells. In some embodiments, K19 is used as a marker for ductal cells.
In some embodiments, a beta-cell exists within pancreatic islands or Islets of Langerhans. An islet
generally comprises around 1500 cells in vivo, for example, 1300-1700 cells. In one embodiment,
a pancreatic organoid comprises at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 3%,
at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30% or more Islets of
Langerhans by mass. In some embodiments, the Islets of Langerhans of the pancreatic organoid
are composed of approximately 65 to 90% beta cells, approximately 15 to 20% alpha-cells,
approximately 3 to 10% delta cells, and approximately 1% PP cells. However, this is by no means
exclusive. For example, in some embodiments, it is desirable to have many beta cells in an
organoid of the invention. Alternatively, an organoid may comprise progenitor cells that may be
transplanted so that they differentiate in vivo.
In some embodiments, a pancreatic organoid expresses one, two or all three of Pdx1, Nkx2.2 and
Nkx6.1. A pancreatic organoid may express one, two, three or all four of NeuroD, Pax6, Pax4 and
Mafa. Pax4 serves as a marker for the presence of insulin producing cells because it is an essential
transcription factor for the differentiation of insulin producing cells from endocrine progenitor
cells during embryonic development. A pancreatic organoid may express Ngn3.
In some embodiments, at least one (for example 1, 2, 3, 4, 5) of the following markers can be
detected in a pancreatic organoid of the invention: insulin (ins1 and/or ins2), glucagon (Gcg),
somatostatin, Pdx1 and NeuroD. In some embodiments, at least one (for example 1, 2, 3, 4, 5) of
the following markers can be detected in a pancreatic organoid of the invention: insulin (ins1
and/or ins2), glucagon (Gcg), somatostatin, Pdx1 and NeuroD and the following markers are not
detected: ptf1a, amy2a4, Pnlip and cela1. In some embodiments, at least one (for example 1, 2, 3,
4, 5, 6, 7, 8 or 9) of the following markers can be detected in a pancreatic organoid of the
invention: Ptf1a, pancreatic amylase (Amy2a4), pancreatic lipase (Pnlip), insulin (ins1 and/or
ins2), glucagon (Gcg), somatostatin, chymotrypsin (cela1), Pdx1 and NeuroD.
In some embodiments, the pancreatic organoids show natural expression of Lgr5. In some
embodiments, the pancreatic organoids show natural expression of at least Lgr5 and one or more
(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) stem cell markers selected
from the group consisting of: CK19, Nestin, CXCR4 , CD133 , DCAMKL-1, CD44, Sord, Sox9,
CD44, Prss23, Sp5, Hnf1α, Hnf4a, Sox9, KRT7 and KRT19, prom1, Cd24a, Lgr4, epcam.
Alternatively or additionally, in some embodiments, pancreatic organoids may be characterised by
natural expression of one or more (for example 1, 2, 3 or 4) of: CK19, Nestin, (insulin, glucagon)
and CXCR4 .
In some embodiments, the pancreatic organoids or cells of the invention express Somatostatin.
Somatostatin is a hormone expressed in differentiated delta cells and so may serve as a marker for
delta cells.
Alternatively or additionally, in some embodiments, pancreatic organoids show natural expression
of one or more early endocrine markers, for example at least one or more (e.g. 1, 2, 3, 4, 5, 6 or 7)
of the following early endocrine markers: Sox9, Hnf1b, Hnf6, Hnf1a, Nkx2.2, Nkx6.1 and Pdx1.
Alternatively or additionally, in some embodiments, pancreatic organoids show natural expression
of one or more early endocrine markers, for example at least one or more (e.g. 1, 2, 3 or 4) of the
following endocrine markers: Foxa2, Hnf6, Hnf1b and Sox9. In some embodiments, although the
pancreatic organoids show natural expression of one or more (e.g. 1, 2, 3 or 4) of the following
endocrine markers: Foxa2, Hnf6, Hnf1b and Sox9, they do not show expression of Ngn3.
Alternatively or additionally, in some embodiments, pancreatic organoids show natural expression
of one or more ductal markers, for example, one or both of keratin 7 and keratin 19. In some
embodiments, the pancreatic organoids show natural expression of one or more ductal markers at a
significant or detectable level. Thus, in some embodiments, the pancreatic organoids have a ductal
phenotype. In some embodiments, pancreatic organoids show expression of one or more (for
example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) or all of the following markers, selected
from the group: Hnf1A, Hnf1B, Hnf4A, HHEX, ONECUT1, ONECUT2, CDH1, FOXA2,
GATA6, CEBPB, CEBPD, CEBPG, Glul, Krt7, Krt19 and MET.
However, the pancreatic organoids may have some ductal features in combination with features of
insulin-producing precursor cells. For example, they may express one or more ductal markers as
shown in Figure 16B. In some embodiments, a pancreatic organoid exhibits a gene expression
profile relative to adult pancreas or liver organoids approximately as shown in Figure 16B. For
example, in some embodiments, these genes are upregulated or down regulated in pancreatic
organoids compared to adult pancreas liver organoids to approximately the same fold ratio as in
figure 16B, for example, less than +/- 3%, less than +/- 5%, less than +/- 10%, less than +/- 20%,
In some embodiments, insulin-positive cells appear from the ductal lining in the pancreatic
organoids.
In some embodiments, one or more (e.g. 1, 2, 3, 4, 5, 6 or 7), preferably all of the following genes
are upregulated in pancreas organoids compared to liver organoids: Aaas, Rps4y2, Atp2c2, Akap2,
Uts2, Sox17, Agr2. For example, in some embodiments, these genes are upregulated in pancreatic
organoids compared to liver organoids to approximately the same fold ratio as in figure 19, for
example, less than +/- 3%, less than +/- 5%, less than +/- 10%, less than +/- 20%.
3 4 5
In one embodiment, a pancreatic organoid comprises at least 10 , at least 10 , at least 10 or more
cells in total. In one embodiment, a pancreatic organoid comprises more than 50%, more than
60%, more than 70% or more than 80% ductal-like endocrine progenitor cells However, lower
percentages of ductal-like endocrine progenitor cells are also envisaged.
Barrett’s Esophagus (BE) organoids
A BE organoid of the invention is Ki67+.
Preferably a BE organoid has a minimal number (e.g. less than 25%, less than 20%, less than 10%,
less than 5%, less than 2%, less than 1% cells) of PAS+ and Mucin+ cells 4 days after withdrawal
of Nicotinamide and SB202190 from the expansion medium to covert it to the differentiation
medium.
In some embodiments, a BE organoid comprises goblet cells. These may be induced by treatment
of the differentiation medium with a gamma-secretase inhibitor such as DBZ (e.g. at 10uM), for
example, for 4 days.
In some embodiments, a Barrett’s Esophagus organoid of the invention comprises Paneth cells.
In some embodiments, a Barrett’s Esophagus organoid of the invention expresses lysozyme.
Gastric organoids
In some embodiments, the gastric organoids of the invention show natural expression of Lgr5. In
some embodiments, gastric organoids of the invention show natural expression of at least Lgr5 and
one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17) of stem cell markers from
the group consisting of: CK19, Nestin, Somatostatin, CXCR4 , CD133 , DCAMKL-1, CD44,
Sord, Sox9, CD44, Prss23, Sp5, Hnf1α, Hnf4a, Sox9, KRT7 and KRT19. Alternatively or
additionally, in some embodiments gastric organoids may be characterised by natural expression
of one or more (for example 1, 2 or 3) of: CD133 , DCAMKL-1 and CD44. Alternatively or
additionally, gastric organoids may be characterised by CD44 and Sox9.
Prostate organoids
In some embodiments, the prostate organoids of the invention, such as mouse prostates, show
natural expression of Lgr5. In some embodiments, the prostate organoids show natural expression
of luminal prostate markers, such as Cytokeratin 18 (CK18) and Cytokeratin 8 (CK8). In some
embodiments, the prostate organoids of the invention show natural expression of Androgen
Receptor (AR). In some embodiments, the prostate organoids express basal markers, such as p63
and/or Cytokeratin 5 (CK5). In some embodiments, when testosterone (e.g. DHT) is added to the
medium, the expression of basal markers, Lgr5 and Tnfrsf19 are downregulated compared to
organoids grown in the absence of testosterone (e.g. DHT). The prostate specific transcription
factor NKX3.1 is expressed in all conditions. Therefore, in some embodiments the prostate
organoids of the invention show natural expression of the prostate specific marker Nkx3.1.
In some embodiments, the prostate organoids of the invention, for example normal or cancer
human prostate organoids, show natural expression of luminal markers, such as CK18, CK8 and/or
B-MSP. In some embodiments, the prostate organoids show natural expression of AR. In some
embodiments, basal epithelial markers, such as CK14, CK5 and/or p63 are expressed. In some
embodiments, TNFRSF19 is expressed. The prostate specific transcription factor NKX3.1 is
expressed in all conditions. Therefore, in some embodiments the prostate organoids of the
invention show natural expression of the prostate specific marker Nkx3.1.
The addition of testosterone (e.g. DHT) to a culture medium according to the invention allows
prostate organoids to grow that maintain a stem cell population allowing up to 3-fold faster growth
(than without testosterone) and most (if not all) differentiated cell types of the prostate (both basal
and luminal cells) are also present. These conditions allow unlimited cell expansion (so far 9
months at 2.5 population doublings a week). Therefore, in some embodiments, a prostate organoid
comprises all differentiated cell types of the prostate, for example both basal and luminal cells. In a
preferred embodiment, a prostate organoid comprises all differentiated cell types, for example both
basal and luminal cells, and stem cells.
In normal tissue, addition of testosterone (e.g. DHT) increases AR expression in all culture
conditions. In some embodiments, prostate organoids have upregulated AR expression compared
to prostate cells grown in the absence of testosterone. In tumour tissue AR expression is not
influenced by testosterone (e.g. DHT) addition. Therefore, in some embodiments, a prostate cancer
organoid does not have increased AR expression relative to in vivo prostate cancer cells. The stem
cell marker LGR5 is expressed under ENRF conditions in prostate organoids from normal tissue.
In prostate organoids obtained from tumour tissue, LGR5 expression is induced with the addition
of testosterone (e.g. DHT). In some embodiments, prostate organoids express LGR5.
Organoid functions
In some embodiments, organoids generated by media and methods of the present invention, mimic
in vivo cell fate decisions in response to external factors. Preferably, cells and organoids generated
according to the invention also possess tissue-specific functions.
Pancreatic organoids
A pancreatic organoid preferably possesses endocrine and exocrine pancreatic functions, such as
expressing one or more (for example 1, 2 or all 3) of insulin, glucagon and somatostatin. The
expression of these hormones is tightly regulated by a set of endocrine pancreas-specific
transcription factors, the most important being Pdx1 and NeuroD. The exocrine pancreas is formed
by acinar and ductal compartments responsible of producing the digestive enzymes amylase,
pancreatic lipase and chymotrypsin, among others. The expression of these genes is also regulated
by specific exocrine pancreatic genes as Ptf1a.
Pancreatic cells and organoids according to the present invention may preferably be capable of
secreting insulin, for example, at a rate of between approximately 1μg per hour per 10 cells and
10μg per hour per 10 cells, for example, between 2μg and 6μg per hour per 10 cells. The level of
insulin secretion can be detected by methods well known in the art, for example, by Western Blot
compared to a reference or by C-peptide Elisa. The preferred method to demonstrate that
pancreatic organoids can secrete insulin is by testing productin of C-peptide. Proinsulin C-peptide
serves as an important linker between the A- and the B- chains of insulin and facilitates the
efficient assembly, folding, and processing of insulin in the endoplasmic reticulum. Equimolar
amounts of C-peptide and insulin are then stored in secretory granules of the pancreatic beta cells
and both are eventually released to the portal circulation. Thus, C-peptide is a preferred marker of
insulin secretion.
Thus, in one embodiment there is provided a pancreatic organoid that secretes insulin following
transplantation in vivo. In some embodiments, following transplantation in vivo, the pancreatic
organoid secretes insulin at a rate of at least 1μg per hour per 10 cells, for example, at least 2μg
6 6 6
per hour per 10 cells, at least 4μg per hour per 10 cells, at least 6μg per hour per 10 cells, at least
8μg per hour per 10 cells or at least 10μg per hour per 10 cells, In some embodiments, the cells
in the pancreatic organoid are not capable of secreting insulin and/or do not express insulin as a
marker when cultured in vitro. However, cells from a pancreatic organoid of the present invention
are preferably capable of secreting insulin in vivo when transplanted into a patient, for example,
into the patient’s pancreas. In some embodiments, the ability to secrete insulin may not be present
immediately upon transplantation, but is present by about one month after transplantation, for
example, by 6 weeks, 2 months or 3 months after transplantation.
If an enriched endocrine cell sample is obtained from a pancreatic organoid of the invention, in
some embodiments, 75-85% of the cells in the enriched endocrine cell sample would be insulin-
secreting cells.
In some embodiments, the invention provides pancreatic organoids for use in treating diabetes. In
some embodiments the pancreatic organoids are expanding organoids, whereas in other
embodiments they may be differentiated organoids. In some embodiments one or more (e.g. 1, 2,
3, 4, 5, 6, 7 etc) whole organoids are transplanted into an animal or patient, whereas in other
embodiments a sample of cells is transplanted into a patient.
Crypt-villus organoids
A crypt-villus organoid preferably possesses secretory and self-renewal functions. For example, a
crypt-villus organoid preferably secretes mucin, enzymatic and hormonal secretions, such as
lysozyme, cholecystokinin, secretin and gastric inhibitory peptide, and other glycoproteins.
Gastric organoids
The human stomach is anatomically and functionally divided into two major regions. The pyloric
antrum close to the intestine mainly produces protective mucus and secretes hormones such as
gastrin. The gastric corpus secretes hydrochloric acid and gastric enzymes such as pepsinogen. The
gastric epithelium of the both regions is organized in invaginations called glands. These glands
harbor the gastric stem cells, progenitor cells and differentiated cells. The precise composition of
the differentiated cells varies according to the function of the anatomic region. In the pyloric
antrum, glands are mainly composed of mucin 6 producing cells and hormone producing endocrine
cells. In the corpus, pepsinogen-producing chief cells and acid-secreting parietal cells are dispersed
between the mucus producing cells and sparse endocrine cells. The surface region between gastric
glands is occupied by mucus producing cells that mainly produce the surface mucin 5.
Gastric organoids resemble the gastric epithelium in structure and function. Although they are
mostly spheric, they can have domains with invaginations that most likely resemble glandular
structures. Staining of mucins and pepsinogen shows that the most abundant cell types in the
gastric organoids are mucin 6 producing mucus cells and pepsinogen producing chief cells (and/or
their progenitors). Accordingly, RT-PCRs indicate the expression of pepsinogen and mucin 6.
Further, expression of gastrin indicates the presence of endocrine cells and the expression of Lgr5
indicates the presence of stem cells.
In some embodiments, gastric organoids have natural expression of one or more (e.g. 1, 2, 3 or 4)
of gastrin, pepsinogen, mucin 6 and/or Lgr5. In some embodiments, gastric organoids comprise
mucin 6 producing mucus cells and pepsinogen producing chief cells and optionally Lgr5+ stem
cells. In some embodiments, gastric organoids comprise endocrine cells. In some embodiments,
gastric organoids are mostly spheric but have domains with invaginations that resemble glandular
structures.
Prostate organoids
In some embodiments, prostatic organoids comprise or consist of two distinct epithelial lineages,
basal cells and luminal cells. In some embodiments basal cells and luminal cells secrete prostatic
fluids.
In vivo the prostatic epithelium is strongly folded, ensuring maximum surface area. The two
epithelial lineages form a simple stratified epithelium with the basal epithelial cells forming the
basal/outer layer and the strongly polarized luminal epithelial cells situated on top forming the
inner/luminal layer. The luminal compartment is essential for the secretory function of the
prostate. The prostatic fluid is alkaline and is composed of several proteins, such as Prostate
Specific Antigen (PSA), Human Kallikrein 2 (KLK2) and β-microseminoprotein (β-MSP). The
primary functions of prostatice fluid are: 1) preparing the milieu of the uturus for the semen, which
is performed by the alkalinity of the fluid and the paracrine functions of β-MSP, and 2) increasing
the fluidity of the seminal fluid, allowing the spermatozoa to swim freely, which is performed by
the proteases PSA and KLK2 which breakdown seminogelins.
The expression of secretory proteins is tightly regulated by the androgen receptor (AR), which
binds to testosterone and subsequently translocates to the nucleus and activates transcription.
Disruptions in AR function show a strong downregulated of secretory proteins on a transcriptional
and protein level.
Figure 43 shows the stratification of the prostate organoids grown under
ENR+Dihydrotestosterone (DHT) conditions, clearly showing Cytokeratin 5+ basal cells forming
an outer layer of cells and the Cytokeratin 8+ luminal cells forming a strongly polarized inner
layer. In some embodiments, a prostate organoid comprises cytokeratin 5+ basal cells and
cytokeratin 8+ luminal cells, optionally wherein the Cytokeratin 5+ basal cells forming an outer
layer of cells and the Cytokeratin 8+ luminal cells forming a strongly polarized inner layer. In
some embodiments, a prostate organoid comprises folded layers of cells, optionally comprising
strong folding. Such folding maximizes the surface area of secretory cells, showing that on a
morphological level prostate organoids resemble the in vivo prostate. In some embodiments, the
morphology of a prostate organoid resembles the in vivo morphology of the prostate.
The prostate organoids cultured in ENR conditions do not any secrete prostatic fluid into the
lumen. By contrast, addition of testosterone (e.g. DHT) to the medium results in secretion of
fluids, for example prostatic fluid, in the organoid lumen. This is due to the activation of the AR-
dependent transcriptional program in prostatic organoids by testosterone (e.g. DHT), which results
in secretion of fluids by CK8+ luminals cells. The data show that prostatic organoids both
morphologicaly and functionally resemble the in vivo prostatic epithelium.
Accordingly, in some embodiments, for example wherein the prostate organoids are cultured in a
culture medium comprising testosterone (e.g. DHT), a prostate organoid secretes fluid, for
example prostatic fluid into the lumen of the organoid. In some embodiments, for example wherein
the prostate organoids are cultured in a culture medium comprising testosterone (e.g. DHT), the
functionality of a prostate organoid resembles the in vivo functionality of the prostate.
Tissue fragments
Within the context of the invention, a tissue fragment is a part of an adult tissue, preferably a
human adult tissue, such as part of a human adult small intestine, colon or pancreas. Further
examples of human adult tissue in the context of this invention include stomach, liver and prostate.
The tissue may be normal (healthy) tissue or it may be diseased or infected tissue. Preferably an
organoid as identified herein is therefore not a tissue fragment. An organoid is preferably obtained
using a cell from an adult tissue, preferably an epithelial stem cell from an adult tissue, optionally
from an adult tissue fragment, more preferably an epithelial stem cell from an adult tissue or adult
tissue fragment expressing Lgr5. Therefore, within the context of this invention, a tissue fragment
preferably comprises Lgr5+ stem cells.
In an embodiment, an organoid is an organoid which is still being cultured using a method of the
invention (preferably using a culture medium of the invention) and is therefore in contact with an
extracellular matrix. Preferably, an organoid is embedded in a non-mesenchymal extracellular
matrix. Within the context of the invention, “in contact” means a physical or mechanical or
chemical contact, which means that for separating said organoid from said extracellular matrix a
force needs to be used. In some embodiments, the extracellular matrix is a gelatinous protein
mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, such as Matrigel (BD
Biosciences). In other embodiments of the invention, organoids may be removed from culture and
used for transplantation or regenerative purposes. Thus the invention provides an organoid of the
invention for use in transplantation into a mammal, preferably into a human.
Survival rate
The inventors show here, for the first time, that addition of an inhibitor of ALK4, ALK5, ALK7 or
p38 kinase, to the previously described stem cell culture medium, improved culture plating
efficiency by at least 50% and by more than 100% in some cases (see table 2). The inventors have
also shown that including both inhibitors (an ALK inhibitor and a p38 inhibitor e.g. A83-01 and
SB-202190) in the culture medium synergistically prolongs the culture period.
Accordingly, in one embodiment of the invention, the stem cells survive for at least 3 months,
preferably at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9
months, or at least 12 months or more.
Speed of proliferation
The speed of proliferation may be assessed in terms of the cell population doubling level. The
population doubling level refers to the total number of times the cells in the population have
doubled since their primary isolation in vitro. The population doubling level can be determined by
cell counting. Alternatively, the speed of proliferation can be assessed by a cellular proliferation
assay, for example in which specific fluorescent probes measure DNA synthesis activity by BrdU
incorporation and cell proliferation state by Ki67 expression (Thermo Scientific* Cellomics,
Millipore).
Further examples of cellular proliferation assays for stem cells are readily available can be found
online or in journals such as Current Protocols. One example of many is:
http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catDisplayStyle&catKey=101
&filterDispName=Cellular Proliferation Assays for Stem
Cells&filterType=1&OP=filter&filter=ft_1101%2Ff_494303*&_bcs_=H4sIAAAAAAAAAH2Ns
QrDMAxEv0ZTsEkdKFmzZC70C4IjakFsGVuOfz%2FK0I6F4x284c48YJxfhffm%0ApQ7gnsMb
y0ke6x8fRDJMC7hV03u3lE6Swh9M1nNUWUlQq1UFJkXgeIvvotnSbn6Lbl1yPshvQpyq%0AD
RIPfQE33RlnKQ21Lvuql7CrAAAA.
The inventors have observed that using the culture media of the invention cells can expand by up
to an average of 5 times a week. For example, growing a single cell for two weeks would give
approximately 25 cells on average. The skilled person will understand that the average population
doubling time of the stem cells described herein may vary according to several factors, such as
passage number, culture conditions, seeding density etc.
In one embodiment, the average population doubling time may be 6 to 48 hours, 12 to 36 hours, 18
to 30 hours, or approximately 24 hours. For example, a stem cell population cultured using a
culture medium of the invention may be expected to double approximately 4-7 times, or
approximately 5 times per week.
In another embodiment, the average population doubling time is 12 to 96 hours, 24 to 72 hours, or
approximately 72 hours. In another embodiment, the cell population doubles on average more than
once, more than twice, more than three times, more than four times or more than five times a
week.
Other properties of organoids of the invention
In a preferred embodiment, an organoid could be cultured during at least 2, 3, 4, 5, 6, 7, 8, 9, 10
weeks or 1, 2, 3, 4, 5, 6 months or longer. In a preferred embodiment, an organoid could be
cultured during at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months
or longer.In another preferred embodiment, an organoid originates from a single cell, preferably
expressing Lgr5, more preferably wherein the single cell comprises a nucleic acid construct
comprising a nucleic acid molecule of interest.
Also described herein is an organoid, preferably comprising at least 50% viable cells, more
preferred at least 60% viable cells, more preferred at least 70% viable cells, more preferred at least
80% viable cells, more preferred at least 90% viable cells. Viability of cells may be assessed using
Hoechst staining or Propidium Iodide staining in FACS.
The viable cells preferably possess tissue-specific functions, or characteristics of tissue-specific
functions, as described above.
The inventors have also shown that organoids generated by media and methods of the present
invention can be frozen and stored at -80 C or below, such as in liquid nitrogen. Frozen organoids
can be thawed and put into culture without losing their 3D structure and integrity and without
significant cell death. Therefore, in one embodiment, the invention provides frozen organoids
o o o o o o
stored at below -5 C, below -10 C, below -20 C, below -40 C, below -60 C, or below -80 C.
The cells and organoids of the present invention differ from any cells and organoids that have been
made previously (WO2009/022907 and WO2010/016766) in that they have better phenotypic
(better differentiation profile including goblet cell conversion upon addition of gamma secretase
inhibitors for the crypt-villus organoids) and karyotypic integrity, as determined by the methods
outlined above, better survival rates and faster speeds of cellular proliferation. Accordingly, for
intestinal, colon and pancreatic embodiments, an organoid of the present invention clearly
represents the human intestinal, colon or pancreas epithelium, with full preservation of phenotypic
and karyotypic integrity and maintenance of proliferation and differentiation.
Uses of stem cells or organoids of the invention
The invention provides the use of an organoid or expanded population of cells of the invention for
use in drug screening, (drug) target validation, (drug) target discovery, toxicology and toxicology
screens, personalized medicine, regenerative medicine and/or as ex vivo cell/organ models, such as
disease models.
Cells and organoids cultured according to the media and methods of the invention are thought to
faithfully represent the in vivo situation. This is true both for expanded populations of cells and
organoids grown from normal tissue and for expanded populations of cells and organoids grown
from diseased tissue. Therefore, as well as providing normal ex vivo cell/organ models, the
organoids or expanded population of cells of the invention can be used as ex vivo disease models.
Organoids of the invention can also be used for culturing of a pathogen and thus can be used as ex
vivo infection models. Examples of pathogens that may be cultured using an organoid of the
invention include viruses, bacteria, prions or fungi that cause disease in its animal host. Thus an
organoid of the invention can be used as a disease model that represents an infected state. In some
embodiments of the invention, the organoids can be used in vaccine development and/or
production.
Diseases that can be studied by the organoids of the invention thus include genetic diseases,
metabolic diseases, pathogenic diseases, inflammatory diseases etc, for example including, but not
limited to: cystic fibrosis, inflammatory bowel disease (such as Crohn’s disease), carcinoma,
adenoma, adenocarcinoma, colon cancer, diabetes (such as type I or type II), Barrett’s esophagus,
Gaucher’s disease, alphaantitrypsin deficiency, Lesch-Nyhan syndrome, anaemia,
Schwachman-Bodian-Diamond syndrome, polycythaemia vera, primary myelofibrosis, glycogen
storage disease, familial hypercholestrolaemia, Crigler-Najjar syndrome, hereditary
tyrosinanaemia, Pompe disease, progressive familial cholestasis, Hreler syndrome, SCID or leaky
SCID, Omenn syndrome, Cartilage-hair hypoplasia, Herpes simplex encephalitis, Scleroderma,
Osteogenesis imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy, Dyskeratosis
congenitor etc.
Traditionally, cell lines and more recently iPS cells have been used as ex vivo cell/organ and/or
disease models (for example, see Robinton et al. Nature 481, 295, 2012). However, these methods
suffer a number of challenges and disadvantages. For example, cell lines cannot be obtained from
all patients (only certain biopsies result in successful cell lines) and therefore, cell lines cannot be
used in personalised diagnostics and medicine. iPS cells usually require some level of genetic
manipulation to reprogramme the cells into specific cell fates. Alternatively, they are subject to
culture conditions that affect karotypic integrity and so the time in culture must be kept to a
minimum (this is also the case for human embryonic stem cells). This means that iPS cells cannot
accurately represent the in vivo situation but instead are an attempt to mimic the behaviour of in
vivo cells. Cell lines and iPS cells also suffer from genetic instability.
By contrast, the organoids of the invention provide a genetically stable platform which faithfully
represents the in vivo situation. The organoids of the invention can also be expanded continuously,
providing a good source of genetically stable cells. In particular, an expanding population can be
“split”, meaning that the organoid is split apart and all cells of the organoid are divided into new
culture dishes or flasks. The divided cells are removed from the organoid and can then themselves
be cultured and expanded to produce new organoids containing further expanded populations that
can then be split again. Splits are also referred to herein as “passages”. An organoid of the
invention may be cultured for 1 or more passages, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,
, 30 or more passages, for example, 20-30 passages, 30-35 passages, 32-40 passages or more. In
some embodiments, an expanding cell population or organoid is split once a month, once every
two weeks, once a week, twice a week, three times a week, four times a week, five times a week,
six times a week or daily. Thus the organoids of the invention can provide an ongoing source of
genetically stable cellular material. In some embodiments, the expanding organoids of the
invention comprise all differentiated cell types that are present in the corresponding in vivo
situation. In other embodiments, the organoids of the invention may be differentiated to provide all
differentiated cell types that are present in vivo. Thus the organoids of the invention can be used to
gain mechanistic insight into a variety of diseases and therapeutics, to carry out in vitro drug
screening, to evaluate potential therapeutics, to identify possible targets (e.g. proteins) for future
novel (drug) therapy development and/or to explore gene repair coupled with cell-replacement
therapy.
The organoids of the invention can be frozen and thawed and put into culture without losing their
genetic integrity or phenotypic chrateristics and without loss of proliferative capacity. Thus the
organoids can be easily stored and transported.
For these reason the organoids or expanded populations of cells of the invention can be a tool for
drug screening, target validation, target discovery, toxicology and toxicology screens and
personalized medicine.
Accordingly, in a further aspect, the invention provides the use of the expanded stem cell
population or organoid, such as intestinal crypt-villus organoids or pancreatic organoids according
to the invention in a drug discovery screen, toxicity assay or in medicine, such as regenerative
medicine. For example, any one of the small intestinal, colon, pancreatic, gastric, liver or prostate
organoids may be used in a drug discovery screen, toxicity assay or in medicine, such as
regenerative medicine.
Mucosal vaccines
An additional important use of the organoids is in the development of mucosal vaccinations.
Mucosal vaccines are vaccines that are administered via the mucosa. This can be any mucosal
surface such as via the nose, mouth, or rectum. They can be administered via an inhaler, a spray or
other external aids. This has several clear benefits over injections such as that no medical staff are
needed for administering the vaccine, which may be important, for example in developing
countries.
In the intestine, M cells (or “microfold cells”) are cells found in the follicle-associated epithelium
of the aggregated lymphoid nodules of the ileum. They transport organisms and particles from the
gut lumen to immune cells across the epithelial barrier, and thus are important in stimulating
mucosal immunity. They have the unique ability to take up antigen from the lumen of the small
intestine via endocytosis or phagocytosis, and then deliver it via transcytosis to dendritic cells (an
antigen presenting cell) and lymphocytes (namely T cells) located in a unique pocket-like structure
on their basolateral side.
Figure 48 shows that mouse organoids can develop into M cells when stimulated with RANK
ligand. Figure 49 shows that it is also possible to generate M cells in human intestinal organoids.
Therefore, in some embodiments of the invention, the expanded cell population comprises M cells.
In some embodiments of the invention, an organoid, for example a small-intestinal organoid,
comprises M cells.
The efficiency of mucosal vaccines can be substantially increased when they are targeted to M
cells. Therefore, the expanded stem cell population or organoid of the invention can be used for
testing the ability of M cells to take up pathogens or antigens and to present them to the immune
system. Therefore, in some embodiments the invention provides the use of the expanded stem cell
population or organoid of the invention in drug screening, for example in vaccine development
and/or vaccine production. For example, in some embodiments the expanded stem cell population
or organoid may be used for the development or production of vaccines against viral, bacterial,
fungal or other parasitic infections, for example (but not limited to) cholera, Respiratory syncytial
virus (RSV), Rotavirus and HIV. In a particular embodiment, the invention provides small
intestinal organoids that have been differentiated in a culture medium of the invention comprising
RANKL, for use in mucosal vaccine development.
Drug screening
For preferably high-throughput purposes, said expanded stem cell population or organoid of the
invention, such as crypt-villus organoids or pancreatic organoids, are cultured in multiwell plates
such as, for example, 96 well plates or 384 well plates. Libraries of molecules are used to identify
a molecule that affects said organoids. Preferred libraries comprise antibody fragment libraries,
peptide phage display libraries, peptide libraries (e.g. LOPAP™, Sigma Aldrich), lipid libraries
(BioMol), synthetic compound libraries (e.g. LOP AC™, Sigma Aldrich) or natural compound
libraries (Specs, TimTec). Furthermore, genetic libraries can be used that induce or repress the
expression of one of more genes in the progeny of the stem cells. These genetic libraries comprise
cDNA libraries, antisense libraries, and siRNA or other non-coding RNA libraries. The cells are
preferably exposed to multiple concentrations of a test agent for a certain period of time. At the
end of the exposure period, the cultures are evaluated. The term "affecting" is used to cover any
change in a cell, including, but not limited to, a reduction in, or loss of, proliferation, a
morphological change, and cell death. Said expanded stem cell population or organoid of the
invention such as crypt-villus organoids or pancreatic organoids can also be used to identify drugs
that specifically target epithelial carcinoma cells, but not said expanded stem cell population or
organoid of the invention, such as crypt-villus organoids or pancreatic organoids.
The inventors have shown that it is possible to take a biopsy from the small intestine and expand it
for just 7-14 days and obtain an organoid which is ready for carrying out a drug screen. The ability
to obtain a useful organoid of the invention in such a short time shows that the organoids would be
highly useful for testing individual patient responses to specific drugs and tailoring treatment
according to the responsiveness. In some embodiments, wherein the organoid is obtained from a
biopsy from a patient, the organoid is cultured for less than 21 days, for example less than 14 days,
less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than
8 days, less than 7 days (etc).
The organoids are also useful for wider drug discovery purposes. For example, Figures 32 to 40
show that small intestinal organoids taken from healthy patients and from cystic fibrosis patients
can be used to test drugs against cystic fibrosis. Specifically, Figures 32 to 40 show that forskolin-
induced swelling of normal small intestinal organoids is dependent upon the cystic fibrosis
transmembrane conductance regulator (CFTR), and thus it is possible to test for correction of
CFTR function using forskolin-induced swelling as a positive read out. Therefore, in some
embodiments, the organoids of the invention could be used for screening for cystic fibrosis drugs.
However, it will be understood by the skilled person that the organoids of the invention would be
widely applicable as drug screening tools for infectious, inflammatory and neoplastic pathologies
of the human gastrointestinal tract and other diseases of the gastrointestinal tract and infectious,
inflammatory and neoplastic pathologies and other diseases of other tissues described herein
including pancreas, liver and prostate. In some embodiments the organoids of the invention could
be used for screening for cancer drugs.
In some embodiments, the expanded cell populations, for example the organoids of the invention
or organoids obtained using media and methods of the invention can be used to test libraries of
chemicals, antibodies, natural product (plant extracts), etc for suitability for use as drugs,
cosmetics and/or preventative medicines. For instance, in some embodiments, a cell biopsy from a
patient of interest, such as tumour cells from a cancer patient, can be cultured using culture media
and methods of the invention and then treated with a a chemical compound or a chemical library. It
is then possible to determine which compounds effectively modify, kill and/or treat the patient’s
cells. This allows specific patient responsiveness to a particular drug to be tested thus allowing
treatment to be tailored to a specific patient. Thus, this allows a personalized medicine approach.
The added advantage of using the organoids for identifying drugs in this way is that it is also
possible to screen normal organoids (organoids derived from healthy tissue) to check which drugs
and compounds have minimal effect on healthy tissue. This allows screening for drugs with
minimal off-target activity or unwanted side-effects.
Drugs for any number of diseases can be screened in this way. For example the organoids of the
invention can be used for screening for drugs for cystic fibrosis, Barrett’s esophagus, carcinomas,
adenocarcinomas, adenomas, inflammatory bowel disease (such as Crohn’s disease), liver disease
etc. The testing parameters depend on the disease of interest. For example, when screening for
cancer drugs, cancer cell death is usually the ultimate aim. For cystic fibrosis, measuring the
expansion of the organoids in response to the drugs and stimuli of CFTR is of interest. In other
embodiments, metabolics or gene expression may be evaluated to study the effects of compounds
and drugs of the screen on the cells or organoids of interest.
Therefore, the invention provides a method for screening for a therapeutic or prophylactic drug or
cosmetic, wherein the method comprises:
culturing an expanded cell population (for example, an organoid) of the invention, for
example with a culture medium of the invention, optionally for less than 21 days;
exposing said expanded cell population (for example, an organoid) of the invention to one
or a library of candidate molecules;
evaluating said expanded cell populations (for example, organoids) for any effects, for
example any change in the cell, such as a reduction in or loss of proliferation, a
morphological change and/or cell death;
identifying the candidate molecule that causes said effects as a potential drug or cosmetic.
In some embodiments, computer- or robot-assisted culturing and data collection methods are
employed to increase the throughput of the screen.
In some embodiments, expanded cell population (for example, an organoid) is obtained from a
patient biopsy. In some embodiments, the candidate molecule that causes a desired effect on the
cultured expanded cell population (for example, an organoid) is administered to said patient.
Accordingly, described herein is a method of treating a patient comprising:
(a) obtaining a biopsy from the diseased tissue of interest in the patient;
(b) screening for a suitable drug using a screening method of the invention; and
(c) treating said patient with the drug obtained in step (b).
In some embodiments, the drug or cosmetic is used for treating, preventing or ameliorating
symptoms of genetic diseases, metabolic diseases, pathogenic diseases, inflammatory diseases etc,
for example including, but not limited to: cystic fibrosis, inflammatory bowel disease (such as
Crohn’s disease), carcinoma, adenoma, adenocarcinoma, colon cancer, diabetes (such as type I or
type II), Barrett’s esophagus, Gaucher’s disases, alphaantitrypsin deficiency, Lesch-Nyhan
syndrome, anaemia, Schwachman-Bodian-Diamond syndrome, polycythaemia vera, primary
myelofibrosis, glycogen storage disease, familial hypercholestrolaemia, Crigler-Najjar syndrome,
hereditary tyrosinanaemia, Pompe disease, progressive familial cholestasis, Hreler syndrome,
SCID or leaky SCID, Omenn syndrome, Cartilage-hair hypoplasia, Herpes simplex encephalitis,
Scleroderma, Osteogenesis imperfecta, Becker muscular dystrophy, Duchenne muscular
dystrophy, Dyskeratosis congenitor etc.
Target discovery
In some embodiments, the organoids of the invention or cells grown using the culture media and
methods of the invention can be used for target discovery. Cells of the organoids originating from
healthy or diseased tissue may be used for target identification. The organoids of the invention
may be used for discovery of drug targets for cystic fibrosis, inflammatory bowel disease (such as
Crohn’s disease), carcinoma, adenoma, adenocarcinoma, colon cancer, diabetes (such as type I or
type II), Barrett’s esophagus Gaucher’s disease, alphaantitrypsin deficiency, Lesch-Nyhan
syndrome, anaemia, Schwachman-Bodian-Diamond syndrome, polycythaemia vera, primary
myelofibrosis, glycogen storage disease, familial hypercholestrolaemia, Crigler-Najjar syndrome,
hereditary tyrosinanaemia, Pompe disease, progressive familial cholestasis, Hreler syndrome,
SCID or leaky SCID, Omenn syndrome, Cartilage-hair hypoplasia, Herpes simplex encephalitis,
Scleroderma, Osteogenesis imperfecta, Becker muscular dystrophy, Duchenne muscular
dystrophy, Dyskeratosis congenitor etc. Cells and organoids cultured according to the media and
methods of the invention are thought to faithfully represent the in vivo situation. For this reason
they can be a tool to find novel (molecular) targets in specific diseases.
To search for a new drug target, a library of compounds (such as siRNA) may be used to transduce
the cells and inactivate specific genes. In some embodiments, cells are transduced with siRNA to
inhibit the function of a (large) group of genes. Any functional read out of the group of genes or
specific cellular function can be used to determine if a target is relevant for the study. A disease-
specific read out can be determined using assays well known in the art. For example, cellular
proliferation is assayed to test for genes involved in cancer. For example, a Topflash assay as
described herein, may be used to detect changes in Wnt activity caused by siRNA inhibition.
Where growth reduction or cell death occurs, the corresponding siRNA related genes can be
identified by methods known in the art. These genes are possible targets for inhibiting growth of
these cells. Upon identification, the specificity of the identified target for the cellular process that
was studied will need to be determined by methods well known in the art. Using these methods,
new molecules can be identified as possible drug targets for therapy.
Target and drug validation screens
Patient-specific organoids obtained from diseased and/or normal tissue can be used for target
validation of molecules identified in high throughput screens. The same goes for the validation of
compounds that were identified as possible therapeutic drugs in high throughput screens. The use
of primary patient material expanded in the organoid culture system can be useful to test for false
positives, etc from high throughput drug discovery cell line studies.
In some embodiments, the expanded stem cell population (for example, organoid of the invention),
such as crypt-villus organoids or pancreatic organoids can be used for validation of compounds
that have been idenfitied as possible drugs or cosmetics in a high-throughput screen.
Toxicity assay
Said expanded stem cell population (for example, organoid of the invention), such as crypt-villus
organoids or pancreatic organoids, can further replace the use of cell lines such as Caco-2 cells in
toxicity assays of potential novel drugs or of known or novel food supplements.
Toxicology screens work in a similar way to drug screens (as described above) but they test for the
toxic effects of drugs and not therapeutic effects. Therefore, in some embodiments, the effects of
the candidate compounds are toxic.
Culturing pathogens
Furthermore, said expanded stem cell population (for example, organoid of the invention), such as
crypt-villus organoids or pancreatic organoids, can be used for culturing of a pathogen such as a
norovirus which presently lacks a suitable tissue culture or animal model.
Regenerative medicine and transplantation
Cultures comprising the expanded stem cell population (for example, organoid of the invention),
such as crypt-villus organoids or pancreatic organoids are useful in regenerative medicine, for
example in post-radiation and/or post-surgery repair of the intestinal epithelium, in the repair of
the intestinal epithelium in patients suffering from inflammatory bowel disease such as Crohn's
disease and ulcerative colitis, and in the repair of the intestinal epithelium in patients suffering
from short bowel syndrome. Further use is present in the repair of the intestinal epithelium in
patients with hereditary diseases of the small intestine/colon. Cultures comprising pancreatic
organoids are also useful in regenerative medicine, for example as implants after resection of the
pancreas or part thereof and for treatment of diabetes such as diabetes I and diabetes II.
In an alternative embodiment, the expanded epithelial stem cells are reprogrammed into related
tissue fates such as, for example, pancreatic cells including pancreatic beta-cells. Thus far, it has
not been possible to regenerate pancreatic cells from adult stem cells. The culturing methods of the
present invention will enable to analyse for factors that trans-differentiate the closely related
epithelial stem cell to a pancreatic cell, including a pancreatic beta-cell.
It will be clear to a skilled person that gene therapy can additionally be used in a method directed
at repairing damaged or diseased tissue. Use can, for example, be made of an adenoviral or
retroviral gene delivery vehicle to deliver genetic information, like DNA and/or RNA to stem
cells. A skilled person can replace or repair particular genes targeted in gene therapy. For example,
a normal gene may be inserted into a nonspecific location within the genome to replace a
nonfunctional gene. In another example, an abnormal gene sequence can be replaced for a normal
gene sequence through homologous recombination. Alternatively, selective reverse mutation can
return a gene to its normal function. A further example is altering the regulation (the degree to
which a gene is turned on or off) of a particular gene. Preferably, the stem cells are ex vivo treated
by a gene therapy approach and are subsequently transferred to the mammal, preferably a human
being in need of treatment.
Since small biopsies taken from adult donors can be expanded without any apparent limit or
genetic harm, the technology may serve to generate transplantable epithelium for regenerative
purposes. The fact that organoids can be frozen and thawed and put into culture without losing
their 3D structure and integrity and without significant cell death further adds to the applicability
of organoids for transplantation purposes. Furthermore, in some embodiments, organoids
embedded in, or in contact with, an ECM can be transplanted into a mammal, preferably into a
human. In another embodiment, organoids and ECM can be transplanted simultaneously into a
mammal, preferably into a human.
The skilled person would understand that an ECM can be used as a 3D scaffold for obtaining
tissue-like structures comprising expanded populations of cells or organoids according to the
invention. Such structures can then be transplanted into a patient by methods well known in the art.
An ECM scaffold can be made synthetically using ECM proteins, such as collagen and/or laminin,
or alternatively an ECM scaffold can be obtained by “decellularising” an isolated organ or tissue
fragment to leave behind a scaffold consisting of the ECM (for example see Macchiarini et al. The
Lancet, Volume 372, Issue 9655, Pages 2023 - 2030, 2008). In some embodiments, an ECM
scaffold can be obtained by decellularising an organ or tissue fragment, wherein optionally said
organ or tissue fragment is from the pancreas, liver, intestine, stomach or prostate.
As mentioned above, the invention provides an organoid or population of cells of the invention for
use in transplantation into a mammal, preferably into a human. Also provided is a method of
treating a patient in need of a transplant comprising transplanting an organoid or population of
cells of the invention into said patient, wherein said patient is a mammal, preferably a human.
Advantageously, the invention enables a small biopsy to be taken from an adult donor and
expanded without any apparent limit or genetic harm and so the technology provided herein may
serve to generate transplantable epithelium for regenerative purposes.
Significantly, the inventors have found that when human pancreatic organoids of the invention are
transplanted under the peri-renal capsule in mice, these cells differentiate to form mature beta cells
that secrete insulin. This is significant as it means that even if the population of cells or organoid
of the invention does not secrete insulin at a detectable level whilst the cells or organoids are being
cultured in vitro, these cells may be useful for transplantation into a patient for the treatment of an
insulin-deficiency disorder such as diabetes.
Thus described herein is a method of treating an insulin-deficiency disorder such as diabetes, or a
patient having a dysfunctional pancreas, comprising transplanting a pancreatic organoid of the
invention or cells from a pancreatic organoid of the invention into the patient.
In some embodiments, the cells or organoid do not express or secrete insulin upon transplantation
into the patient but differentiate within the patient such that they secrete insulin. For example, the
ability to secrete insulin may not be detectable immediately upon transplantation, but may be
present by about one month after transplantation, for example, by 6 weeks, 2 months or 3 months
after transplantation.
The patient is preferably a human, but may alternatively be a non-human mammal, such as a cat,
dog, horse, cow, pig, sheep, rabbit or mouse.
Thus, described herein are methods of treatment of a human or non-human animal patient through
cellular therapy. Such cellular therapy encompasses the application of the stem cells or organoids
of the invention to the patient through any appropriate means. Specifically, such methods of
treatment involve the regeneration of damaged tissue. In accordance with the present description, a
patient can be treated with allogeneic or autologous stem cells or organoids. “Autologous” cells
are cells which originated from the same organism into which they are being re-introduced for
cellular therapy, for example in order to permit tissue regeneration. However, the cells have not
necessarily been isolated from the same tissue as the tissue they are being introduced into. An
autologous cell does not require matching to the patient in order to overcome the problems of
rejection. “Allogeneic” cells are cells which originated from an individual which is different from
the individual into which the cells are being introduced for cellular therapy, for example in order to
permit tissue regeneration, although of the same species. Some degree of patient matching may
still be required to prevent the problems of rejection.
Generally the cells or organoids of the invention are introduced into the body of the patient by
injection or implantation. Generally the cells will be directly injected into the tissue in which they
are intended to act. Alternatively, the cells will be injected through the portal vein. A syringe
containing cells of the invention and a pharmaceutically acceptable carrier is also contemplated
herein. A catheter attached to a syringe containing cells of the invention and a pharmaceutically
acceptable carrier is also contemplated herein.
The skilled person will be able to select an appropriate method and route of administration
depending on the material that is being transplanted (i.e. population of cells, single cells in cell
suspension, organoids or fragments of organoids) as well as the organ that is being treated.
As discussed above, cells of the invention can be used in the regeneration of tissue. In order to
achieve this function, cells may be injected or implanted directly into the damaged tissue, where
they may multiply and eventually differentiate into the required cell type, in accordance with their
location in the body. Alternatively, the organoid can be injected or implanted directly into the
damaged tissue. Tissues that are susceptible to treatment include all damaged tissues, particularly
including those which may have been damaged by disease, injury, trauma, an autoimmune
reaction, or by a viral or bacterial infection. In some embodiments of the invention, the cells or
organoids of the invention are used to regenerate the colon, small intestine, pancreas, esophagus or
gastric system.
For example, in one embodiment, the cells or organoids of the invention are injected into a patient
using a Hamilton syringe.
The skilled person will be aware what the appropriate dosage of cells or organoids of the invention
will be for a particular condition to be treated.
In one embodiment the cells or organoids of the invention, either in solution, in microspheres or in
microparticles of a variety of compositions, will be administered into the artery irrigating the tissue
or the part of the damaged organ in need of regeneration. Generally such administration will be
performed using a catheter. The catheter may be one of the large variety of balloon catheters used
for angioplasty and/or cell delivery or a catheter designed for the specific purpose of delivering the
cells to a particular local of the body. For certain uses, the cells or organoids may be encapsulated
into microspheres made of a number of different biodegradable compounds, and with a diameter of
about 15 μm. This method may allow intravascularly administered cells or organoids to remain at
the site of damage, and not to go through the capillary network and into the systemic circulation in
the first passage. The retention at the arterial side of the capillary network may also facilitate their
translocation into the extravascular space.
In another embodiment, the cells or organoids may be retrograde injected into the vascular tree,
either through a vein to deliver them to the whole body or locally into the particular vein that
drains into the tissue or body part to which the cells or organoids are directed. For this embodiment
many of the preparations described above may be used.
In another embodiment, the cells or organoids of the invention may be implanted into the damaged
tissue adhered to a biocompatible implant. Within this embodiment, the cells may be adhered to
the biocompatible implant in vitro, prior to implantation into the patient. As will be clear to a
person skilled in the art, any one of a number of adherents may be used to adhere the cells to the
implant, prior to implantation. By way of example only, such adherents may include fibrin, one or
more members of the integrin family, one or more members of the cadherin family, one or more
members of the selectin family, one or more cell adhesion molecules (CAMs), one or more of the
immunoglobulin family and one or more artificial adherents. This list is provided by way of
illustration only, and is not intended to be limiting. It will be clear to a person skilled in the art,
that any combination of one or more adherents may be used.
In another embodiment, the cells or organoids of the invention may be embedded in a matrix, prior
to implantation of the matrix into the patient. Generally, the matrix will be implanted into the
damaged tissue of the patient. Examples of matrices include collagen based matrices, fibrin based
matrices, laminin based matrices, fibronectin based matrices and artificial matrices. This list is
provided by way of illustration only, and is not intended to be limiting.
In a further embodiment, the cells or organoids of the invention may be implanted or injected into
the patient together with a matrix forming component. This may allow the cells to form a matrix
following injection or implantation, ensuring that the cells or organoids remain at the appropriate
location within the patient. Examples of matrix forming components include fibrin glue liquid
alkyl, cyanoacrylate monomers, plasticizers, polysaccharides such as dextran, ethylene oxide-
containing oligomers, block co-polymers such as poloxamer and Pluronics, non-ionic surfactants
such as Tween and Triton'8', and artificial matrix forming components. This list is provided by
way of illustration only, and is not intended to be limiting. It will be clear to a person skilled in the
art, that any combination of one or more matrix forming components may be used.
In a further embodiment, the cells or organoids of the invention may be contained within a
microsphere. Within this embodiment, the cells may be encapsulated within the centre of the
microsphere. Also within this embodiment, the cells may be embedded into the matrix material of
the microsphere. The matrix material may include any suitable biodegradable polymer, including
but not limited to alginates, Poly ethylene glycol (PLGA), and polyurethanes. This list is provided
by way of example only, and is not intended to be limiting.
In a further embodiment, the cells or organoids of the invention may be adhered to a medical
device intended for implantation. Examples of such medical devices include stents, pins, stitches,
splits, pacemakers, prosthetic joints, artificial skin, and rods. This list is provided by way of
illustration only, and is not intended to be limiting. It will be clear to a person skilled in the art,
that the cells may be adhered to the medical device by a variety of methods. For example, the cells
or organoids may be adhered to the medical device using fibrin, one or more members of the
integrin family, one or more members of the cadherin family, one or more members of the selectin
family, one or more cell adhesion molecules (CAMs), one or more of the immunoglobulin family
and one or more artificial adherents. This list is provided by way of illustration only, and is not
intended to be limiting. It will be clear to a person skilled in the art, that any combination of one or
more adherents may be used.
Methods of the invention
Also described herein is a method for expanding a population of stem cells, wherein the method
comprises:
a) providing a population of stem cells;
b) providing a culture medium according to the invention;
c) contacting the stem cells with the culture medium; and
d) culturing the cells under appropriate conditions.
Also described herein is a method for expanding isolated tissue fragments, wherein the method
comprises:
a) providing an isolated tissue fragment;
b) providing a culture medium according to the invention;
c) contacting the isolated tissue fragment with the culture medium; and
d) culturing the cells under appropriate conditions.
A method for ‘expanding’ a population of cells or isolated tissue fragments is one that involves
maintaining or increasing the number of stem cells in an initial population to generate an expanded
population of stem cells which retain their undifferentiated phenotype and self-renewing
properties. However, it may also include the production of differentiating progeny, which may, for
example, form tissue-like structures contributing to organoid formation. Hence, there are herein
provided methods for obtaining an organoid, such as a small intestinal (crypt-villus) organoid, a
colon organoid, a pancreatic organoid, a gastric organoid, a prostatic organoid, a liver organoid, an
adenocarcinoma organoid, a carcinoma organoid or a Barrett’s Esophagus organoid, comprising
culturing stem cells or tissue fragments comprising said stem cells in a culture medium of the
invention. Also described herein is a method for expanding a single stem cell or a population of
stem cells, preferably to generate an organoid, wherein the method comprises culturing the single
stem cell, population of stem cells or tissue fragment in a culture medium according to the
invention. In some embodiments, the method for obtaining an organoid comprises culturing the
stem cells or tissue fragments with a first “expansion” medium, followed by culturing the stem
cells or tissue fragments with a second “differentiation” medium. In some embodiments, the
differentiation medium does not comprise certain components of the expansion medium, for
example, the differentiation medium does not comprise Wnt, Rspondin, nicotinamide, a TGF-beta
inhibitor and/or a p38 inhibitor.
In some embodiments, the method for expanding a single stem cell or a population of stem cells,
preferably to generate an organoid, comprises expanding the single stem cell, population of stem
cells or tissue fragment in a first culture medium according to the invention, and optionally,
differentiating the expanded cells or tissue fragments in a second culture medium according to the
invention.
Also described herein is a method for expanding a single stem cell or a population of stem cells,
preferably to generate an organoid, wherein the method comprises:
providing a stem cell, a population of stem cells or an isolated tissue fragment;
providing a culture medium according to the invention;
contacting the stem cells with the culture medium;
culturing the cells under appropriate conditions.
In some embodiments, the method comprises bringing the stem cell, the population of stem cells or
the isolated tissue fragment and the culture medium into contact with an extracellular matrix or a
3D matrix that mimics the extracellular matrix by its interaction with the cellular membrane
proteins such as integrins, for example a laminin-containing extracellular matrix such as
Matrigel (BD Biosciences). In some embodiments, the culture medium is diffused into the
extracellular matrix.
In some embodiments, described is a method for expanding a single stem cell or a population of
stem cells or tissue fragment, preferably to generate an organoid, wherein the method comprises:
culturing the stem cell, population of stem cells or tissue fragment in a first expansion
medium;
continuing to culture the stem cell, population of stem cells or tissue fragment and
replenishing the medium with a differentiation medium, wherein the differentiation
medium does not comprise one or more of, preferably all of the factors selected from:
a TGF-beta inhibitor, a p38 inhibitor, nicotinamide and Wnt.
In some embodiments, described is a method for expanding a single stem cell or a population of
stem cells, preferably to generate an organoid of a tissue of interest, comprising:
expanding stem cells or tissue fragments from said tissue of interest in a culture
medium of the invention that is suitable for said tissue of interest; and optionally
differentiating the expanded stem cells or tissue fragments in a culture medium of the
invention that is suitable for said tissue of interest.
Isolated stem cells are preferably cultured in a microenvironment that mimics at least in part a
cellular niche in which said stem cells naturally reside. Said cellular niche is mimicked by
culturing said stem cells in the presence of biomaterials, such as matrices, scaffolds, and culture
substrates that represent key regulatory signals controlling stem cell fate. Said biomaterials
comprise natural, semi-synthetic and synthetic biomaterials, and/or mixtures thereof. A scaffold
provides a two-dimensional or three dimensional network. Suitable synthetic materials for said
scaffold comprise polymers selected from porous solids, nanofibers, and hydrogels such as, for
example, peptides including self-assembling peptides, hydrogels composed of polyethylene glycol
phosphate, polyethylene glycol fumarate, polyacrylamide, polyhydroxyethyl methacrylate,
polycellulose acetate, and/or co-polymers thereof (see, for example, Saha et al , 2007 Curr Opin
Chem Biol 1 1(4) 381-387, Saha et al , 2008 Biophysical Journal 95 4426-4438, Little et al , 2008
Chem Rev 108, 1787-1796). As is known to a skilled person, the mechanical properties such as,
for example, the elasticity of the scaffold influences proliferation, differentiation and migration of
stem cells. A preferred scaffold comprises biodegradable (co)polymers that are replaced by natural
occurring components after transplantation in a subject, for example to promote tissue regeneration
and/or wound healing. It is furthermore preferred that said scaffold does not substantially induce
an immunogenic response after transplantation in a subject. Said scaffold is supplemented with
natural, semi-synthetic or synthetic ligands, which provide the signals that are required for
proliferation and/or differentiation, and/or migration of stem cells. In a preferred embodiment, said
ligands comprise defined amino acid fragments. Examples of said synthetic polymers comprise
Pluronic® F 127 block copolymer surfactant (BASF), and Ethisorb® (Johnson and Johnson).
A cellular niche is in part determined by the stem cells and surrounding cells, and the extracellular
matrix (ECM) that is produced by the cells in said niche. In one method described, isolated crypts
or epithelial stem cells are attached to an ECM. ECM is composed of a variety of polysaccharides
(mostly heparin sulphate proteoglycans), water, elastin, and glycoproteins, wherein the
glycoproteins comprise collagen, entactin (nidogen), fibronectin, and laminin. ECM is secreted by
connective tissue cells. Different types of ECM are known, comprising different compositions
including different types of glycoproteins and/or different combination of glycoproteins. Said
ECM can be provided by culturing ECM-producing cells, such as for example fibroblast cells, in a
receptacle, prior to the removal of these cells and the addition of isolated crypts or epithelial stem
cells. Examples of extracellular matrix-producing cells are chondrocytes, producing mainly
collagen and proteoglycans, fibroblast cells, producing mainly type IV collagen, laminin,
interstitial procollagens, and fibronectin, and colonic myofibroblasts producing mainly collagens
(type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic acid, fibronectin, and tenascin-C.
Alternatively, said ECM is commercially provided. Commercially provided ECMs are typically
synthetic ECMs. Examples of commercially available extracellular matrices are extracellular
matrix proteins (Invitrogen) and Matrigel™ (BD Biosciences). The use of an ECM for culturing
stem cells enhanced long-term survival of the stem cells and the continued presence of
undifferentiated stem cells.
An example of an ECM for use in a method of the invention comprises at least two distinct
glycoproteins, such as two different types of collagen or a collagen and laminin. Said ECM can be
a synthetic hydrogel extracellular matrix or a naturally occurring ECM. A most preferred ECM is
provided by Matrigel™ (BD Biosciences), which comprises laminin, entactin, and collagen IV.
Therefore, in some embodiments, the ECM for use in a method of the invention is a 3D matrix that
mimics the extracellular matrix by its interaction with the cellular membrane proteins such as
integrins.
Thus in some embodiments, a method as described comprises bringing the stem cell, the
population of stem cells or the isolated tissue fragment and the culture medium into contact with
an extracellular matrix, for example a laminin-containing extracellular matrix such as MatrigelTM
(BD Biosciences). In some embodiments, the culture medium is diffused into the extracellular
matrix.
Compositions and other forms of the invention
Also described herein is a composition comprising a culture medium according to the invention
and stem cells. Also described herein is a composition comprising a culture medium according to
the invention and organoids. Also described herein is a composition comprising a culture medium
according to the invention and an extracellular matrix.
Also described herein is a composition comprising a culture medium of the invention, an
extracellular matrix and stem cells as described herein. The invention also provides a composition
comprising a culture medium of the invention, an extracellular matrix and one or more organoids
of the invention. Also described herein is a culture medium supplement that can be used to
produce a culture medium as disclosed herein. A ‘culture medium supplement’ is a mixture of
ingredients that cannot itself support stem cells, but which enables or improves stem cell culture
when combined with other cell culture ingredients. The supplement can therefore be used to
produce a functional cell culture medium of the invention by combining it with other cell culture
ingredients to produce an appropriate medium formulation. The use of culture medium
supplements is well known in the art.
Also described herein is a culture medium supplement that comprises an inhibitor as described
herein. The supplement may contain any inhibitor (or combination of inhibitors) disclosed herein.
The supplement may also contain one or more additional cell culture ingredients as disclosed
herein, e.g. one or more cell culture ingredients selected from the group consisting of amino acids,
vitamins, inorganic salts, carbon energy sources and buffers.
A culture medium or culture medium supplement may be a concentrated liquid culture medium or
supplement (e.g. a 2x to 250x concentrated liquid culture medium or supplement) or may be a dry
culture medium or supplement. Both liquid and dry culture media or supplements are well known
in the art. A culture medium or supplement may be lyophilised.
A culture medium or supplement will typically be sterilized prior to use to prevent contamination,
e.g. by ultraviolet light, heating, irradiation or filtration. A culture medium or culture medium
supplement may be frozen (e.g. at -20°C or -80°C) for storage or transport. In some embodiments,
the culture medium may be stored as a liquid (e.g. at approximately 4 C). In some embodiments,
the culture medium may be split and stored as two components: a frozen component (e.g. at
between approximately -20°C and approximately -80°C) and a liquid component (e.g. at
approximately 4 C). In particular, temperature-sensitive or time-sensitive degradable material is
preferably included in the frozen component, whereas less sensitive material (for example DMEM
or FCS) can be storred in the liquid form and thus included in the liquid component for storage and
shipping.
The invention also provides a hermetically-sealed vessel containing a culture medium of the
invention or culture medium supplement as described. Hermetically-sealed vessels may be
preferred for transport or storage of the culture media or culture media supplements disclosed
herein, to prevent contamination. The vessel may be any suitable vessel, such as a flask, a plate, a
bottle, a jar, a vial or a bag.
Also described herein is a kit comprising a culture medium, culture medium supplement and/or a
composition as described herein. In some embodiments, the kit further comprises at least one other
additional component, for example selected from the list comprising: an ECM (for example,
Matrigel ), a population of cells and an organoid.
General
“GI” numbering is used above. A GI number, or “GenInfo Identifier”, is a series of digits assigned
consecutively to each sequence record processed by NCBI when sequences are added to its
databases. The GI number bears no resemblance to the accession number of the sequence record.
When a sequence is updated (e.g. for correction, or to add more annotation or information) then it
receives a new GI number. Thus the sequence associated with a given GI number is never
changed.
The term “comprising” encompasses “including” as well as “consisting” e.g. a composition
“comprising” X may consist exclusively of X or may include something additional e.g. X + Y.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially
free” from Y may be completely free from Y. Where necessary, the word “substantially” may be
omitted from the definition of the invention.
The term “about” in relation to a numerical value x is optional and means, for example, x+10%.
Unless specifically stated, a process comprising a step of mixing two or more components does not
require any specific order of mixing. Thus components can be mixed in any order. Where there are
three components then two components can be combined with each other, and then the
combination may be combined with the third component, etc.
Various aspects and embodiments of the invention are described below in more detail by way of
example. It will be appreciated that modification of detail may be made without departing from the
scope of the invention.
In this specification where reference has been made to patent specifications, other external
documents, or other sources of information, this is generally for the purpose of providing a context
for discussing the features of the invention. Unless specifically stated otherwise, reference to such
external documents is not to be construed as an admission that such documents, or such sources of
information, in any jurisdiction, are prior art, or form part of the common general knowledge in the
art.
DESCRIPTION OF THE DRAWINGS
Figure 1 Mouse colon culture
a. left: Axin2 expression is under the control of the Wnt signaling pathway. Colon crypt organoids
of Axin2-LacZ reporter mice cultured with EGF, Noggin, and R-spondin (ENR) for 3 days.
Absence of LacZ stain indicates that no active Wnt signal is present in the colon organoids under
ENR growth condition. Inset shows active Wnt signalling visualized by LacZ expression (dark
stain) in freshly isolated colon crypts from the Axin2-LacZ reporter mice. right: Axin2-LacZ mice
derived colon crypts cultured with ENR + Wnt3A (WENR) for 10 days. Dark stain indicates LacZ
expression in these organoids.
b. left: Lgr5-GFP-ires-CreER colon crypts cultured with ENR for 3 days. Absence of GFP
fluorescence indicates loss of Lgr5 expression in the colon organoids under ENR growth
condition. Inset shows Lgr5-GFP expression in freshly isolated colon crypts from Lgr5-GFP-ires-
CreER mice. right: Lgr5-GFP-ires-CreER colon crypt cultured with WENR for 10 days
demonstrates the presence of Lgr5 stem cells.
c. Culture efficiency is determined under three different conditions: ENR, WENR full crypts, and
WENR crypts after mild enzymatic digestion (WENR digested). Colon crypts were isolated from
proximal colon (black columns) or distal colon (white columns). *:p<0.05.
d, e: 4 days after removal of Wnt3A from the WENR culture medium results in organoid
differentiation. d. Chromogranin A (ChA) in enteroendocrine cells;Mucin2 (muc2) in Goblet
cells and the counter stain with DAPI can be seen. e. Mature enterocytes are visualized by Alkaline
phosphatase staining.
f. Relative mRNA expression of mature epithelial cell markers (Vil1 (Villin1), Alpi (Alkalin
phosphatase), Chga (Chromogranin A), Muc2 (Mucin2)) are shown. WENR cultured colon crypt
organoids are cultured for 4 days in WENR (hatched pattern) or ENR (black) condition. Freshly
isolated colon crypts (white) are used for control. Scale bar in a, b, d, e: 50 μm. Error bars indicate
s.e.m. n=3.
Figure 2 Human colon culture
a. The effect of nicotinamide on human colon crypt organoids. The majority of human colon crypt
organoids die within a few days in WENR+gastrin (WENRg) condition (left panel). Addition of
nicotinamide (middle panel: WENRg+nic) improves culture efficiency and lifespan of human
colon organoids. * p<0.001. nic: nicotinamide.
b. The effect of small molecule inhibitor for Alk4/5/7 (A83-01) and for the MAP kinase p38
(SB202190) on human colon crypt organoids. Left panel: Human colon organoids cultured in
WENRg + nicotinamide containing medium form cystic structures 3-4 weeks after culture. Middle
panel: Human colon organoids retain their characteristic budding structure under the Human
Intestinal Stem Cell Culture (“HISC”) condition (WENRg+nicotinamide+A83-01+SB202190).
Right panel: A83-01 and SB202190 synergistically increase number of passages of the human
colon organoids. * p<0.001. N.S.=statistically not significant. Error bars indicate s.e.m. n=5.
c. Proliferating cells visualized by the incorporation of EdU are confined to the
budding structures. DAPI is used as a counterstain
d. Representative picture of a karyotype from a 3-month-old human colon crypt organoid. Scale:
100 μm.
e. Heat-map of the expression profile of cultured human intestinal organoids. The heat-map is a
comparison of human small intestinal crypts and human small intestinal villi. Genes more highly
expressed in the crypt are dark grey (top-half of heat-map), genes more highly expressed in the
villus are light grey (bottom-half of the heat-map). Organoids cultured in-vitro clearly exhibit a
similar expression profile to freshly isolated small intestinal crypts and express known stem cell
markers. (lane 1: human small intestinal organoids #1, lane 2: human small intestinal organoids #2,
lane 3: human colon organoids, lane 4: freshly isolated human small intestinal crypts. The four
samples are compared to human smallintestinal villus).
Figure 3 Human intestinal organoid cell type composition
(a-c) Human organoids differentiate into the different cell types of the intestine after
withdrawal of Nicotinamide and SB202190. Markers of the different cell types were used
to demonstrate differentiation. (a) Top panel: Alkaline phosphatase staining for mature
enterocytes, Middle panel: PAS staining for goblet cells, Bottom panel: Synaptophysin staining for
enteroendocrine cells. (b) In each case, the light areas indicate staining. Mucin2 (Muc2) staining in
the middle panel represents goblet cells and Chromogranin A (ChgA) in the left-hand panel
represents enteroendocrine cells (see arrow and inset). DAPI is used as a counterstain (right panel).
(c) Lysozyme (Lysz) is stained in the left-hand panel to show Paneth cells. DAPI is used as a
counterstain (right panel).
(d-f) Goblet cell differentiation (Muc2) is blocked by SB202190 treatment of organoids (d), while
the Notch inhibitor DBZ increases goblet cell number in the human organoids (f). Proliferating
cells are represented by EdU incorporation (middle panel) are increased in SB202190 treated
organoids (d) or decreased in DBZ treated organoids (f).
Organoids are cultured under the following conditions for 5 days: a) top: ENRg+A83-
01+SB202190+Nicotinamide, a) middle and bottom, b), c) WENRg+A83-01, d)WENRg+A83-
01+SB202190, e) WENRg+A83-01, f) WENRg+A83-01+DBZ.
Scale bar:20 μm (a), 50 μm (b-f). a, b, d-f: human colon crypt organoids, c: human small intestinal
organoids.
Figure 4 Adeno(carcino)ma cultures
a. Lgr5-GFP-ires-CreER/APCfl/fl crypts cultured with EGF (E) (top) or EGF+Noggin
(EN) (bottom) for 10 days. b. Relative mRNA expression of Lgr5 and Axin2. Freshly
isolated adenoma cells (white) were cultured with EGF (hatched) or EGF+Noggin (black).
c. Culture efficiency of organoids from sorted Lgr5-GFPhi, Lgr5-GFPlo, Lgr5-GFP-ve cells.
*p<0.01. one way ANOVA. Error bars indicate s.e.m. n=3
d. Time course culture of human colon adenocarcinoma cells.
Figure 5 Culture of Barrett’s esophagus and treatment with Notch inhibitor.
a. Isolated epithelium from Barrett’s esophagus (BE) cultured with HISC condition for 7 days
forms cystic structures. b. Addition of FGF10 significantly increases the number of passages for
BE organoids. Error bars indicate s.e.m. n=3 c. Representative time course of a BE organoid. d.
Paraffin sections from BE organoids. Nicotinamide and SB202190 are withdrawn for 4 days with
(right) or without (left) the Notch inhibitor DBZ added to the medium. Proliferating cells (Ki67
stain) disappear and PAS+ goblet cells increase with DBZ treatment.
Figure 6 Axin2 mRNA expression is recovered in mouse colon
organoids under the presence of Wnt-3A
Isolated colonic crypts are analysed for Axin2 mRNA expression after 3 days or 7 days culture
with ENR (hatched) or WENR (black). Freshly isolated colon crypts were used as control. Error
bars indicate s.e.m. n=3
Figure 7 Relative mRNA expression of mature epithelial cell markers
Freshly isolated small intestinal crypts (white) are cultured in HISC condition for 14 days,
followed by a culture with the indicated culture condition for 4 days. mRNA expression
of ALPI (Alkaline phosphatase), VIL1 (Villin 1), LYZ (Lysozyme), CHGB (Chromogranin
B) and MUC2 (Mucin2) was analysed. Culture condition: HISC (black), ENRg+A83-
01+SB202190+Nicotinamide, WENRg+A83-01, ENRg+A83-01, ENRg. Freshly isolated small
intestinal crypts were used as control (set as 1.0 for ALPI, VIL1 and LYZ, as 5.0 for CHGB and
MUC2. Error bars indicate s.e.m. n=3.
Figure 8 Sorted Lgr5-GFP- cells form Lgr5-GFP+ organoids
Single sorted Lgr5-GFP- APCfl/fl adenoma cells are cultured with EGF+Noggin (EN) or
EGF (E) for 7 days. Adenoma organoids derived from Lgr5-GFP- cells recovered Lgr5-
GFP expression under EN condition but not under E condition (a, c: bright, b, d: GFP
autofluorescence).
Figure 9 Histochemical analysis of adenoma/colon cancer organoids
Mouse small intestinal adenoma organoids (Left panel) and human colon cancer organoids (Right
panel) were analyzed with indicated histochemical (HE, PAS and Alkaline phosphatase) or
immunohistochemical (Chromogaranin A, Ki67 and Caspase3) stainings.
Figure 10 Paneth cells in BE organoids
Lysozyme+ Paneth cells were observed in differentiated BE organoids.
Figure 11 List of reagents used for organoid culture
Figure 12 List of reagents used for optimization of human intestinal
organoid culture
Figure 13 List of small molecule inhibitors used for optimization of
human intestinal organoids culture
Figure 14 List of the 25 most up- and down-regulated genes
mRNA from human small intestinal organoids or colon organoids are compared with that
from freshly isolated small intestinal villi by microarray. The 25 most upregulated and
downregulated genes are shown. Hatched lines highlight genes which were in the top 70
most upregulated and downregulated genes in freshly isolated human small intestinal crypts vs.
villi.
Figure 15. Summary of proliferation, differentiation and apoptosis status of each organoid
culture condition
Figure 16: Microarray comparison of mouse pancreatic organoids
A – Microarray clustering analysis, comparing RNA from the pancreas organoids (cultured in the
conditions described in Example 2) with adult pancreas, adult liver and newborn liver. From left
to right: i) pancreas organoid; ii) adult pancreas; iii) adult liver (sample 1 [S1] and sample 2 [2]);
iv) adult liver S2; and v) newborn liver.
B – Raw signal data from the microarray analysis, comparing the expression levels of selected
ductal markers, transcription factors necessary for Ngn3 expression and endocrine markers in adult
liver, adult pancreas, pancreas organoids and liver organoids in expansion media.
Figure 17: The effect of Noggin on the expansion of pancreatic organoids
A – Bar charts showing gene expression analysis of pancreatic organoids cultured in EGFRA so,
that have never been cultured with Noggin (black) with organoids cultured in EGFRAN , so have
always been cultured with Noggin (white). The effect of culturing the pancreatic organoids in
EGFRA for 2 days and then withdrawing Noggin and culturing for a further 2 or 4 days (light
grey) and the effect of culturing the pancreatic organoids in EGFRA for 2 days and then adding
Noggin and culturing for a further 2 or 4 days (dark grey) on gene expression is also shown.
mRNA levels (arbitrary units) are presented on the Y axis. mRNA of the following early
endocrine markers is analysed in the main figure: Sox9, Hnf1b, Hnf6, Hnf1a, Nkx2.2, Nkx6.1 and
Pdx1. mRNA of the following ductal markers in analysed in the inset part: keratin 7 (Krt7) and
keratin 19 (Krt19).
B – Bar chart showing the effect of Noggin on the expression of Lgr5 in pancreatic organoids in
the expansion culture medium. Data are provided for pancreatic organoids cultured in EGFRA that
have never been cultured with Noggin (black) with organoids cultured in EGFRAN and so have
always been cultured with Noggin (white). The effect of culturing the pancreatic organoids in
EGFRAN and then withdrawing Noggin and culturing for a further 6 days (light grey) and the
effect of culturing the pancreatic organoids in EGFRA and then adding Noggin and culturing for a
further 6 days (dark grey) on Lgr5 gene expression is also shown. mRNA levels (arbitrary units)
are presented on the Y axis.
Figure 18: Human insulin producing cells develop from ex vivo expanded, in vivo
transplanted progenitor cells
A – Growth of human pancreas tissue from progenitor cells (pancreas stem cells) at P0: (Day 1);
P0: (Day 5); P1: (Day 12) and P3: (Day 24), where “P” refers to the number of passages.
Figures 18B and C show transplantation of human pancreatic organoids under the murine peri-
renal capsule.
B – 3 hours after transplantation of the pancreatic organoid cells into the recipient mice: DAPI
(nuclear marker) staining in the upper picture indicates all cells; K19 (ductal marker) staining in
the lower picture shows all transplanted cells and insulin (beta cell marker) in the lower picture
indicates insulin-producing cells.
C – 1 month after transplantation of the pancreatic organoid cells into the recipient mice: DAPI
(nuclear marker) staining in the upper picture (in blue) indicates all cells; CK19 (ductal marker)
expression in the middle picture (in green) indicates all transplanted cells and insulin (beta cell
marker) in the lower picture (in red) indicates insulin-producing cells. A selection of the insulin-
producing cells are encircled but all clearly stained cells are thought to be insulin positive.
Figure 19: Pancreatic organoid gene expression
This table shows the pancreatic gene expression of the most upregulated genes when compared to
liver organoids.
Figure 20: Mouse liver organoid culture shows stable karyotyping after long-term culture.
A – DIC images of liver organoids maintained in EGF (E) and R-spondin 1 (R),
supplemented with FGF10, HGF and Nicotinamide (left figure, ER) or maintained in the same
combination supplemented with Noggin (N) and Wnt3Aconditioned media (W) (right figure,
ENRW) for a period of 24 months.
B – Karyotype analysis of mouse liver organoids after 8 months in culture. Normal
chromosomal counts (n=40, left panel figure) and polyploidy, a typical hepatocyte feature, were
found (n=80, right panel figure)
Figure 21: Supplemental factors FGF10, HGF and Nicotinamide; effect on liver organoid
growth and differentiation.
A – Diagram depicting the genes differentially expressed during the 3 stages of liver
development, from hepatoblast to mature hepatocyte.
B – Scheme showing the protocol used. Cultures were seeded in expansion medium
(ERFHNic: EGF (E) and R-spondin 1 (R), supplemented with FGF10, HGF and Nicotinamide;
ERFHNic is indicated as ‘ER’ in Figure 8B) 2 days prior the experiment. Two days later, culture
media was changed to either EGF (E) alone or EGF supplemented with R-spondin 1 (ER) with or
without additional supplements chosen from FGF10 (F) or HGF (H) or Nicotinamide (Nic) or a
combination of these at the concentrations stated in the text. Five days later cultures were split and
replated at 1:4 ratio for each condition. Under these conditions, cultures have been split and
replated every 7 days for a total period of 10 weeks
C – First day after first split in each of the culture conditions tested. Results shows that EGF
and Rspondin 1 combined with FGF10 or HGF or Nicotinamide or a combination of these are
essential to achieve at least 1 passage.
D – After long-term culture, the combination of ER supplemented with FNic or ER
supplemented with FHNic, both result in high passage numbers. After passage 10, the growth rate
is better for the culture condition including the 3 supplemental factors; ERFHNic.
E – RT-PCR analysis showing the expression of different hepatocyte markers (CYP3A11,
Alb, FAH) and cholangiocyte marker (K19) 5 days after the withdrawal of certain factors (starting
point was ERFHNic). Note that only the condition EF showed expression of all hepatocyte
markers tested. HPRT was used as a housekeeping gene to normalize for gene expression.
Figure 22: Table showing the quantification of different hepatocyte and cholangiocyte
specific transcription factors in cells from three different liver culture conditions and in
adult liver tissue. Also shown is the expression of the key components of the Notch and TGF-beta
signalling pathways. E=EFHNic, ER=ERFHNic, ENRW=ENRWFHNic.
Figure 23: Differentiation protocol
A – Scheme showing the protocol used. Cultures were seeded in expansion medium
(ERFHNic: EGF (E) and R-spondin 1 (R), supplemented with FGF10, HGF and Nicotinamide;
ERFHNic is indicated as ‘ER’ in Figure 10A) 2 days prior to the experiment. Two days later,
culture media was changed to EGF (E) supplemented with either A8301 (A), or DAPT (D), or
FGF10 (F) or HGF (H) or Nicotinamide (Nic) or R-spondin 1 (R) or Wnt3A or Noggin (N) or a
combination of these at the concentrations shown. RNA was isolated at several time points. Mouse
liver tissue was taken as positive control (+) whereas water was taken as negative control (-).
B – RT-PCR analysis showing the expression of the hepatocyte markers CYP3A11, Alb,
FAH, tbx3, TAT and Gck 7 days after differentiation conditions. Note that only the condition
EADF showed an expression of all hepatocyte markers tested. HPRT was used as a housekeeping
gene to normalize for gene expression.
C – Time course expression analysis after differentiation conditions. At days 2, 5 and 8 days
after differentiation, the expression of the hepatocyte markers CYP3A11, Alb, FAH, and the
cholangyocyte marker K19, was analysed by RTPCR. Note that the expression of the liver markers
CYP3A11 and FAH starts at day 5 and peaks at day 8 after. HPRT was used as a housekeeping
gene to normalize for gene expression. A; A8301, D; DAPT, F; FGF10, H; HGF, De;
Dexamethasone
D – Titration experiment showing the expression of the hepatocyte markers CYP3A11, Alb,
FAH, tbx3, TAT, G6P and Gck 7 days after different concentrations of the differentiation
compounds A and D. HPRT was used as a housekeeping gene to normalize for gene expression.
E – Immunofluorescent staining for the liver markers K19, Albumin and hepatocyte surface
marker
F – Xgal staining on Albcreert2LacZ mice liver-derived organoids. Albumin positive cells
(arrows) were detected after EADF differentiation in tamoxifen induced Albcreert2LacZ derived
cultures.
Figure 24: Prostaglandin signalling pathway (Antagonism of the prostaglandin D receptors DP
and CRTH2 as an approach to treat allergic diseases. Roy Pettipher, Trevor T. Hansel & Richard
Armer Nature Reviews Drug Discovery 6, 313-325 (April 2007)).
Figure 25: Liver organoids cultured in (A) basal medium comprising hEGF (100ng/ml,
Invitrogen); human noggin (hnoggin) (25ng/ml, peprotech); gastrin (10nM, sigma); hFGF10
(peprotech); nicotinamide (10mM, sigma); A8301 (500nM, Tocris); hHGF (50ng/ml, peprotech);
Rspo conditioned media (10%); (B) basal medium + PGE2 (50nM); (C) basal medium +
CHIR99021 (3uM); (D) basal medium + CHIR99021 (3uM) + PGE2 (50 nM).
Figure 26: Liver organoids cultured in basal medium (as described for Figure 25) with and
without Arachidonic acid.
Figure 27: Gene expression profile of mouse liver organoids under differentiation conditions
resemble the adult and newborn liver profile
A – Gene clusters showing the genes similarly expressed (a) or similarly shut down (b) between
the differentiation condition EADF and adult or newborn liver.
B – Gene clusters showing the genes differentially expressed between the liver organoids and adult
or newborn liver (a) and the genes similarly expressed between EADF and newborn liver (b).
C - Raw signal data from a microarray analysis, comparing the expression levels of selected ductal
markers, transcription factors necessary for Ngn3 expression and endocrine markers in adult liver,
adult pancreas, pancreas organoids and liver organoids in expansion media.
Figure 28: Mouse liver signature genes
Table showing a) markers expressed in mouse liver stem cells; b) markers not expressed in mouse
liver stem cells; c) hepatocyte and cholangiocyte markers expressed in mouse liver stem cell
signature for mouse liver organoids in expansion media; d) hepatocyte and cholangiocyte markers
not expressed in mouse liver stem cell signature for mouse liver organoids in expansion media; e)
reprogramming genes expressed in mouse liver organoids; f) reprogramming genes not expressed
in mouse liver organoids. The results were obtained using a liver microarray using the Universal
Mouse Reference RNA (Strategene, Catalog #740100) as a reference RNA. If the absolute figures
detected were less than 100, the gene was consider as undetected.
Figure 29: Human liver signature genes
Table showing results of liver mircroarray of human organoids. From left to right, the results are
shown for a) expansion medium EM1, b) expansion medium EM2, c) differentiation medium, d)
adult liver.
The numbers (log2) in the left four columns are the result of a comparison between the sample and
a reference (commercial) RNA sample which is used for all arrays. The relative expression of
mRNA in each sample compared to the RNA present in the reference sample is shown. . The
reference RNA used was Universal Human Reference RNA (Stratagene, Catalog #740000). Thus,
negative numbers in these columns do not relate to real expression levels it just means there is less
of that RNA then in the Reference sample. The 4 columns on the right are absolute figures. If they
are below 100, they are considered as undetected.
Figure 30: Morphology of liver organoids. (A) Upper panels: paraffin section of a mouse liver
showing the different domains (PT= portal triad, CV= central vein). Lower panels: Paraffin section
of a liver organoid showing different domains b (single layered epithelia) and h (stratified
epithelia) (B) Right pannel: Ecadherin staining in the liver organoids. Two different domains can
be identified. Domain b, formed by a single layered epithelia that resembles the bile duct structures
in the liver. This bile duct domain is formed by highly polarized cells that shows positive staining
for pancytokeratin (PCK) (lower panel). Left panels show the presence of a second domain within
the liver organoids. This h domain is formed by a stratified epithelia with non-polarized cells. The
cells are organized around a central lumen and express the hepatocyte marker Alb. Magnification
x.
Figure 31: H&E staining of pancreas organoids
Mouse pancreas organoids were cultured in expansion conditions EGFNRA83-01
[(EGF(50ng/ml), Gastrin (50nM), Noggin (10%), Rspondin (5%), FGF10 (100ng/ml) and A8301
(50nM)] during 8 passages (~10 weeks). The organoids were removed from the matrigel using BD
Cell Recovery Solution following manufacturer’s instructions and fixed with 4%
paraformaldehyde at room temperature during 1h. Then, the organoids were washed three times
with cold PBS, dehydrated with increased concentration of alcohol and embedded in paraffin. 3um
parafine sections were stained with Hematoxyline-Eosine to analyze the histology of the pancreas
organoids.
A strong variability in the shape and structure of the organoids was observed. Some of the
organoids are cystic structures formed by a monolayer of polarized epithelial cells. Other
organoids show the same monolayer of epithelial cells and some stratified areas where cells are
smaller in size and with a round shape. In some organoids invaginations occupying the inner space
of the cystic structure were observed.
The stainings show that some organoids comprise mostly monolayers of epithelial cells (left
bottom), whereas other organoids comprise stratified regions and/or pseudo stratified regions
and/or folded monolayers (right bottom). Most pancreatic organoids comprise regions of stratified
cells and monolayers (sometimes folded).
Figure 32. Quantification of forskolin-induced murine small intestine organoid swelling. (a)
Light microscopy analysis of organoids stimulated with forskolin or DMSO. Representative
examples for the indicated time points after start of stimulation are shown. The red line indicates
the internal organoid lumen. (b) Fluorescence confocal image of a calcein-green-labeled organoid
with object recognition (green line) by image analysis software. (c) Representative example of a
forskolin-stimulated calcein-green-labeled organoid. Differential interference contrast (DIC) and
fluorescence was imaged using live cell confocal microscopy. Surface area relative to t=0 is
indicated in the top-left corner. (d) The surface area relative to t=0 (normalized area) of 11
individual organoids in a single well. The average is indicated in black (mean ± s.e.m.). (e) Dose-
dependent increase of surface area by forskolin (5μM (n=4 number of organoids analyzed), 5x10
2 -4
μM (n=11), 5x10 μM (n=10), DMSO n=9)). Scale bars (a-c) 30μm. All results are representative
for at least three independent experiments.
Figure 33. Forskolin-induced swelling of murine organoids is CFTR dependent. (a)
Normalized swelling curves of forskolin-stimulated calcein-green-labeled organoids pre-incubated
with DMSO (n=8), CFTR- 172 (n=7), GlyH-101 (n=9) or both CFTR- 172 and GlyH-101
inh inh
(n=11) (mean ± s.e.m.). (b) Representative confocal microscopy images of calcein-green labeled
wild type or CFTR-deficient organoids in response to forskolin. Scale bars 50μm. (c)
Quantification of forskolin-induced swelling in wild type (n=6) or CFTR-deficient (n=11)
organoids (mean ± s.e.m.) (d,e) Similar to b,c but for wild type (n=8) and CFTR-F508del (n=12)
organoids. Scale bars 30μm. (f) Absolute size of wild type or CFTR-deficient organoids quantified
in (c) at t=0 (mean ± s.e.m.). (g) Forskolin-stimulated swelling of calcein-green labeled CFTR-
F508del organoids cultured at 37°C with (n=20) or without (n=15) CFTR inhibition or cultured at
27°C for 24 hours with (n=31) or without (n=27) CFTR inhibition (mean ± s.e.m.). (h) Forskolin-
induced swelling of calcein-green labeled CFTR-F508del organoids pre-incubated for 24 hours
with DMSO with (n=15) or without (n=18) CFTR inhibition or pre-incubated with the CFTR
corrector compound VRT-325 with (n=14) or without (n=26) CFTR inhibition (mean ± s.e.m.). (i)
Normalized forskolin-induced swelling of CFTR-F508del organoids pre-treated for 24 hours with
DMSO (n=16), VRT-325 (n=18), Corr-4a (n=20) or both correctors (n=20) (mean ± s.e.m.). All
results are representative for at least three independent experiments.
Figure 34. Forskolin-induced swelling in human healthy control or cystic fibrosis organoids.
(a) Quantification of forskolin-induced organoid swelling pre-incubated with DMSO, CFTR -
172, GlyH-101 or both CFTR -172 and GlyH-101 (n=5, n=7, n=8, n=10) (mean ± s.e.m.). (b)
Forskolin-stimulated swelling of organoids derived from 4 individual healthy controls (1-4: n=30,
n=18, n=13, n=42) and 1 CF CFTR-F508del homozygous patient (n=30) (mean ± s.e.m.). (c)
Normalized swelling of forskolin-induced calcein-green labeled CFTR-F508del organoids cultured
for 24 at 37°C, or at 27°C with or without CFTR inhibition (n=10 for all conditions) (mean ±
s.e.m.). (d) Representative confocal microscopy images of calcein-green labeled HC-derived or CF
patient-derived organoids in response to forskolin upon pharmalogical manipulation of CFTR.
Scale bars 60μm. (e) Normalized forskolin-induced swelling of CFTR-F508del organoids pre-
treated for 24 hours with DMSO, VRT-325, Corr-4a, or both correctors (n=10 for all conditions)
(mean ± s.e.m.). (f) CF patient-derived organoid swelling in response to forskolin with or without
24 hour pre-treatment of corrector VX-809, VX-770 stimulation (simultaneous with forskolin) or
combined VX-809 and VX-770 treatment (n=10 for all conditions) (mean ± s.e.m.). (g) Forskolin-
induced swelling of organoids upon DMSO treatment (control) or combined compound treatment
from e and f, compared to HC organoids (n=10 for all conditions) (mean ± s.e.m.). Each line in (d)
represents organoid swelling averaged form at least three independent experiments per individual.
Results from all other figures are representative for at least three independent experiments.
Figure 35. Light microscopy analysis of wild type murine organoids stimulated with forskolin or
DMSO. Representative examples for the indicated timepoints after start of stimulation are shown.
The forskolin-induced swelling (FIS) of organoids was reversed upon removal of forskolin by
washing.
Figure 36. CFTR mRNA is expressed in mouse and human organoids. The bars show real-time
PCR cycle threshold (CT) values representing mRNA levels of CFTR, β2m or GAPDH isolated
from CFTR-F508del (left graph) or CFTR-/- (middle graph) organoids and their corresponding
wild types, or human organoids.
Figure 37. Gradual forskolin-induced swelling prevents organoid collapse. Normalized surface
area increase of individual forskolin-stimulated (a) wild type, (b) CFTR-F508del (temperature-
rescued) and (c) human (5% Wnt3a-conditioned medium, WCM) organoids. The averaged
forskolin-induced swelling of different organoid types was analyzed up to 10 (Fig. 1d,e + 2a,c,e),
(Fig. 2g) or 40 (Fig. 2h,i + 2a-c) minutes (dashed line).
Figure 38. Increased FIS by treatment of corrector compounds is CFTR dependent. Forskolin-
induced swelling of calcein-green labeled human CFTR-F508del organoids pre-incubated for 24
hours with DMSO, or with both VRT-325 and Corr-4a with or without CFTR inhibition (n=10 for
all conditions) (mean ± s.e.m.). Results are representative for at least three different experiments.
Figure 39. Cholera toxin-induced organoid swelling in human organoids is CFTR dependent.
Forskolin and cholera toxin induce swelling of HC-derived organoids, but not of CFTR-F508del
organoids. The cholera toxin response is delayed compared to forskolin because of its apical
extracellular function. (n=10 for all conditions) (mean ± s.e.m.). Results are representative for at
least three different experiments.
Figure 40. Human organoids in normal or reduced Wnt3a culture conditions. (a) Light microscopy
images of human organoids cultured at normal (50%, left panel) or reduced (5%, right panel)
Wnt3a conditioned medium (WCM) concentrations. Scale bars 400 μm. (b) Representative
examples of forskolin-induced swelling at normal or reduced Wnt3a conditions. Scale bars 50 μm.
The dashed line depicts the internal organoid lumen (c) Quantification of forskolin-induced
organoid swelling at normal Wnt3a levels pre-incubated with DMSO, CFTR -172, GlyH-101 or
both CFTR -172 and GlyH-101 (n=29, n=41, n=26, n=15) (mean ± s.e.m.). (d) Quantification of
forskolin-induced swelling of low-passage budding organoids cultured at 50% (n=9) or 5% (n=12)
Wnt3a conditioned medium (WCM) concentrations averaged from two independent experiments
(mean ± s.e.m.). All results are representative for at least three independent experiments.
Figure 41. H&E stains of prostate organoids
Tissue fragments of mouse prostate epithelium were embedded in matrigel. The expanding cells
were split weekly. The culture can be maintained for extended period of time without loosing
genetic stability or proliferation capacity. Figure 41 shows a comparison of the prostate epithelium
of the prostate itself and the organoid cultures after periods of three months. The mouse prostate
organoids grow in media containing ENR (EGF, Noggin and Rspondin) in the presence or absence
of testosterone. Human culture requires the addition of a TGF-beta inhibitor. The H&E of fixation
and embedding in paraffin demonstrates the different levels of stratification and folding of the
epithelium in vivo. The cultured prostate organoids show similar diversity in folding and
stratification.
Figure 42. CK8 (a differentiation marker) stains of prostate organoids
Tissue fragments of mouse prostate epithelium were embedded in matrigel. The expanding cells
expand were split weekly. The culture can be maintained for extended period of time without
loosing genetic stability or proliferation capacity. Figure 42 shows the presence of CK8 expressing
luminal cells. The cultures are grown in media containing ENR (EGF, Noggin and Rspondin) in
the presence or absence of testosterone. Human culture requires the addition of a TGF-beta
inhibitor. The addition of testosterone allows for the differentiation into CK8 positive luminal
cells while at the same time stimulating stem cell maintenance and expansion. Testosterone also
increases the stratification and folding of the epithelium.
Figure 43. Mouse prostate after 25 weeks in culture.
Prostate organoids grown in the EGF, Noggin, Rspondin, NAC, B27, Glutamin/max, pen/strep,
Ad-DMEM/F12 + testosterone. The shape of the organoid is determined by origin of tissue
(position in the prostate before isolation). The prostate consists of different lobes or regions. The
different regions display specific epithelial structures (stratification and folding), After in vitro
culturing the organoids appear to maintain the different macroscopic structure (stratified or folded)
of the part of the prostate from which it originated.
Figure 44. PCR of 3 week human prostate culture
Normal and cancerous prostatic epithelium was isolated and grown for three weeks in ENR
FGF10, ENRF+DHT, WENRF, WENRF + DHT, ENR, ENR + DHT culture conditions. All
culture conditions contained A83, P38i and Nicotinamide.
Fig 44(a): RNA was isolated and RT-PCR was performed for markers of prostatic epithelium. In
both normal and tumor tissue the luminal markers CK18, CK8 and B-MSP are expressed. All
culture conditions express the AR. In normal tissue addition of DHT increases AR expression in
all culture conditions. In tumor tissue AR expression is not influenced by DHT addition. In all
culture conditions basal epithelial markers CK14, CK5 and p63 are expressed. Putative stem cell
marker LGR5 is expressed under ENRF conditions in normal tissue. In tumor tissue LGR5
expression is induced with the addition of DHT in all culture conditions. TNFRSF19, also a
putative stem cell marker, is expressed in all conditions in normal and tumor tissue. The prostate
specific transcription factor NKX3.1 is expressed in all conditions. Addition of testosterone
increases growth/ doublings while maintaining markers for the different cell type of the prostate
(basal and luminal).
Fig 44(b): Two representative pictures of human organoids grown under ENRF+ 1nM DHT
conditions
Lane 1: Line 1 ENRF Normal tissue
Lane 2: Line 1 ENRF + 1 nM DHT Normal tissue
Lane 3: Line 2 WENRF Normal tissue
Lane 4: Line 2 WENRF + 1 nM DHT Normal tissue
Lane 5: Line 3 ENR Normal tissue
Lane 6: Line 3 ENR + 1 nM DHT Normal tissue
Lane 7: Whole prostate Normal tissue
Lane 8: H O
Lane 9: Line 1 ENRF Tumor tissue
Lane 10: Line 1 ENRF + 1 nM DHT Tumor tissue
Lane 11: Line 2 WENRF Tumor tissue
Lane 12: Line 2 WENRF + 1 nM DHT Tumor tissue
Lane 13: Line 3 ENR Tumor tissue
Lane 14: Line 3 ENR + 1 nM DHT Tumor tissue
Lane 15: Whole prostate Tumor tissue
Lane 16: H O
Figure 45. PCR of mouse prostate organoid
Three biologically independent lines were cultured under ENR or ENR + 1 nM DHT conditions.
RNA was isolated and RT-PCR was performed for markers of prostatic epithelium. In both culture
conditions luminal prostate markers Cytokeratin 18 (CK18) and Cytokeratin 8 (CK8) are broadly
expressed. Androgen Receptor (AR) is expressed in both conditions. Basal markers p63 and
Cytokeratin 5 (CK5) are expressed in both culture conditions, but upon addition of DHT basal
markers are downregulated. Putative stem cell markers Lgr5 and Tnfrsf19 are downregulated upon
addition DHT. However under these conditions stemness is maintained while differentiated cells
are also present. These conditions allow unlimited cell expansion (for now 9 months at population
doublings 2,5 a week). All cultures are positive for the prostate specific marker Nkx3.1. Addition
of testosterone increase growth/ doublings up to 3 fold while maintain markers for the different
cell type of the prostate (basal and luminal).
Lane 1: Line 1 ENR
Lane 2: Line 1 ENR + 1 nM DHT
Lane 3: Line 2 ENR
Lane 4: Line 2 ENR + 1 nM DHT
Lane 5: Line 3 ENR
Lane 6: Line 3 ENR + 1 nM DHT
Lane 7: Whole mouse prostate
Lane 8: H O
Figure 46: Stomach (gastric) organoids
Human stomach organoids. Tissue was isolated from the corpus. The cells were culture in the
stomach organoid medium (EGF, Noggin, Rspondin, Wnt, Nicotinamide, FGF10, Gastrin, TGF-
beta inhibitor (A8301). Cells are split weekly.
Fig 46(a): This picture was taken after 2 months of culturing.
Fig 46(b): H&E and different antibody staining after fixation and paraffin sectioning of a culture
after 2 weeks of culturing. It shows the presence of the following cells: PAS for Mucin producing
cells; Muc5Ac for Surface mucous pit cells; Muc6 for mucous neck cells. The H&E stain shows a
single layer of polarized epithelium.
Figure 47: Isolation of prostatic tissue
See example 8 (Photos from UCSF)
Figure 48: A) Mouse organoids were cultured in the presence of different doses recombinant
mouse RANKL for 72h. mRNA expression levels for RANK, the transciption factor SpiB and the
M cell-specific markers GP2 and AnnexinV were determined by qPCR. B) Confocal analysis of
GP2 (see arrow) and AnnexinV (see arrow) expression in mouse organoids cultured with 100ng/ml
RANKL for 72h.
Figure 49: Human organoids were cultured in the presence of different doses of recombinant
human RANKL for 7 days. mRNA expression levels for RANK, SipB and the M cell-specific
marker GP2 were determined by qPCR. EM: Expansion medium; DM: differentiation medium.
EXAMPLE 1:
To address the need for improved culture media and methods for human epithelial stem cells, the
inventors investigated signalling pathways that are known to be subverted in certain cancers e.g.
colorectal cancer. It was hypothesised that these pathways, which affect cell fate in cancer, may
also play a role in determining cell fate under in vitro cell culture conditions.
In a first screening experiment, a series of vitamins, hormones and growth factors were tested in
combination with standard stem cell culture media. Gastrin and nicotinamide were identified as
resulting in significantly improved culture conditions. Incorporating these factors into the standard
culture conditions, a second screening experiment was performed, in which certain small molecule
inhibitors related to relevant signalling pathways, such as ERK, p38, JNK, PTEN, ROCK, and
Hedgehog, were tested. In the present state of the art, there would be no reasonable way to predict
what the outcome of each of these additional compounds would be on the culture medium
properties.
Table 2: List of reagents used for optimization of human intestinal organoids culture
First screening (WENR**)
Concentratio Activity*
Description Source n
Hormones, vitamins etc
Hydrocortison Sigma 500nM 0
Gastrin*** Sigma 1uM 1+
Exendin4 GLP1 analog Sigma 100nM 0
Nicotinamide Vitamin B derivative Sigma 10mM 3+
L-Ascorbic acid Vitamin C Sigma 10uM 0
anti-oxidant mixture Sigma 1x 0
Lipid mixture Sigma 1x 0
PGE2 Sigma 10uM (Cystic)
Cholera Toxin Sigma 100nM (Cystic)
Growth factors
Peprotec
BDNF h 100ng/ml 0
Peprotec
GDNF h 100ng/ml 0
Peprotec
FGF2 h 100ng/ml 0
Peprotec
FGF10 h 100ng/ml 0
Peprotec
Follistatin h 100ng/ml 0
Peprotec
Cyr61 h 1ug/ml 0
Millipor
LIF e 1000U/ml 0
Second screening (WENR+gastrin+Nicotinamide)
Small molecule inhibitors
PD98059 ERK inhibitor Sigma 10uM 1-
SB203580 p38 inhibitor Sigma 1-10uM 2+
SB202190 p38 inhibitor Sigma 1-10uM 2+
SP600125 JNK inhibitor Sigma 10uM 0
PS48 PDK1 activator Sigma 5uM 0
Y27632 ROCK inihibitor Sigma 10uM cystic
Cyclopamine Hedgehog inhibitor Sigma 100nM 1-
Azacytidin DNA methylase inhibitor Stemolecule 1-
Dorsomorphin BMP inhibitor Stemolecule 0
A83-01 ALK4,5,7 inhibitor Tocris 50n-1uM 3+
VO-OHpic trihydrate PTEN inhibitor Sigma 500nM 3-
Pifithrin-α p53 inhibitor Sigma 0
BIX01294 G9a HMTase inhibitor Stemolecule 1-
*Activity scale (plating efficiency was compared with control after 4 days culture):
0 = no change; 1+ = <50% increase; 2+ = 50-100% increase; 3+ = >100% increase;
1- = 0-50%; 2- = 50-100% decrease; 3- = >100% decrease.
** WENR comprises EGF+Noggin+R-spondin+Wnt-3a
*** Highlighted in bold are the compounds which showed the greatest improvement to the
culture medium.
In summary, the inventors have established long-term culture conditions under which single crypts
or stem cells derived from murine small intestine (SI) expand over long periods of time. Growing
crypts undergo multiple crypt fission events, whilst simultaneously generating villus-like epithelial
domains in which all differentiated cell types are present. The inventors have now adapted the
culture conditions to grow similar epithelial organoids from mouse colon and human SI and colon.
Based on the murine small intestinal culture system, the inventors optimized the murine and
human colon culture system. They found that addition of Wnt3A to the growth factor cocktail
allowed mouse colon crypts to expand indefinitely. Further addition of nicotinamide, a small
molecule Alk inhibitor and a p38 inhibitor was preferable for long-term human SI and colon
culture. The culture system also allowed growth of murine Apc adenomas, human colorectal
cancer and human esophageal metaplastic Barrett’s epithelium. The culture technology should be
widely applicable as a research tool for infectious, inflammatory and neoplastic pathologies of the
human gastrointestinal tract. Moreover, regenerative applications may become feasible with ex
vivo expanded intestinal epithelia.Self-renewal of the small intestinal and colonic epithelium is
driven by the proliferation of stem cells and their progenitors located in crypts. Although multiple
culture systems have been described (Evans GS et al. J Cell Sci 1992;101 ( Pt 1):219-31;
Fukamachi H. J Cell Sci 1992;103 ( Pt 2):511-9; Perreault N & Jean-Francois B. Exp Cell Res
1996;224:354-64; Whitehead RH et al. Gastroenterology 1999;117:858-65), only recently have
long-term culture systems become available that maintain basic crypt physiology. Two different
protocols were published which allow long-term expansion of murine small intestinal epithelium.
Kuo and colleagues demonstrated long-term growth of small fragments containing epithelial as
well as stromal elements in a growth factor-independent fashion (Ootani A et al. Nat Med
2009;15:701-6). The inventors designed a culture system for single stem cells by combining
previously defined insights in the growth requirements of intestinal epithelium. Wnt signalling is a
pivotal requirement for crypt proliferation (Korinek V et al. Nat Genet 1998;19:379-83; Pinto D et
al. Genes Dev 2003;17:1709-13; Kuhnert F et al. Proc Natl Acad Sci U S A 2004;101:266-71) and
the Wnt agonist R-spondin1 induces dramatic crypt hyperplasia in vivo (Kim KA et al. Science
2005;309:1256-9). Second, EGF signalling is associated with intestinal proliferation (Dignass AU
& Sturm A. Eur J Gastroenterol Hepatol 2001;13:763-70). Third, transgenic expression of Noggin
induces expansion of crypt numbers (Haramis AP et al. Science 2004;303:1684-6). Fourth,
isolated intestinal cells undergo anoikis outside the normal tissue context (Hofmann C et al.
Gastroenterology 2007;132:587-600). Since laminin (α1 and α2) is enriched at the crypt base
(Sasaki T et al. Exp Cell Res 2002;275:185-), the inventors explored laminin-rich Matrigel to
support intestinal epithelial growth. Matrigel-based cultures have successfully been used for
growth of mammary epithelium (Stingl J et al. Breast Cancer Res Treat 2001;67:93-109). Under
this culture condition (R-spondin1, EGF, and Noggin in Matrigel), the inventors obtained ever-
expanding small intestinal organoids, which displayed all hallmarks of the small intestinal
epithelium in terms of architecture, cell type composition and self-renewal dynamics.
Despite extensive efforts, long-term adult human intestinal epithelial cell culture has remained
difficult. There have been some long-term culture models, but these techniques and cell lines have
not gained wide acceptance, possibly as a result of inherent technical difficulties in extracting and
maintaining viable cells (Rogler G et al. Scandinavian journal of gastroenterology 2001;36:389-98;
Buset M et al. In vitro cellular & developmental biology: journal of the Tissue Culture Association
1987;23:403-12; Whitehead RH et al. In vitro cellular & developmental biology: journal of the
Tissue Culture Association 1987;23:436-42; Deveney CW et al. The Journal of surgical research
1996;64:161-9; Pang G et al. Gastroenterology 1996;111:8-18; Latella G et al. International
journal of colorectal disease 1996;11:76-83; Panja A. Laboratory investigation; a journal of
technical methods and pathology 2000;80:1473-5; Grossmann J et al. European journal of cell
biology 2003;82:262-70). Encouraged by the establishment of murine small intestinal culture, the
inventors aimed to adapt the culture condition to mouse and human colonic epithelium. The
inventors now report the establishment of long-term culture protocols for murine and human
colonic epithelium, which can be adapted to primary colonic adenoma/adenocarcinoma and
Barrett’s esophagus.
Results
Establishment of a mouse colon culture system
In an attempt to establish a mouse colon culture system, the inventors explored our small intestinal
culture condition (here termed ENR: EGF+Noggin+R-spondin). In our experience, initial growth
of colon epithelium is often observed under the ENR culture condition, but is invariably abortive.
Organoid formation was studied using epithelium isolated from the distal part of the mouse colon.
Under ENR conditions, the plating efficiency of single distal colonic crypts was much lower than
that of small intestine (1-3% vs >90%) and these organoids could not be passaged. Recently, the
inventors have shown that Paneth cells produce several Wnt ligands (Gregorieff A et al.
Gastroenterology 2005;129:626-38), and that the production of Wnt by these Paneth cells is
essential to maintain intestinal stem cells (Sato T et al. Nature;469:415-8). To determine the Wnt
signalling status in colon organoids, the inventors cultured colon crypts from Axin2-lacZ mice, (a
faithful Wnt reporter) (Lustig B et al. Mol Cell Biol 2002;22:1184-93) or Lgr5-GFP knock-in
mice (Lgr5 being a Wnt-dependent stem cell marker)(Barker N et al. Nature 2007;449:1003-7).
Freshly isolated colon crypts readily expressed Axin2-LacZ or Lgr5-GFP at their bottoms, but they
lost expression of the Wnt reporters shortly after initiation of culture (Figure 1a,b and Fig. 6).
By contrast, small intestinal organoids constitutively expressed the Wnt reporters at their budding
structures (Sato T et al. Nature;469:415-8; Sato T et al. Nature 2009;459:262-5). These findings
suggested that colon organoids produce insufficient amounts of Wnt ligands to maintain colon
stem cells. To overcome this, the inventors added recombinant Wnt3a or Wnt3a-conditioned
medium to ENR culture medium (WENR medium). This increased plating efficiency of crypts in
the order of 10-fold. Colon crypts formed organoids structures with numerous Axin2-LacZ
(Figure 1a) or Lgr5-GFP+ (Figure 1b) buds, implying that Wnt activation was restored. Freshly
isolated colon crypts contain fully mature cells in their upper parts, and the inventors reasoned that
these mature cells may interfere with organoid growth. When the inventors mildly digested colon
crypts into small clusters of cells, thus physically separating proliferative crypt bottoms from
differentiated upper crypt regions, most of fragments derived from crypt top died, yet cell clusters
from colon crypt base efficiently formed organoids (Figure 1c).
Mouse small intestinal epithelium grown under ENR conditions generates all differentiated
epithelial cell types concomitant with stem cell self-renewal. The inventors have shown previously
that the addition of Wnt3A to these cultures interferes with intestinal differentiation and yields
organoids that largely consist of undifferentiated progenitors (Sato T et al. Nature;469:415-8). This
is not unexpected given the central role of Wnt signalling in the maintenance of the
undifferentiated crypt progenitor state (van de Wetering M et al. Cell 2002;111:241-50).
Consistent with this observation, colonic organoids in WENR condition failed to differentiate
properly. Upon withdrawal of Wnt-3A, the inventors observed differentiation along all epithelial
lineages (Figure 1d-f). Of note, single sorted Lgr5+ colonic epithelial stem cells can form
organoids when cultured in the presence of Y-27632 for the first two days.
Establishment of human colon culture system
Encouraged by the success of the improved mouse colon crypt culture, the inventors applied the
culture condition to human colon crypts. Although these crypts initially survived, most
subsequently disintegrated within 7 days. To increase the plating efficiency of human colon crypts,
the inventors screened candidate growth factors, hormones and vitamins (list in Fig 12). Among
these, the inventors found that gastrin and nicotinamide (Precursor of NAD , and found to
suppress Sirtuin activity (Denu JM. Trends Biochem Sci 2005;30:479-83)) improved culture
efficiency (Fig 12). The effect of gastrin on plating efficiency was marginal. However, the
hormone did not interfere with intestinal differentiation and we decided to include gastrin
(hereafter shortened to ‘g’) in all human intestinal culture conditions. Importantly, nicotinamide
(10 mM) was essential for prolongation of culture period beyond the initial 7 days (Figure 2 a).
Under this culture condition, human colonic organoids could be expanded for at least 1 month.
From 1 month onward, the colonic organoids changed their morphology from budding organoids
structure into cystic structures (Figure 2b left). Coinciding with the morphological conversion,
proliferation progressively decreased. Occasionally, cystic organoids regained their proliferative
potential. However, all organoids eventually arrested growth within 3 months. A two-phase growth
arrest has been observed in other primary culture systems, such as mammary epithelial cells or
keratinocytes, and has been referred to as mortality stage 1 (M1; senescence) and mortality stage 2
(M2; crisis) (Shay et al., 2006). Multi-lineage differentiation was not observed in the human
intestinal organoids cultured in this condition even after the withdrawal of Wnt (data not shown).
The inventors assumed that growth arrest occurred because of inadequate culture conditions rather
than a cell-intrinsic property of senescence/replicative aging. The inventors therefore extended our
attempts to optimized the culture condition. The inventors screened various small molecule
modulators of MAP kinases, of signaling molecules mutated in colon cancer, and of histone
modifiers (Fig 12) under the WENR+gastrin+nicotinamide culture condition. The inventors found
that two small molecule inhibitors, A83-01 (Alk4/5/7 inhibitor; nM) and SB202190 (p38
inhibitor; 10 uM) significantly improved the plating efficiency. Other TGF-beta receptor 1 (ALK
5) inhibitors that were also tested and showed the same results as A83-01 were LY364947,
SB431542, SB505124. It would be expected that other ALK inhibitors would also work in the
same way. Furthermore, the combination of the two compounds synergistically prolonged the
culture period. The inventors demonstrated that all of ten tested samples expanded for at least 6
months with weekly 1:5 split. Under this culture condition, the human colonic organoids displayed
budding organoid structures, rather than the cystic structures seen under the previous culture
condition (Figure 2b). The proliferating cells were confined to the buds (Figure 2c). Metaphase
spreads of organoids more than 3 months old consistently revealed 46 chromosomes in each cell
(20 cells each from three different donors; Figure 2d). The inventors sequenced the whole exome
(all exons) of the colon organoids after two months in culture. The number of mutations in the
organoids was extremely low. In fact in four parallel organoid cultures originating from one clone,
only one mutation was found which was present in all cultures and therefore likely originated from
the parental tissue.
These results implied that Alk receptor and p38 signalling negatively regulate long-term
maintenance of human intestinal epithelial cells. The inventors refer to the optimized culture
condition as the HISC (Human intestinal stem cell culture) condition.
Human intestinal organoids mimic in vivo differentiation
Under the HISC condition, the inventors failed to observe differentiated cells. As was seen in the
mouse colon organoids, withdrawal of Wnt was required for mature enterocyte differentiation in
human organoids (Figure 3a top panel and Fig.7). However, goblet and enteroendocrine cell
differentiation remained blocked. We found that Nicotinamide and SB202190 strongly inhibited
this differentiation, while withdrawal of the two reagents enabled the organoids to produce mature
goblet and enteroendocrine cells (Figure 3a (middle and bottom panel), 3b and Fig. 7.
The same differentiation inhibitory effects of Wnt, Nicotinamide and SB202190 were observed in
human small intestinal organoids. Lysozyme+ Paneth cells were observed in small intestinal
organoids, but not in colonic organoids (Figure 3d). It has been reported that p38 inhibitor
treatment in vivo inhibits goblet cell differentiation and increases intestinal epithelial proliferation
(Otsuka M. Gastroenterology 2010;138:1255-65, 1265 e1-9). Indeed, the inventors observed the
same phenotype in the p38 inhibitor treated intestinal organoids (Figure 3d vs. e).
The inventors further examined the response of human intestinal organoids to Notch-inhibition.
The inventors have previously shown that Notch inhibition with either γ-secretase inhibitors
(dibenzazepine; DBZ) or by conditional targeting of the Notch pathway transcription factor CSL
depleted intestinal stem cells, terminated intestinal epithelial proliferation and induced goblet cell
hyperplasia in vivo (van Es JH et al. Nature 2005;435:959-63). Indeed, upon treatment with DBZ,
the intestinal organoids ceased their proliferation and most cells converted into goblet cells within
3 days (Figure 3g vs f).
Establishment of APC-deficient adenoma and colon adenocarcinoma
Recently, the inventors reported efficient mouse intestinal adenoma formation from Lgr5 stem
flox/flox
cells in Lgr5-GFP-ires-CreERT2 x APC mice upon Tamoxifen-induced Cre activation
(Barker N et al. Genes Dev 2008;22:1856-64). The inventors isolated the intestinal adenomas 10
days after induction and optimized the culture condition. The adenomas efficiently formed cystic
organoid structure without budding. Since APC loss constitutively activates the Wnt pathway, the
inventors expected that R-spondin1 would become dispensable for adenoma organoid growth. This
was indeed observed. Furthermore, Noggin, which is essential for long-term culture of normal
small intestine, was dispensable in adenoma organoids. Interestingly, the inventors observed a loss
of Lgr5-GFP but not Axin2-LacZ in adenomatous organoids 7 days after withdrawal of Noggin
(Figure 4a,b and data not shown). Similar observations were made for normal intestinal organoids
when grown in ER-medium (Sato T et al. Nature 2009;459:262-5). This indicated that Noggin,
most likely through inhibition of BMP signals, is required to maintain Lgr5 expression, but is not
hi low
required for expansion of adenoma organoids. Freshly isolated Lgr5 (but not Lgr5 ) cells
isolated from intestinal crypts can initiate organoid growth in vitro (Sato T et al. Nature
2009;459:262-5). To determine the existence of a similar Lgr5-hierarchy within adenomas, the
hi low -ve
inventors isolated Lgr5-GFP , GFP and GFP cells from EN-cultured organoids and examined
their organoid formation ability. After a 7 day culture, Lgr5-GFP showed the highest organoid-
low –ve
forming efficiency. Yet, Lgr5-GFP or also formed organoids with considerable efficiency
-ve hi
(Figure 4c). Of note, sorted GFP adenoma cells could give rise to Lgr5-GFP organoids ((Fig.
8)).
Many colorectal cancer cell lines have been isolated over the past four decades. Typically, such
cell lines emerge as rare, clonal outgrowths after primary cultures of colon tumors enter tissue-
culture crisis. Currently, no robust culture system exists which allows the consistent culture of
primary human colon cancer samples without culture crisis and the consequent clonal outgrowth of
culture-adapted cells. As a next step, the inventors applied intestinal adenoma culture conditions to
human colorectal cancer samples. As expected, colon cancer cells required neither R-spondin nor
Noggin. EGF was dispensable in most colon cancer organoids, while some colon cancer organoids
decelerated their proliferation after withdrawal of EGF. Distinct from mouse intestinal adenoma,
colorectal cancer organoids in the culture condition grew as irregular compact structures rather
than as simple cystic structures (Figure 4d).
The inventors examined the proliferation/differentiation status of adenoma and colon cancer
organoids. As expected, most of cells were Ki67+. Consistent with the strong inhibitory effect of
Wnt on enterocyte differentiation (Figure 1f and Fig. 7), alkaline phosphatase staining was not
observed in both types of organoids (Fig. 9). In contrast, we occasionally observed PAS+ goblet
cells and chromogranin A+ endocrine cells in adenoma organoids and in some colon cancer
organoids (Fig. 9).
Culturing human metaplastic Barrett’s epithelium
Barrett’s Esophagus is marked by the presence of columnar epithelium in the lower esophagus,
replacing the normal squamous cell epithelium as a result of metaplasia (Odze RD. Nat Rev
Gastroenterol Hepatol 2009;6:478-90). The histological hallmark of Barrett’s Esophagus is the
presence of intestinal goblet cells in the esophagus. Exploiting the similarity between Barrett and
intestinal epithelium, the inventors subjected small Barrett’s epithelium (BE) biopsies to the
human colon culture condition. Under these culture conditions, normal esophageal squamous cells
transiently proliferated for 1 week, but the organoids could not be passaged. Barrett’s Esophagus
epithelium could be maintained for up to 1 month under HISC conditions (Figure 5a). The BE
organoids formed cystic organoid structures indistinguishable from that of senescent human colon
organoids, and typically underwent growth arrest 1 month after the culture. Addition of FGF10 to
the HISC condition enabled the BE organoids to form budding structures and significantly
prolonged the culture duration (> 3 months) (Figure 5 b, c). In contrast to human intestinal
organoids, BE organoids remained Ki67+ with a minimal number of PAS+ and Mucin+ cells 4
days after withdrawal of Nicotinamide and SB202190. Treatment with the γ-secretase inhibitor
DBZ (10 uM) for 4 days after the withdrawal blocked proliferation and induced goblet cell
differentiation (Fig.5 d-g). This supported our previous suggestion that local delivery of such
inhibitors may represent a useful therapeutic strategy for the removal of Barrett’s Esophagus
lesions by differentiation therapy (Menke V et al. Disease models & mechanisms 2010;3:104-10).
Of note, we occasionally observed Lysozyme+ Paneth cells (Fig. 10), which indicates that BE
organoids preserve multilineage differentiation.
Discussion
The protocols developed here allow robust and long-term culture of primary human epithelial cells
isolated from small intestine, colon, adeno(carcino)mas and Barrett’s Esophagus (table 3).
Table 3: List of components of the organoid culture systems
Reagent name Supplier Cat No. Solvent Stock solution Final conc.
Matrigel, GFR, phenol
free BD bioscience 356231
Advanced DMEM/F12 Invitrogen 12634-028
GlutaMAX-I Invitrogen 35050-079 200 mM 2 mM
HEPES 1M Invitrogen 15630-056 10 mM
10000/10000 100/100
Penicillin/Streptomycin Invitrogen 15140-122 U/ml U/ml
N2 supplement Invitrogen 17502-048 100x 1x
B27 supplement Invitrogen 17504-044 50x 1x
500 mM=81.5
N-Acetylcysteine Sigma-Aldrich A9165-5G DW mg/ml 1 mM
EDTA Sigma-Aldrich 431788-25g DW mM=14.6g/100ml 2 mM
Mouse recombinant
noggin Peprotech 250-38 100ug PBS/BSA 100 mg/ml 100ng/ml
mouse recombinant
EGF Invitrogen PMG8043 PBS/BSA 500 mg/ml 50 ng/ml
human recombinant R-
spondin Nuvelo PBS/BSA 1 mg/ml 1 mg/ml
human recombinant
FGF10 Peprotech 100-26 PBS/BSA 100 mg/ml 100 ng/ml
mouse recombinant
Wnt-3A Millipore GF-160 PBS 10 mg/ml 100 ng/ml
mM=1g/338
Y-27632 Sigma-Aldrich Y0503 PBS ml 10 mM
A01 Tocris 2939 DMSO 500 mM 500 nM
SB202190 Sigma-Aldrich S7067 DMSO 30 mM 10 mM
Nicotinamide Sigma-Aldrich DW 1M 10 mM
[Leu15]-Gastrin I Sigma-Aldrich G9145 PBS/BSA 100 mM 10 nM
DNase Sigma-Aldrich DN25-1g PBS 200000 U/ml 2000 U/ml
TrypLE express Invitrogen 12605-036
Collagenase type XI Sigma-Aldrich C9407
Dispase Invitrogen 17105-041
70um Cell strainer BD falcon 352350
All stock solutions and aliquoted Matrigel are stored in -20 C
In contrast to murine small intestine, murine colonic epithelial cells require Wnt ligand in the
culture medium. The inventors have previously reported that CD24 Paneth cells produce Wnt-
3/11, which are essential for stem cell maintenance in small intestine (Sato T, et al. Nature
2011;469:415-8). Wnt-6 and -9b mRNA are expresses at the bottom of colon crypts (Gregorieff A,
et al. Gastroenterology 2005;129:626-38.). It remains undetermined whether this local Wnt
production by colon crypt base cells is sufficient to activate canonical Wnt signal in vivo or there is
another source of Wnt ligand in colon mucosa. The difference between human and mouse
intestinal organoid culture conditions was unexpectedly large. A83-01 inhibits ALK4/5/7,
receptors that are detected in both murine and human crypts by microarray. The inventors are
currently investigating the mechanism by which ALK signal regulates human organoid growth.
The inventors have not observed cellular transformation in long-term cultures and no chromosomal
changes become obvious under the optimized culture conditions. Furthermore, the organoids can
undergo a considerably higher number of cell division than reported for other adult human
epithelial culture system (Dey D et al. PloS one 2009;4:e5329; Garraway IP et al. The Prostate
2010;70:491-501). It is generally believed that somatic cells are inherently limited in their
proliferative capacity, a phenomenon called replicative aging (Walen KH. In vitro cellular &
developmental biology. Animal 2004;40:150-8). Most normal human cells are believed to count
the number of times they have divided, eventually undergoing a growth arrest termed cellular
senescence. This process may be triggered by the shortening of telomeres, and the consequent
activation of DNA damage signals (M1), or telomere attrition (M2). In the absence of the two
small molecule kinase inhibitors, human intestinal organoids underwent growth arrest after 10-20
population doublings. By contrast, the replicative capacity in the optimized culture condition was
extended at least up to 100 population doublings upon addition of the inhibitors, which exceeded
the Hayflick limit (Hayflick L. The Journal of investigative dermatology 1979;73:8-14). This
result clearly indicates that the senescent phenotype seen in the first culture system reflects
inadequate growth conditions, rather than inherent replicative aging.
The culture techniques can be used to study basic aspects of stem cell biology and the control of
differentiation, exemplified by depletion of stem cells and goblet cell differentiation upon Notch
inhibitor treatment. Moreover, the organoid culture platform may be used for pharmacological,
toxicological or microbiological studies on pathologies of the intestinal tract, as the organoids
represent more closely the intestinal epithelium than often-used colon cancer cell lines such as
CaCo2 or DLD1. Lastly, since small biopsies taken from adult donors can be expanded without
any apparent limit or genetic harm, the technology may serve to generate transplantable epithelium
for regenerative purposes.
EXAMPLE 2 – Culturing mouse pancreatic organoids
The use of a TGF-beta inhibitor was also tested in a culture medium for mouse pancreatic
organoids. The expansion medium that was used was DMEM/F12 media (supplemented with P/S,
Glutamax, 10mM Hepes, B27, N2 and N-Acetylcysteine), EGF (50ng/ml), R-spondin (10%),
Noggin (100ng/ml), FGF10 (100ng/ml), A8301(TGF-beta inhibitor, 500nM) and Gastrin (10µM).
This differs slightly from that of the above-described HISC culture used in Example 2 in that there
is no Wnt agonist (other than Rspondin) or Nicotinamide and FGF10 is added. However, these
culture media share a number of key components (ENR + gastrin + TGF-beta inhibitor), the
addition of the TGF-beta inhibitor being advantageous in both cases. Pancreas organoids grown in
these conditions could be expanded for > 3months and passaged at least 5 times.
Microarray experiments were carried out for the pancreas organoids grown in the above-described
expansion medium and the results were compared to the adult pancreas, adult liver and newborn
liver (see figure 16A). The pancreas organoid clearly clusters with the adult pancreas, rather than
with the liver samples, demonstrating a good phenotypic similarity with the adult pancreas.
Figure 16B shows the raw signal from the microarray experiment comparing expression levels in
pancreas organoids, adult pancreas, adult liver and liver organoids for ductal markers, endocrine
markers and transcription factors necessary for Ngn3 expression (Ngn3 is a transcription factor
that is associated with the specification of endocrine lineages). The high levels of expression of
Krt19, Krt7 and other ductal markers in the pancreas organoids, show that the pancreas organoids
clearly have a ductal phenotype. These pancreatic organoids were originally grown from ductal
preparations. The essential transcription factors for Ngn3 expression (Foxa2, Hnf6, Hnf1b, Sox9)
were all also expressed in the pancreas organoids, although expression of Ngn3 itself was not
detected under expansion conditions.
The expression levels of genes important for the generation of insulin-producing cells are low.
However, it is clear that in the expansion medium, proliferation and expression patterns of the
pancreatic organoids closely resemble those seen in early progenitor endocrine cells.
The pancreas is mainly formed by three different cell types: acinar cells, ductal cells and endocrine
cells. In a total RNA sample of adult pancreas, 90% of the RNA comes from acinar cells, so the
expression levels of endocrine markers are very diluted in a total pancreas sample. Therefore,
further experiments are planned for each specific cell type. For example, the inventors plan to
carry out a microarray comparison between pancreas organoids, enriched acinar cell preparation,
enriched ductal cell preparation and enriched endocrine cell preparation, to have a better
estimation of the mRNA levels of the important genes in our pancreas organoids compared with
the levels present in insulin producing cells. For example, in an enriched endocrine cell sample,
75-85% of the cells present would be insulin-secreting cells).
EXAMPLE 3 – The effect of Noggin on the Expansion Medium
To investigate the role of the BMP inhibitor, Noggin, in the expansion medium, the inventors
compared mRNA levels of early endocrine markers and ductal markers in pancreatic organoids
that have always been cultured in EGFRA medium so have never been cultured in the presence of
Noggin with the level of expression of the same markers in organoids that have always been
cultured in EGFRAN medium (i.e. always in the presence of Noggin). The inventors also
compared mRNA levels of these markers in pancreatic organoids from which Noggin was added
or removed from the cultures respectively. Specifically, one sample of pancreatic organoids was
cultured in EGFRA medium and then Noggin was added and the organoids were cultured for a
further 2 or 4 days. Another sample of pancreatic organoids was cultured in EGFRAN medium
and then Noggin was removed and the organoids were cultured for a further 2 or 4 days. The gene
expression was compared and the results are shown in Figure 17A. It was found that Noggin
reduces the expression of keratin 7 and keratin 19 (ductal markers) showing that Noggin blocks the
differentiation towards the ductal phenotype (the keratin levels in white and dark grey samples are
lower than in the black samples). Expression levels of some transcription factors essential for the
generation of insulin producing cells (i.e. Sox9, Hnf6, Hnf1a, Pdx1, Nkx2.2, Nkx6.1 and Hnf1b)
were unaffected by Noggin. Although Noggin prevents the cultures from acquiring a full ductal
phenotype, which will likely prevent future differentiation to insulin producing cells, the inventors
include Noggin in the expansion medium because it allows the cells to expand whilst maintaining
some ductal features in combination with features of insulin-producing precursor cells.
The effect of the presence or absence of Noggin, or its addition or withdrawal to EGFRA medium
on Lgr5 gene expression was assessed using pancreatic organoids obtained from pancreatic ducts.
The results in Figure 17B show that pancreas organoids cultured with Noggin express 2 fold more
Lgr5 than pancreas organoids cultured without Noggin (compare white bar second from left with
black bar on left). Addition (dark grey) or withdrawal (light grey) of Noggin was also shown to
affect Lgr5 levels. It is unclear whether the increase in Lgr5 gene expression in the presence of
Noggin is due to an increased number of Lgr5+ cells or due to an increased level of Lgr5
expression per cell. However, the present inventors show here that BMP inhibitors, such as
Noggin, promote expression of Lgr5 and, therefore, result in more proliferative organoids. Thus,
BMP inhibitors are shown to be an advantageous component of the expansion media.
This is surprising, because in the literature it is described that BMP activity is useful for
differentiation culture of pancreatic cells. This conclusion is based on the observations that BMP
signalling is required for the differentiation into both the ductal (see keratin7 and 19 expression)
and endocrine cells. Thus, the skilled person would expect the inclusion of a BMP inhibitor, such
as Noggin, to be disadvantageous in an expansion medium. However, the inventors surprisingly
found that the use of a BMP inhibitor was advantageous because it resulted in more proliferative
organoids and higher expression of Lgr5.
EXAMPLE 4 – Transplantation of Human pancreatic organoids under the kidney capsule in
mice
Pancreatic organoids, that had been expanded using the protocol described in example 1 (see
Figure 18A), were transplanted under the renal capsule of immunodeficient mice.
Just before transplantation, organoids were treated with cell recovery solution (BD#354253, BD
Biosciences) to get rid of matrigel residues. Organoids were washed several times with PBS and
pelleted.
Transplantation of these organoids under the renal capsule of immunodeficient recipients was
carried out using an NIH recommended procedure for islet transplantation under the kidney
capsule (“Purified Human Pancreatic Islets, In Vivo Islets Function”, Document No. 3104, A04,
Effective Date 7th July 2008, DAIT, NIAID, NIH). A week before the transplantation,
hyperglycemia was chemically induced in the recipient mice (NOD/SCID/IL2RgammaKO a.k.a.
NSG) with a high dose 130mg/kg streptozotocin injection. Blood glucose levels were monitored
and mice having a blood glucose above 18mmol/l were considered hyperglycaemic.
For transplantion, the hyperglycemic recipient was anesthetized and a small incision was made in
the left flank to expose the left kidney. Approximately 2.5 – 3.0 mm of organoids were collected
in a siliconized PE50 transplantation tube and transplanted under the kidney capsule using a
Hamilton syringe. After cauterizing the damaged capsule the kidney was placed back into the
abdominal cavity. The peritoneum and the skin were then closed with 5-0 silk sutures.
One mouse was sacrificed three hours post-transplantation and the graft was analyzed for mature
beta cell and progenitor markers. In this mouse, no insulin-producing cells could be seen in the
murine peri-renal capsule (Figure 18B).
A further mouse was allowed to recover in the cage with a heat pad, under close supervision.
Bodyweights and blood glucose levels of the transplanted mouse were monitored for 1 month.
After one month the mouse was sacrificed and the graft was analyzed for mature beta cell and
progenitor markers.
1 month after transplantation, a number of insulin-producing cells could be identified. These
insulin-producing cells are all the stained cells in Figure 18C, a selection of which are circled for
enhanced clarity. In particular, insulin-positive cells appeared from the ductal lining, whereas no
insulin-positive cells were seen in initial preparations.
The finding that the insulin producing cells are present 1 month after transplantation but are not
present 3 hours after transplantation demonstrates that the insulin producing cells largely or only
arise after transplantation.
These results show that cells taken from pancreatic organoids of the present invention, cultured
with the media and methods of the present invention, can be transplanted into mice and can
promote the growth of insulin-producing cells in the pancreas. Excitingly, human pancreatic
organoids could be transplanted. This opens a number of exciting possibilities for using
transplanted organoid cells to promote insulin production e.g. for treatment of diabetes.
EXAMPLE 5 – Liver organoid culture comprising TGF-beta inhibitor
Under ER or ENRW conditions liver organoid cultures self-renew, and can be maintained and
expanded in a weekly basis, for up to 1 year (figure 20A). The karyotypic analysis after 1 year
shows no evidence of chromosomal aberrations. More than 66% of the cells analysed presented
normal chromosomal counts and 13% of them also showed polyploidy, a characteristic trait of
hepatocytes (Figure 20B).
The combination of EGF (50 ng/ml) and R-spondin 1 (1ug/ml) supplemented with FGF10
(100ng/ml), HGF (25-50ng/ml) and Nicotinamide (1-10mM), were preferable for the long term
maintenance of the cultures. Under these conditions, we obtained long-lived cell cultures that
express biliary duct and some hepatoblast or immature-hepatocyte markers (Glul, Albumine).
However, the number of cells positive for these hepatocyte markers was very low. Under these
culture conditions, no mature hepatocyte markers (e.g. p450 Cytochromes) were detected. These
results suggest that the culture conditions described here facilitate the expansion of liver
progenitors able to generate hepatocyte-like cells, albeit at lower numbers, but not fully mature
hepatocytes (Fig.21A).
To enhance the hepatocytic nature of the cultures and obtain mature hepatocytes in vitro, we first
determined whether the three supplemental factors (FGF10, HGF and Nicotinamide) added to EGF
and Rspondin1 were exerting either a positive or negative effect on the hepatocyte expression, as
well as on the self-renewal of the culture. We generated liver organoid cultures and cultured them
either with EGF or EGF and Rspondin1 plus FGF10 or HGF or Nicotinamide or the combination
of these, and we split the cultures once a week for a total period of 10 weeks. At each time-point
we also analysed the expression of several mature hepatocyte markers (FAH, CYP3A11) and
hepatoblast markers (albumin) (Figure 21B).
It was observed that Rspondin1 and Nicotinamide combined with FGF10 are essential for the
growth and self-renewal of the liver cultures (Figure 21C&D). Rspondin1 and Nicotinamide both
inhibit the expression of the mature marker CYP3A11 and yet promote the expression of the
hepatoblast marker albumin. The addition of either FGF10 or HGF to media containing only EGF
(without Rspondin1 and without nicotinamide), facilitated the expression of the mature marker
CYP3A11, albeit at very low levels (figure 21E). To identify additional compounds that might
facilitate hepatocyte differentiation, we used two different approaches, both based upon base
conditions of: EGF + HGF and/or FGF10.
The first approach involved testing a series of compounds in addition to the EGF + FGF10 or HGF
condition. A complete list of the compounds analysed is shown in table 4.
Table 4
Compounds Signal Concentratio Result
Alb CYP3
Exendin4 Glucagon like 0.1-1uM
Sigma E7144
peptide 2 analog
Retinoic Acid RAR-RXR receptor Sigma 25nM
ligand
Retinoic Acid +
Exendin 4
Sonic Hedgehog Invitrogen 500-100ng/ml
C25II
BMP4 BMP signaling Peprotech 120- 20ng/ml
DAPT Gamma-secretase Sigma D5942 10 nM
inhibitor
A8301 Alk5/4 /7 inhibitor Tocris 50 nM
Bioscience
2939
DAPT + A8301 +++ +++
FGF4 FGFR1,2 ligand Peprotech 50ng/ml
FGF1 FGFR1,2,3,4 ligand Peprotech 450- 100ng/ml
Dexamethasone Sigma D4902 10 µM-1mM
25MG
Oncostatin M R&D systems 10-1000 ng/ml
(OSM)
495-MO-025
FGF4+OSM+Dexa
IGF peprotech 100ng/ml
Valproic acid histone deacetylase Stemgent 04- 250 µM
inhibitor and 0007
regulator of ERK,
PKC wnt/β-catenin
pathways
Sodium Butyrate histone deacetylase Stemgent 04- 250 µM
inhibitor 0005
BIX01294 G9a HMTase Stemgent 04- 1 µM
inhibitor 0002
RG 108 DNA Stemgent 04- 1 µM
methyltransferase 0001
inhibitor
TSA 100 nM + -
Hydrocortisone glucocorticoid Sigma H6909 5nM
Oncostatin M 10-1000 ng/ml
R&D systems
(OSM) 495-MO-025
ARA Sigma A 0937 500 nM
Diacylglycerol Sigma D 5919 500nM-50nM + +
R 59022
kinase inhibitor
Arterenol andrenoreceptor sigma 500nM-50nM-
bitrartre: ---- agonist A 0937 5nM
LIF 10
PD 035901 MEK1 inhibitor Axon 500nM
Medchem cat
n 1386
CHIR99021 GSK3 inhibitor Axon 3uM
Medchem cat
n 1408
DMSO 1%
L-Ascobic acid Sigma 1mM
077K13021
VEGF Peprotech
Matrigel 50%
Matrigel 20%
VEGF+DEXA
The second approach took into account knowledge from published developmental studies
regarding the expression of the transcription factors essential to achieve biliary and hepatocyte
differentiation in vivo. A comparative analysis of the expression of transcription factors in the
organoids under E or ER or ENRW conditions supplemented with FGF10, HGF and Nicotinamide
is shown in figure 21. All the transcription factors required for Hepatocyte specification were
present, besides tbx3 and prox1. However, we also noticed that the expression of specific biliary
transcription factors was highly upregulated in the cultures containing Rspondin1 (R), indicating
that the culture gene expression was unbalanced towards a more biliary cell fate.
Notch and TGF-beta signaling pathways have been implicated in biliary cell fate in vivo. In fact,
deletion of Rbpj (essential to achieve active Notch signalling) results in abnormal tubulogenesis
(Zong Y. Development 2009) and the addition of TGFb to liver explants facilitates the biliary
differentiation in vitro (Clotman F. Genes and Development 2005). Since both Notch and TGFb
signalling pathways were highly upregulated in the liver cultures (Figure 22) we reasoned that
inhibition of biliary duct cell-fate might trigger the differentiation of the cells towards a more
hepatocytic phenotype. A8301 was selected as an inhibitor of TGFb receptor ALK5, 4, and 7 and
DAPT as inhibitor of the gamma-secretase, the active protease essential to activate the Notch
pathway. We first cultured the cells for 2 days in the expansion conditions (ER media) and at day 2
(figure 23A) we started the differentiation conditions by adding the combination of the different
compounds. Media was changed every other day, and the expression of differentiated markers was
analysed 8-9 days later. The ER and ENRW conditions were used as negative control.
The combination of EGF + FGF10 with DAPT and A8301 resulted in surprisingly large
enhancement of expression of the hepatocyte markers analysed (CYP3A11, TAT, Albumin)
(figure 23B). The effect was already detectable by day 5 and peaked at days 8-9 (figure 23C). The
maximal concentration efficiency was achieved at 10uM (DAPT) and 50 nM (A8301) (figure 23D)
respectively. The addition of dexamethasone (a known hepatocyte differentiation molecule) did
not result in any improvement in gene expression. The combination of EGF, FGF10, A8301 and
DAPT not only enhances the expression but also increases the number of hepatocyte-like cells, as
assessed by immunofluorescent against the hepatocyte markers albumin and 2F8, and Xgal
staining on AlbCreLacZ derived organoids (figure 23E & F). Therefore, we can conclude that the
aforementioned differentiation protocol facilitates the generation of hepatocyte-like cells in vitro
from liver stem cell cultures.
METHODS
Reagents
Reagents used in the culture experiments are shown in Table 4.
fl/fl
MiceLgr5-EGFP-ires-creERT2 mice (Barker N et al. Nature 2007;449:1003-7), APC (Sansom
OJ et al. Genes Dev 2004;18:1385-90), Axin2-lacZ mice (Lustig B et al. Mol Cell Biol
2002;22:1184-93), C57B/6 wild type mice (6-12 week old) were genotyped as previously
described and were used for experiments. Lgr5-EGFP-ires-creERT2 mice were crossed with
fl/fl
APC mice. Cre enzyme activity was induced by intraperitoneal injections of Tamoxifen (2
mg/mouse). The mice were euthanized 4 weeks after Tamoxifen induction. Murine small intestines
and colons were opened longitudinally, washed with cold PBS and further processed for crypt
isolation. Regions containing intestinal adenomas were identified using a stereomicroscope, cut
out with a scalpel and washed with cold PBS.
Human tissue materials
Surgically resected intestinal tissues were obtained from 30 patients from the Diaconessen
Hospital Utrecht or the UMCU Hospital.
Patient material was collected from 20 patients with colon cancer (9 cecum-ascending colon, 7
sigmoid colon, 4 rectum; 33-86 years old), 5 patients with screening colonoscopy (33-63 years old)
and 5 patients with Barrett’s esophagus (45-78 years old). For normal tissue a distance of more
than 3 cm to the tumors was kept. The intestinal tissues were washed and stripped of the
underlying muscle layers. The tissue was chopped into around 5 mm pieces, and further washed
with cold PBS. Endoscopic biopsies (Intestinal or esophageal) were obtained from the UMCU
hospital. For each case, at least 5 biopsy samples were collected and stored in cold PBS. This study
was approved by the ethical committee of DHU and UMCU, and all samples were obtained with
informed consent.
Crypt/adenoma isolation and cell dissociation
Intestinal fragments (murine normal colon, human normal small intestine and colon) were further
washed with cold PBS until the supernatant was clear. Next, the tissue fragments were incubated
in 2mM EDTA cold chelation buffer (distilled water with 5.6 mM Na2HPO4, 8.0 mM KH2PO4,
96.2 mM NaCL, 1.6 mM KCl, 43.4 mM Sucrose, 54.9 mM D-Sorbitiol, 0.5 mM DL-
Dithiothreitol) for 30 min on ice (Gregorieff A Gastroenterology 2005(129)626-638). After
removal of the EDTA buffer, tissue fragments were vigorously resuspended in cold chelation
buffer using a 10-ml pipette to isolate intestinal crypts. The tissue fragments were allowed to settle
down under normal gravity for 1 min and the supernatant was removed for inspection by inverted
microscopy. The resuspension/sedimentation procedure was typically 6-8 times, and the
supernatants not containing crypts were discarded. The supernatants containing crypts were
collected in 50 ml-falcon tubes coated with bovine serum albumin. Isolated crypts were pelleted,
washed with cold chelation buffer and centrifuged at 150–200 g for 3 min to separate crypts from
single cells.
Murine colonic crypts were pelleted and resuspended with TrypLE express (Invitrogen) and
incubated for 15 min at 37 C. In this dissociation condition, colonic crypts were mildly digested,
thereby physically separating colonic crypt bottoms from the top of the colon crypts.
Intestinal fragments containing adenomas from Tamoxifen-induced Lgr5-EGFPires-
creERT2/APCfl/fl mice were incubated in 2 mM EDTA chelation buffer for 60 min on
ice. Following washing with cold chelation buffer, most of the normal intestinal epithelial cells
were detached, while adenoma cells remained attached to the mesenchyme. Next, the adenoma
fragments were incubated in digestion buffer (DMEM with 2.5% fetal bovine serum,
Penicillin/Stroptomycin (Invitrogen), 75 U/ml collagenase type IX (Sigma), 125 g /ml dispase
type II (Invitrogen)) for 30 min at 37 C. The adenoma fragments were allowed to settle down
under normal gravity for 1 min and the supernatant was collected in a 50ml-falcon tube, pelleted
and washed with PBS. Isolated adenoma cells were centrifuged at 150–200 g for 3 min to separate
adenoma from single cells.
Biopsy samples from Barrett’s epithelium and human colon cancer samples, chopped into 5 mm
pieces, were washed with PBS several times. The tissue fragments were incubated in digestion
buffer for 60 min at 37 C. After the digestion, tissue fragments were manually picked under the
microscope.
For sorting experiments, isolated crypts were dissociated with TrypLE express (Invitrogen)
including 2,000 U/ml DNase (Sigma) for 60 min at 37 °C. Dissociated cells were passed through
-μm cell strainer (CellTrics) and washed with PBS. Viable epithelial single cells were gated by
forward scatter, side scatter and pulse-width, and negative staining for propidium iodide or 7-ADD
(eBioscience).
Culture of intestinal crypts, adenomas, Barrett’s epithelium and colon cancer
Isolated intestinal crypts, Barrett’s epithelium and colon cancer cells were counted using a
hemocytometer. Crypts, fragments of epithelium or single cells were embedded in matrigel on ice
(growth factor reduced, phenol red-free; BD bioscience) and seeded in 48-well plates (500
crypts/fragments or 1000 single cells per 25 μl of matrigel per well). The matrigel was
polymerized for 10 min at 37 C, and 250μl /well basal culture medium (Advanced DMEM/F12
supplemented with penicillin/streptomycin, 10mM HEPES, Glutamax, 1× N2, 1× B27 (all from
Invitrogen) and 1 mM N-acetylcysteine (Sigma)) was overlaid containing the following optimized
growth factor combinations: murine EGF for murine intestinal adenomas, ENR (murine EGF,
murine noggin, human R-spondin-1) for murine small intestinal crypts, WENR (recombinant
human Wnt-3A or Wnt-3A conditioned medium+ENR) for murine colonic crypts, HISC (human
intestinal stem cells: WENR+gastrin+nicotinamide+A83-01+SB202190) for human small
intestinal/colonic crypts, HISC+human FGF10 for Barrett’s epithelium. Colon cancer cells show a
heterogenous behaviour and require either no addition of growth factors, murine EGF and/or A83-
01 and/or SB202190. For cell sorting experiments, Y-27632 (10 μM; Sigma) was included in the
medium for the first 2 days to avoid anoikis. Reagents and concentrations of each growth factor
are indicated in Fig. 12. An overview of the optimized combinations of growth factors and small
molecule inhibitors for each organ is given in Fig. 12.
Image analysis
The images of organoids were taken by either confocal microscopy with a Leica SP5, an inverted
microscope (Nikon DM-IL) or a stereomicroscope (Leica, MZ16-FA). For immunohistochemistry,
samples were fixed with 4% paraformaldehyde (PFA) for 1h at room temperature, and paraffin
sections were processed with standard techniques. Immunohistochemistry was performed as
described previously. For whole-mount immunostaining, crypt organoids were isolated from
Matrigel using Recovery solution (BD bioscience), and fixed with 4% PFA, followed by
permeabilization with 0.1% Triton X-100. The primary antibodies were: mouse anti-Ki67 (1:250,
Monosan), rabbit anti-Muc2 (1:100, Santa Cruz), rabbit anti-lysozyme (1:1,000, Dako), rabbit anti-
synaptophysin (1:100, Dako) and anti-chromogranin A (1:100, Santa Cruz). The secondary
antibodies were peroxidase-conjugated antibodies or Alexaconjugated antibodies.EdU
staining followed the manufacturer’s protocol (Click-IT; Invitrogen). DNA was stained with DAPI
(Molecular Probes). Three-dimensional images were acquired with confocal microscopy and
reconstructed with Volocity Software (Improvision).
Microarray analysis and Real-time PCR analysis
The data was deposited in the GEO database under the accession number GSE28907.
EXAMPLE 6 – Liver organoid culture comprising Prostaglandin-2 or Arachidonic Acid
In vitro survival, growth and expansion of liver organoids was potently enhanced by addition of
prostaglandin E2 (PGE2) or Arachidonic acid (AA) to the basal medium.
Figures 25 and 26 show that addition of PGE2 at 50nM (also seen to work in the range 10-500nM)
or addition of AA at 10ug/ml (also works at 100ug/ml, though not so well), results in a greater
number of larger organoids than using the basal medium alone. Importantly, the addition of PGE2
or AA allows for a longer expansion time. This means that organoids can be expanded for more
population doublings before there growth decreases or slows down. Without PGE2 a growth
reduction is seen after 5 weeks of culturing at 5 fold expansion per week. With PGE2 there is no
growth reduction before at least 8 weeks at 5 fold expansion per week. PGE2 was seen to have a
slightly greater effect than AA. The basal medium used was: hEGF (100ng/ml, Invitrogen); human
noggin (hnoggin) (25ng/ml, peprotech); gastrin (10nM, sigma); hFGF10 (peprotech); nicotinamide
(10mM, sigma); A8301 (500nM, Tocris); hHGF (50ng/ml, peprotech); Rspo conditioned media
(10%).
PGE2 and AA are both in the same prostaglandin signalling pathway (see Figure 24), along with
phospholipids, prostaglandin G2 (PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2),
prostaglandin D2 (PGD2). It would be expected that addition of any other activating component of
this pathway would have the same beneficial effect on the culture media.
Addition of PGE2 or AA is particularly beneficial for expansion culture media. However, they
may in some circumstances also be included in differentiation media.
EXAMPLE 7 – GSK3 inhibitors are effective Wnt agonists in the culture media
CHIR99021, a GSK3 inhibitor, was shown to be an effective Wnt agonist for the culture media. In
particular, it was shown to be a suitable replacement for Wnt in the culture media for colon and
liver organoids.
Furthermore, as an extension to Example 6, Figure 25 shows that human liver cells grown in the
presence of both CHIR99021 (Wnt agonist) and PGE2 result in more and larger organoids than
cells grown with either the Wnt agonist or PGE2 alone and certainly more/larger organoids than in
only the basal medium.
Therefore, GSK3 inhibitors could be used in the culture media instead of, or in addition to, other
Wnt agonists, such as Wnt or Rspondin1-4.
It is surprising that CHIR99021 was such an effective Wnt replacement because GSK3 is involved
in a number of different pathways, not only the Wnt pathway. This finding opens up the possibility
of designing other Wnt agonists targeting GSK3, which might be useful in culture media.
EXAMPLE 8 – Prostate
Isolation of Prostatic epithelium (Murine protocol).
The numbered steps correspond to figure 47.
i) Sacrifice male mouse at minimally 8 weeks of age to isolate a mature prostate; isolate the
urogenital sinus from the mouse.
ii) Remove seminal vesicles by breaking/cutting bloodvessels and connective tissue and making a
incision at the base at the urethra
iii) Remove the bladder by breaking/cutting it near the base at the urethra
iv) Remove remaining vesicles & fat tissue by gentle tugging and cutting. What you should have
left it the prostate lobes (6 of them) and a pink structure in the middle, which is the urethra;
v) Remove urethra, easily recognized by the pink color (stained dark in the picture). Carefully pull
the prostate lobes, so they are no longer attached to the urethra; isolate each lobe individually, just
by pulling them apart, or continue with the whole prostate.
Next, mince the prostate (lobes) in small pieces; digest the prostate in 1 ml 10 mg/ml Collagenase
II (dissolved in ADMEM/F12) for 1 ½ hours at 37 C; after collagenase digestion only “fingerlike”
structures of epithelial cells should remain.
- Wash in ADMEM/F12
- Let the chunks settle down and draw off supernatant (centrifugation at low speed gets rid
of most the mesenchyme)
- Centrifuge 50xG 5 min 4’C
- Resuspend in 1 ml Trypsin (TLE) and digest for approximately 30 min 37’C.
Pipette up and down every 10 minutes to ensure digestion
- Wash in ADMEM/F12
- Either start culture in ENR or ENR+ 1nM DiHydroTestosterone (seed approximately 5000
cells per well) (0.1nM-10uM) we donot know an upper limit
- Or continue with isolation of specific celltype via FACS
Results
Prostatic epithelial cells cultured in ENR + DiHydro Testosterone, according to the methods
described described above, can be maintained for 35 weeks so far. In the presence of testosterone,
the cultures expand the same as without testosterone. However, with testosterone all cell types are
present including stem cells, transit amplifying cells and differentiated cells i.e. there is increased
differentiation whilst maintaining a stem cell population. Prostate organoids grown in the presence
of testosterone also look more like the in vivo organ (see figures 41 and 42). Furthermore, IHC and
RT-PCR shows that prostate organoids grown in the presence of testosterone contain both basal
and luminal cells.
In the description in this specification reference may be made to subject matter which may not be
within the scope of the claims of the current specification. That subject matter is readily
identifiable by a person skilled in the art and may assist in putting into practice the invention as
defined in the claims of this specification.
Claims (47)
1. A culture medium for expanding or differentiating a population of adult stem cells, wherein said culture medium comprises: 5 i. an agonist of Lgr5; and ii. one or more TGF-beta inhibitor, wherein the inhibitor is a TGF-beta inhibitor if it can inhibit TGF-beta signalling in a cellular assay in which cells are stably transfected with a reporter construct comprising the human plasminogen activator inhibitor-1 (PAI-1) promoter. 10
2. The culture medium of claim 1, wherein the one or more TGF-beta inhibitor binds to and reduces the activity of one or more serine/threonine protein kinases selected from the group consisting of ALK5, ALK4 and ALK7.
3. The culture medium of claim 1 or claim 2, wherein the one or more TGF-beta inhibitor is selected from the group consisting of 15 A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-2511.
4. The culture medium of any one of the preceding claims, wherein the TGF-beta inhibitor is added at a concentration of between 1 nM and 100 μM, between 10 nM and 100 μM, between 100 nM and 10 μM, or approximately 1 μM, for example, wherein the total concentration of the one or more inhibitor is between 10 nM and 100 μM, between 100 nM and 10 μM, or 20 approximately 1 μM.
5. The culture medium of any one of the preceding claims, wherein the agonist of Lgr5 is any one of Rspondin 1-4.
6. The culture medium of any one of the preceding claims, wherein the culture medium comprises one or more additional components selected from: a BMP inhibitor, a Wnt agonist, 25 a receptor tyrosine kinase ligand, nicotinamide, a p38 inhibitor, a Rock inhibitor, gastrin, an activator of the prostaglandin signalling pathway and testosterone.
7. The culture medium of claim 6, wherein the culture medium is for culturing human intestinal stem cells, human small intestinal crypts or human colonic crypts and wherein the culture medium comprises a basal medium and additionally comprises a BMP inhibitor, Rspondin, a 30 TGF-beta inhibitor, a Wnt agonist, EGF, a p38 inhibitor, gastrin and nicotinamide.
8. The culture medium of claim 6, wherein the culture medium is for culturing pancreatic stem cells, and wherein the culture medium comprises a basal medium, any one of Rspondin 1-4, Noggin, a TGF-beta inhibitor, EGF, FGF10, gastrin.
9. The culture medium of claim 8, wherein the culture medium for culturing pancreatic stem cells 35 further comprises exendin 4 and Wnt-3a.
10. The culture medium of claim 6, wherein the culture medium is for culturing prostate cells, and wherein the culture medium comprises a basal medium, any one of Rspondin 1-4, Noggin, a TGF-beta inhibitor, nicotinamide, EGF, testosterone.
11. The culture medium of claim 10, wherein the culture medium for culturing prostate cells 5 further comprises Wnt-3a and FGF10.
12. The culture medium of claim 6, wherein the culture medium is for culturing gastric cells, and wherein the culture medium comprises a basal medium, Noggin, any one of Rspondin 1-4, a TGF-beta inhibitor, Wnt-3a, EGF, gastrin, nicotinamide, FGF-10.
13. The culture medium of claim 12, wherein the culture medium for culturing gastric cells further 10 comprises a p38 inhibitor.
14. The culture medium of any one of claims 7 to 13, wherein the culture medium is for culturing cancer cells, and wherein one or more of the following are excluded from the medium: Wnt- 3a, EGF, Noggin, Rspondin, TGF-beta inhibitor, p38 inhibitor, nicotinamide, gastrin, FGF10 and HGF.
15 15. A composition comprising a culture medium according to any one of claims 1 to 14 and an extracellular matrix or a 3D matrix that mimics the extracellular matrix by its interaction with the cellular membrane proteins.
16. The composition of claim 15, wherein the extracellular matrix is a laminin-containing extracellular matrix. 20
17. A hermetically-sealed vessel containing a culture medium or composition according to any one of the preceding claims.
18. Use of a culture medium according to any one of claims 1 to 14 for expanding or differentiating a stem cell, population of stem cells, tissue fragment or organoid.
19. A method for expanding a single adult stem cell, a population of adult stem cells or a tissue 25 fragment, wherein the method comprises culturing the single stem cell or population of stem cells in a culture medium according to any one of claims 1 to 14.
20. A method according to claim 19, wherein the method comprises: providing a culture medium according to any one of claims 1 to 14; contacting an adult stem cell, a population of adult stem cells or an isolated tissue 30 fragment with the culture medium; and culturing the cells under appropriate conditions.
21. A method according to claim 20, wherein the method comprises bringing the stem cell, the population of stem cells or the isolated tissue fragment and the culture medium into contact with an extracellular matrix or a 3D matrix that mimics the extracellular matrix by its 35 interaction with the cellular membrane proteins.
22. The method according to claim 21, wherein the extracellular matrix is a laminin-containing extracellular matrix.
23. The method according to any one of claims 19 to 22, whereby an organoid is obtained.
24. A method according to any one of claims 19 to 23, wherein the method comprises: 5 culturing the adult stem cell, population of adult stem cells or tissue fragments in a first expansion medium; continuing to culture the adult stem cell, population of adult stem cells or tissue fragments and replenishing the medium with a differentiation medium, wherein the differentiation medium does not comprise one or more of, preferably all of the factors 10 selected from: a TGF-beta inhibitor, a p38 inhibitor, nicotinamide and Wnt.
25. The method according to any one of claims 19 to 24, wherein a Rock inhibitor is added to the culture medium for the first 1, 2, 3, 4, 5, 6 or 7 days, optionally every second day.
26. An organoid or population of cells obtained by the method of any one of claims 19 to 25.
27. An organoid or population of cells according to claim 26, wherein the organoid or population 15 of cells has been cultured for at least 3 months, for example at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 9 months, or at least 12 months or more.
28. An organoid or population of cells according to any one of claims 26 or 27, wherein the organoid or population of cells expands at a rate of at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold or at least 10 fold per week. 20
29. An organoid or population of cells according to any one of claims 26 to 28 which is frozen and o o o o o o stored at below -5 C, below -10 C, below -20 C, below -40 C, below -60 C, below -80 C, o o o below -100 C, or below -150 C, for example at approximately -180 C.
30. A composition comprising: iii) one or more organoids or population of cells according to any one of 25 claims 26 to 29; and iv) a culture medium according to any one of claims 1 to 14 and/or an extracellular matrix.
31. An organoid according to any one of claims 26 to 29 or a population of cells according to any one of claims 26 to 29 or a composition according to claim 15, claim 16 or claim 30 for use in 30 drug screening, target validation, target discovery, toxicology, toxicology screens, personalized medicine, regenerative medicine or ex vivo cell/organ models.
32. The organoid, population of cells or composition for use according to claim 31, wherein the regenerative medicine or personalized medicine comprises transplantation of said organoid, population of cells or composition into a mammal.
33. The organoid, population of cells or composition for use according to claim 32, wherein the mammal is a human.
34. Use of an organoid according to any one of claims 26 to 29 or a population of cells according to any one of claims 26 to 29 as a disease model. 5
35. A method for screening for a therapeutic or prophylactic drug or cosmetic, wherein the method comprises: culturing an organoid or population of cells according to any one of claims 26 to 29; exposing said organoid or population of cells to one or a library of candidate molecules; evaluating said organoid or population of cells for any effects; and identifying the candidate molecule that causes said effects as a potential drug or cosmetic. 10
36. The method of claim 35, wherein the organoid or population of cells according to any one of claims 26 to 29 is cultured in a culture medium according to claims 1 to 14.
37. The method of claim 35 or 36, wherein evaluating said organoid or population of cells for any effects comprises evaluating any change in a cell of the organoid or population.
38. The method of claim 37 wherein evaluating any change in a cell of the organoid or population 15 comprises evaluating a reduction in or loss of proliferation, a morphological change and/or cell death.
39. The composition of claim 15 wherein the cellular membrane proteins are integrins.
40. The method of claim 21 wherein the cellular membrane proteins are integrins.
41. A culture medium as defined in claim 1 substantially as herein described with reference to any 20 example thereof.
42. A composition as claimed in claim 15, claim 16 or claim 30 substantially as herein described with reference to any example thereof.
43. A hermetically-sealed vessel as claimed in claim 17 substantially as herein described with reference to any example thereof. 25
44. A use as claimed in claim 18 substantially as herein described with reference to any example thereof.
45. A method as defined in claim 19 for expanding a single stem cell, a population of stem cells or a tissue fragment substantially as herein described with reference to any example thereof.
46. An organoid or population of cells as defined in claim 26 substantially as herein described 30 with reference to any example thereof.
47. A method as claimed in claim 35 for screening for a therapeutic or prophylactic drug or cosmetic substantially as herein described with reference to any example thereof.
Applications Claiming Priority (13)
Application Number | Priority Date | Filing Date | Title |
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US201161520569P | 2011-06-10 | 2011-06-10 | |
US61/520,569 | 2011-06-10 | ||
US201161571663P | 2011-06-30 | 2011-06-30 | |
GBGB1111244.8A GB201111244D0 (en) | 2011-06-30 | 2011-06-30 | Culture media for stem cells |
US61/571,663 | 2011-06-30 | ||
GB1111244.8 | 2011-06-30 | ||
US201161513461P | 2011-07-29 | 2011-07-29 | |
US13/194,866 US9752124B2 (en) | 2009-02-03 | 2011-07-29 | Culture medium for epithelial stem cells and organoids comprising the stem cells |
US13/194,866 | 2011-07-29 | ||
US61/513,461 | 2011-07-29 | ||
US201261594295P | 2012-02-02 | 2012-02-02 | |
US61/594,295 | 2012-02-02 | ||
PCT/IB2012/052950 WO2012168930A2 (en) | 2011-06-10 | 2012-06-11 | Culture media for stem cells |
Publications (2)
Publication Number | Publication Date |
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NZ619314A NZ619314A (en) | 2016-01-29 |
NZ619314B2 true NZ619314B2 (en) | 2016-05-03 |
Family
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