WO2023118101A1

WO2023118101A1 - Stacked bmp inhibition for neural induction of pluripotent stem cells

Info

Publication number: WO2023118101A1
Application number: PCT/EP2022/086932
Authority: WO
Inventors: Jonathan NICLIS; Josefine Rågård CHRISTIANSEN
Original assignee: Novo Nordisk A/S
Priority date: 2021-12-21
Filing date: 2022-12-20
Publication date: 2023-06-29

Abstract

An in vitro method for differentiating a population of human pluripotent stem cells (hPSCs) into a population of neural cells, wherein neuronal differentiation in said hPSCs is induced by the inhibition of the Small Mothers Against Decapentaplegic (SMAD) signaling pathway by culturing the population of hPSCs in a culture medium comprising two or more inducing factors consisting of two or more inhibitors of the SMAD signaling pathway, wherein a. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP ligand and b. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP receptor, and c. none of the inhibitors of the SMAD signaling pathway is a direct inhibitor of the TGFβ pathway.

Description

STACKED BMP INHIBITION FOR NEURAL INDUCTION OF PLURIPOTENT STEM CELLS

TECHNICAL FIELD

The present invention relates generally to the field of stem cells, such as human pluripotent stem cells. Specifically, methods are provided for obtaining a population of stem cell-derived neural cells.

BACKGROUND

Human pluripotent stem cells have the potential to revolutionize the treatment of various intractable diseases and disorders of the human body. Treatments include cellreplacement therapy of neural conditions such as stroke, spinal cord injury and Parkinson’s disease. For such treatments to become viable, however, it requires the development of in vitro methods of directed differentiation to artificially produce stem cell- derived neuroectodermal cells suitable for delivery to the central nervous system (CNS).

In vitro differentiating human pluripotent stem cells (hPSCs) into the neuroectodermal lineage choice has become well-established and several methods are described. The present standard methodologies for robustly and at high efficiency differentiating hPSCs into the neuroectodermal germ layer typically employ the combined artificial suppression of the transforming growth factor beta (TGFB) pathway and suppression of the bone morphogenetic protein (BMP) pathway, of which only the latter is normally suppressed in human development. Generally, it has been believed that simplifying the approach by the suppression of the BMP pathway only, thereby mimicking natural development, is inefficient. In analogy, suppressing only the TGFB pathway has also been believed to be inefficient for neural induction of hPSCs. In addition, the artificial suppression of the TGFB pathway alone is postulated to have off-target effects that must be accepted or circumvented when this method is used.

Accordingly, the prevailing approach for in vitro neural induction is by the combined inhibition of the TGFB and BMP pathways leading to the inhibition of the two downstream Small Mothers Against Decapentaplegic (SMAD) pathways. However, the efficiency and reproducibly, while high, is not as efficient as possible. Higher efficiencies are valuable for numerous purposes and particularly for stem cell manufacturing for cell replacement therapies, where the quality of the product is of utmost importance for safety, ethical and regulatory issues. It is therefore an object of the present invention to provide an efficient and robust method for differentiating hPSCs into the neuroectodermal lineage that mimics the natural development as stringently as possible, thus avoiding unwanted off-target effects, such as unvoluntary introducing developmental blockages. Thus, producing cells that can after transplantation give rise to derivatives of different tissues of the nervous system.

SUMMARY

In its broadest aspect, the present invention relates to an in vitro method for differentiating a population of human pluripotent stem cells (hPSCs) into a population of neural cells, wherein neuronal differentiation in said hPSCs is induced by the inhibition of the Small Mothers Against Decapentaplegic (SMAD) signaling pathway by culturing the population of hPSCs in a culture medium comprising two or more inducing factors consisting of two or more inhibitors of the SMAD signaling pathway, wherein a. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP ligand and b. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP receptor, and c. none of the inhibitors of the SMAD signaling pathway is a direct inhibitor of the TGFB pathway.

As described herein, the present inventors have found an efficient method of differentiating hPSCs into neural cells, the method only involving suppression of the bone morphogenetic protein (BMP) pathway. The suppression is obtained by the use of two or more inhibitors of the BMP pathway, wherein at least one of the inhibitors is an inhibitor of a BMP ligand and at least one of the inhibitors is an inhibitor of a BMP receptor and none of the inhibitors is a direct inhibitor of the TGFB pathway.

Alternatively worded, the invention relates to an in vitro method for differentiating a population of human pluripotent stem cells (hPSCs) into a population of neural cells, wherein neuronal differentiation in said hPSCs is induced by the inhibition of the BMP associated Small Mothers Against Decapentaplegic (SMAD) signaling pathway by culturing the population of hPSCs in the presence of two or more inhibitors of the BMP associated SMAD protein signaling pathway, wherein d. at least one inhibitor of the SMAD protein signaling pathway is an inhibitor of a BMP ligand and e. at least one inhibitor of the SMAD protein signaling pathway is an inhibitor of a BMP receptor.

The method of the invention is thus defined as a stacked single SMAD inhibition procedure. As mentioned above, in a method of the present invention, the TGFB pathway is not suppressed by use of a direct inhibitor of the TGFB pathway.

The one or more inhibitor of a BMP ligand is typically selected from crossveinless, USAG- 1 , noggin, follistatin including three isoforms: FST288, FST303, FST315, chordin and greml. Such an inhibitor typically interferes with a protein selected from the group consisting of ALK1, ALK2, ALK3 and ALK6.

The one or more inhibitor of a BMP receptor is selected from LDN-193189, dorsomorphin, LDN-212854, LDN-214117, and DMH1.

In some embodiments, the inhibitor of a BMP ligand is noggin.

In some embodiments, the inhibitor of a BMP receptor is LDN-193189.

In some embodiments, the inhibitor of a BMP ligand is noggin and the inhibitor of a BMP receptor is LDN-193189.

The differentiation of hPSCs takes place in a suitable culture medium. In some embodiments, the culture medium does not comprise any other added inducing factors; thus, the only inducing factors are the at least one inhibitor of a BMP ligand and the at least one inhibitor of a BMP receptor.

Typically, the culture medium does not comprise an inhibitor of nitric oxide synthase (NOS).

In presently preferred embodiments of the invention, the hPSCs are contacted simultaneously with the two or more inhibitors of the SMAD signaling pathway. In general, the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway for 5 to 11 days, such as 6, 8, 9 or 10 days. Contacting the hPSCs with the two or more inhibitors of the SMAD pathway normally starts at day 0, i.e., at the day and/or the moment when the culturing starts.

As seen from the examples herein, the cultured hPSCs may be differentiated towards various subtypes of neural cells, such as, but not limited to, ventral midbrain cells or dorsal forebrain cells. Typically, the culturing is performed for 9 days, when the neural cells obtained are ventral midbrain cells, and the culturing is performed for 11 days, when the neural cells obtained are dorsal forebrain cells.

In the method of the invention, the two or more inhibitors of the SMAD signaling pathway are added at least every 48 hours, preferably at least every 24 hours or less.

In the method of the invention, the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway in an effective amount. The concentration of the inhibitor of a BMP ligand such as Noggin in the culture medium is from 50 to 1000 ng/ml, or from 75 to 500 ng/ml such as about 100 ng/ml.

The concentration of the inhibitor of a BMP receptor such as LDN-193189 is from 10 to 1000 nM, from 50 to 500 nM, or from 75 to 200 nM such as about 100 nM.

The two or more inhibitors of the SMAD signaling pathway are added at least every 48 hours, preferably at least every 24 hours or less.

The method of the invention provides a population of neural cells, wherein i) at least 90% are positive for SOX2, and/or ii) at least 95% of the population of neural cells are positive for one or more of SOX2, NES, PAX6, FOXA2, OTX2, and SOX1, and/or iii) less than 5% of the population of neural cells are positive for POLI5F1 , NANOG and CD9, and/or iv) less than 1% of the population of neural cells are positive for POLI5F1 , NANOG and CD9.

The neural cells obtained may be non-native, such as artificial cells.

The method of the invention may comprise further steps for differentiating the resulting population of neural cells into neurons, or another cell or tissue type of the nervous system.

In embodiments, the method results in a population of neural cells, wherein the neuronal cells are suitable for further differentiation into neurons, or another cell or tissue type of the nervous system.

One aspect of the invention further relates to a composition comprising a population of hPSCs and a culture medium comprising two or more inducing factors consisting of two or more inhibitors of the SMAD signaling pathway, wherein i. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP ligand and ii. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP receptor, and iii. none of the inhibitors of the SMAD signaling pathway is a direct inhibitor of the TGFB pathway.

The details described for the method aspect are also applicable for the composition aspect. The two or more inhibitors of SMAD signaling pathway in the culture medium in the composition are typically noggin and LDN-193189.

The composition of the inventions does not comprise an inhibitor of nitric oxide synthase (NOS).

In a further aspect a cell population or composition is disclosed according to the present invention for use as a medicament, such as in the treatment of a neurological condition. It is believed that transplantation of homogeneous populations of cells increases the safety and efficacy profile of cell therapy and decreases the risks of undesirable side-effects from undesirable cell types. A treatment with a cell population or composition according to the present invention provides neurons and/or precursors thereof, thereby making it suitable for prevention or treatment of a condition requiring the administration of such cells.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 gives an overview of ventral midbrain NSC (neural stem cell) differentiation procedure with standard dual SMAD inhibition method and the stacked single SMAD inhibition procedure according to the invention. In the dual SMAD inhibition method two different inhibitors are used; one of the inhibitors is an inhibitor of the TGFB pathway, whereas the other is an inhibitor of the BMP SMAD pathway. At day 0, hPSC are subjected to differentiation. Figure A describes the dual SMAD inhibition, where the inhibitor of the TGFB pathway is SB431542 and the inhibitor of the BMP SMAD pathway is Noggin. Figure B describes the stacked single SMAD inhibition, where the inhibitor of a BMP ligand is Noggin, and the inhibitor of a BMP receptor is LDN193189. At day 9 FGF8b is added followed at day 11 of ascorbic acid and BDNF. Figure C shows schematically the developing of neural tube.

Figure 2 shows the differentiation to ventral midbrain NSC. It is seen that the stacked single SMAD inhibition procedure is just as efficient as the dual SMAD inhibition method. All hPSC markers were downregulated to <1% while expression of the pan-NSC marker SOX2 was maintained at >90%. The data is obtained by flow cytometry from n=3 at day 9.

Figure 3 shows histogram of ventral midbrain NSC analyzed at day 9 by flow cytometry. The numbers in figure A show the percentage of SOX2 positive and negative events. From the figure B it is seen that there is no statistically significant difference between the results obtained using the dual SMAD or the stacked single SMAD inhibition procedure.

Figure 4 shows representative dot plots of ventral midbrain NSC analyzed by flow cytometry on day 9 using antibodies against the pluripotency markers OCT3/4 and Nanog. Figure A relates to the dual SMAD inhibition procedure whereas figure B relates to the stacked single SMAD inhibition procedure. It is seen that the two inhibition procedures give equivalent results.

Figure 5 shows that stacked single SMAD inhibition upregulates ventral midbrain NSC, and it is equivalent to the dual SMAD inhibition procedure as assessed with antibodies against the ventral midbrain NSC markers, OTX2 and FOXA2.

Figure 6 shows representative dot plots of ventral midbrain NSC analyzed by flow cytometry on day 9 using antibodies against the ventral midbrain NSC markers, OTX2 and FOXA2. The results show that the dual SMAD inhibition procedure is equivalent with the stacked single SMAD inhibition procedure.

Figure 7 gives an overview of dorsal forebrain NSC differentiation procedure with standard dual SMAD inhibition method and the stacked single SMAD inhibition procedure according to the invention. In the dual SMAD inhibition method two different inhibitors are used; one of the inhibitors is an inhibitor of the TGFB pathway, whereas the other is an inhibitor of the BMP SMAD pathway. At day 0, hPSCs are subjected to differentiation. Figure A describes the dual SMAD inhibition, where the inhibitor of the TGFB pathway is SB431542 and the inhibitor of the BMP SMAD pathway is Noggin. Figure B describes the stacked single SMAD inhibition, where the inhibitor of a BMP ligand is Noggin, and the inhibitor of a BMP receptor is LDN193189. Figure C shows schematically the developing of neural tube.

Figure 8 shows the differentiation to dorsal forebrain NSC (neural stem cells). It is seen that the stacked single SMAD inhibition procedure is just as efficient as the dual SMAD inhibition method. All hPSC markers were downregulated to <1% while expression of the pan-NSC marker SOX2 was maintained at >90%. The data is obtained by flow cytometry from n=3 at day 11.

Figure 9 shows representative dot plots of dorsal forebrain NSC analyzed by flow cytometry on day 11 using antibodies against the pluripotency markers OCT3/4 and Nanog. Figure A relates to the dual SMAD inhibition procedure whereas Figure B relates to the stacked single SMAD inhibition procedure. It is seen that the two inhibition procedures give equivalent results.

Figure 10 shows that stacked single SMAD inhibition upregulates dorsal forebrain NSC, and it is equivalent to the dual SMAD inhibition procedure as assessed with antibodies against the dorsal forebrain NSC markers, OTX2 and PAX6. Figure 11 shows representative dot plots of dorsal forebrain NSC analyzed by flow cytometry on day 11 using antibodies against the dorsal forebrain NSC markers, OTX2 and PAX6. The results show that the dual SMAD inhibition procedure is equivalent with the stacked single SMAD inhibition procedure.

Figure 12 gives an overview of ventral spinal cord NSC (neural stem cell) differentiation procedure with standard dual SMAD inhibition method and the stacked single SMAD inhibition procedure according to the invention. In the dual SMAD inhibition method two different inhibitors are used; one of the inhibitors is an inhibitor of the TGFB pathway, whereas the other is an inhibitor of the BMP SMAD pathway. At day 0, hPSC are subjected to differentiation. (A) describes the dual SMAD inhibition, where the inhibitor of the TGFB pathway is SB431542 and the inhibitor of the BMP SMAD pathway is Noggin. (B) describes the stacked single SMAD inhibition, where the inhibitor of a BMP ligand is Noggin and the inhibitor of a BMP receptor is LDN193189. At day 9 FGF8b is added followed at day 11 of ascorbic acid and BDNF. (C) shows schematically the developing of neural tube.

Figure 13 shows ventral spinal cord differentiation equivalently reduces pluripotency markers using a dual or stacked single SMAD inhibition method. Control human embryonic stem cells express more than 98% of the pluripotency marker CD9 (A), dual SMAD inhibition spinal cord differentiated samples express <1 % CD9 (B), and stacked single SMAD inhibition spinal cord differentiated samples express <1% CD9 (C). Data determined using Flow Cytometry after 12 days of in vitro differentiation.

Figure 14 Both standard dual SMAD inhibition (A-B) and stacked single SMAD inhibition (C-D) downregulates a forebrain/midbrain NSC marker (OTX2; A,C) and upregulates a ventral spinal cord NSC marker (OLIG2; B,D) in a ventral spinal cord differentiation procedure. Representative flow cytometry histogram plots at day in vitro 12.

DETAILED DESCRIPTION

Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, biophysics, molecular biology, cell biology, genetics, immunology, and pharmacology, known to those skilled in the art.

It is noted that all headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

General definitions

Throughout this application the terms “method” and “protocol” when referring to processes for differentiating cells may be used interchangeably. As used herein, “a” or “an” or “the” can mean one or more than one. Unless otherwise indicated in the specification, terms presented in singular form also include the plural situation. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

As used herein, the term “day” and similarly day in vitro (DIV) in reference to the protocols refers to a specific time for carrying out certain steps during the differentiation procedure.

In general, and unless otherwise stated “day 0” refers to the initiation of the protocol, this be by for example but not limited to plating the stem cells or transferring the stem cells to an incubator or contacting the stem cells in their current cell culture medium with a compound prior to transfer of the stem cells. Typically, the initiation of the protocol will be by transferring undifferentiated stem cells to a different cell culture medium and/or container such as, but not limited to, by plating or incubating, and/or with the first contacting of the undifferentiated stem cells with a compound or compounds that affects the undifferentiated stem cells in such a way that a differentiation process is initiated.

When referring to “day X”, such as day 1 , day 2 etc., it is relative to the initiation of the protocol at day 0. One of ordinary skill in the art will recognize that unless otherwise specified the exact time of the day for carrying out the step may vary. Accordingly, “day X” is meant to encompass a time span such as of +/-10 hours, +/-8 hours, +/-6 hours, +/-4 hours, +1-2 hours, or +/-1 hours.

As used herein, the phrase “from at about day X to at about day Y” refers to a day at which an event starts from. The phrase provides an interval of days on which the event may start from. For example, if “cells are contacted with a differentiating factor from at about day 3 to at about day 5” then this is to be construed as encompassing all the options: “the cells are contacted with a differentiating factor from about day 3”, “the cells are contacted with a differentiating factor from about day 4”, and “the cells are contacted with a differentiating factor from about day 5”. Accordingly, this phrase should not be construed as the event only occurring in the interval from day 3 to day 5. This applies mutatis mutandis to the phrase “to at about day X to at about day Y”.

Hereinafter, the methods according to the present invention are described in more detail by non-limiting embodiments and examples. A method is provided for obtaining neuroectodermal lineage cells from pluripotent stem cells.

By “stem cell” is to be understood an undifferentiated cell having differentiation potency and proliferative capacity (particularly self-renewal competence) but maintaining differentiation potency. The term “stem cell” includes categories such as pluripotent stem cell, multipotent stem cell, unipotent stem cell and the like according to their differentiation potentiality. As used herein, the term “pluripotent stem cell” (PSC) refers to a stem cell capable of being cultured in vitro and having a potency to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm) and/or extraembryonic tissue (pluripotency). As used herein, the term “multipotent stem cell” means a stem cell having a potency to differentiate into plural types of tissues or cells, though not all kinds and is typically restricted to one germ layer. As used herein, the term “unipotent stem cell” means a stem cell having a potency to differentiate into only one particular tissue or cell. A pluripotent stem cell can be induced from fertilized egg, clone embryo, germ stem cell, stem cell in a tissue, somatic cell and the like. Examples of the pluripotent stem cell (PSC) include embryonic stem cell (ESC), EG cell (embryonic germ cell), induced pluripotent stem cell (iPSC) and the like. Muse cell (Multi-lineage differentiating stress enduring cell) obtained from mesenchymal stem cell (MSC), and GS cell produced from reproductive cell (e.g., testis) are also encompassed in the pluripotent stem cell. As used herein, the term “induced pluripotent stem cell” (also known as iPS cells or iPSCs) means a type of pluripotent stem cell that can be generated directly from adult cells. By the introduction of products of specific sets of pluripotency-associated genes non-pluripotent cells can be converted into pluripotent stem cells. Pluripotent embryonic stem cells may also be derived from parthenotes as described in e.g., WO 2003/046141. Additionally, embryonic stem cells can be produced from a single blastomere or by culturing an inner cell mass obtained without the destruction of the embryo. Embryonic stem cells are available from given organizations and are also commercially available. Preferably, the methods and products of the present invention are based on hPSCs, i.e. , stem cells derived from either induced pluripotent stem cells or embryonic stem cells, including parthenotes.

In a preferred embodiment, the method is carried out in vitro. By the term “in vitro" is meant that the cells are provided and maintained outside of the human or animal body. In an embodiment, the cells are non-native. By the term “non-native” is meant that the cells although derived from pluripotent stem cells, which may have human origin, is an artificial construct, that does not exist in nature. In general, it is an object within the field of stem cell therapy to provide cells, which resemble the cells of the human body as much as possible. However, it may never become possible to mimic the development which the pluripotent stem cells undergo during the embryonic and fetal stage to such an extent that the mature cells are indistinguishable from native cells of the human body. Inherently, in an embodiment of the present invention, the cells are artificial. As used herein, the term “artificial” may comprise material naturally occurring in nature but modified to a construct not naturally occurring. This includes human stem cells, which are differentiated into non- naturally occurring cells mimicking the cells of the human body. Preferably, the neural cells are stem cell-derived. More preferably, the neural cells are stem cell-derived from pluripotent stem cells. In a further embodiment, the neural cells are stem cell-derived from human embryonic stem cells (hESCs) and/or human induced pluripotent stem cells (hiPSCs).

As used herein, the term “neural” refers to the nervous system. As used herein, the term “neural cell” refers to cells mimicking a cell type, which are naturally part of the ectoderm germ layer, more specifically the neuroectoderm and is meant to encompass cells at any stage of development within this germ layer, such as neural stem cells all the way through to neurons, i.e., cell stages such as neural stem progenitor cell stage and neural blast/intermediate progenitor cell stage. Accordingly, specific embodiments referring to neural cells may apply equally to embodiments comprising e.g., only neural stem progenitor cells or only neural blast cells or a mixture thereof. Neural cells may be derived from embryonic stem cells and induced pluripotent stem cells, and other pluripotent cells (i.e., parthenogenic stem cells) and via direct conversion (also referred to transdifferentiation) methodologies.

As used herein, the term “neural stem progenitor cell” (NSPC) refers to cells having the capacity to self-renew, proliferate and/or differentiate into one or more than one cell type. Neural progenitor cells can therefore be unipotent, bipotent or multipotent. Neural stem progenitor cells (NSPCs) are therefore mitotic and typically differentiate terminally into neurons and glial cells as well as other cell types that reside in the CNS including meningeal cells.

As used herein, the term “neural blast cell” (NBC) refers to neural cells that are further differentiated than neural stem progenitor cells (NSCPs), typically have the capacity to further differentiate (i.e., to neurons) and do not self-renew and are postmitotic. The term “neural stem precursor blast cell” (NSPBC) is here within used to collectively describe a pure population of either NSCs, neural precursor cells or neural progenitor cells (collectively referred to as NPCs) with NSC and NPCs typically expressing transcription factors such as SOX2, NES, PAX6, SOX1 , OTX2, OTX1, NKX6.1 , FOXA2 or LMX1A, or neural blast cells (NBCs) that typically express transcription factors such as TBR2 or SOX4 or ASCL1 , or a mixed population of any of these cell types.

As used herein, the terms “neuron” and “nerve cell” may be used interchangeably referring to neural cells which are post-mitotic have fully developed/terminally differentiated, typically from a pluripotent stem cell into a specialized cell which can transmit nerve impulses. When referring to neural cell as “mitotic” is meant proliferative cells that are in the process of dividing/proliferating or capable of doing so. It follows that when referring to neural cells as “post-mitotic” is meant cells that cannot divide/proliferate.

The neural cells according to the present invention may have a specific regional identity, such as cells specific to the forebrain, midbrain, hindbrain, etc. As used herein, the term “forebrain” refers to the rostral region of the neural tube and CNS that gives rise to structures including the cortex and striatum. As used herein, the term “midbrain” refers to the medial region of the neural tube and CNS (on the rostro-caudal axis) that gives rise to structures including the substantia nigra, As used herein, the terms “hindbrain” and “spinal cord” refer to the caudal region of the neural tube that is caudal to the isthmus organiser.

The method according to the present invention is typically defined by a series of method steps. As used herein, the term “step” in relation to the method is to be understood as a stage, where something is undertaking and/or an action is performed. It will be understood by one of ordinary skill in the art when the steps to be performed and/or the steps undertaking are concurrent and/or successive and/or continuous.

Differentiation of stem cells into neural cells

In the step of culturing hPSCs, the cells may be obtained from any suitable source as referred to in the above. By the term “culturing” is meant that the hPSCs are cultured in a cell culture medium, which is suitable for viability in their current state of development. Culturing the stem cells typically implies a transfer of the stem cells into a different environment such as by seeding onto a new substrate or suspending in an incubator. One of ordinary skill in the art will readily recognize that stem cells are fragile to such a transfer and the procedure requires diligence and that maintaining the stem cells in the origin cell culture medium may facilitate a more sustainable transfer of the cells before replacing the cell culture medium with another cell culture medium more suitable for the differentiation process. As used herein, the term "differentiation" refers broadly to the process wherein cells progress from an undifferentiated state or a state different from the intended differentiated state to a specific differentiated state, e.g., from an immature state to a less immature state or from an immature state to a mature state, which may occur continuously as the method is performed. The term "differentiation" in respect to pluripotent stem cells refers to the process wherein cells progress from an undifferentiated state to a specific differentiated state, i.e. , from an immature state to a less immature state or to a mature state. Changes in cell interaction and maturation occur as cells lose markers of undifferentiated cells or gain markers of differentiated cells. Loss or gain of a single marker can indicate that a cell has "matured or fully differentiated”. Examples of cell types where efficient two-dimensional protocols are available span the four major domains of the CNS including(a) forebrain cortical glutamatergic neurons (Shi, Kirwan et al. 2012), (b) midbrain dopaminergic neurons (Niclis, Gantner et al. 2017), (c) hindbrain serotonergic neurons (Lu, Zhong et al. 2016), and (d) spinal cord motor neurons (Amoroso, Croft et al. 2013). The time required for the step of differentiating the hPSCs into neural stem precursor blast cells depends on the protocol used. All protocols aforementioned, the majority in the field of neural differentiation and the understood “gold standard” is to utilize the dual SMAD pathway inhibition strategy which simultaneously suppresses the BMP and TGFB pathways that typically activate SMAD proteins 1/5/8 and 2/3, respectively. The first report on this concept states the highly efficient neural conversion of human ES and iPS cells is attained with the dual SMAD inhibition method is required (Chambers et al Studer 2009).

The present inventors have found the above-mentioned dual SMAD inhibition method is not required for highly efficient neural conversion of hPSCs and instead the suppression of the BMP pathway alone and particularly at multiple levels in a stacked manner (i.e., interfering with both the pathway ligand and receptor) is sufficient. A person skilled in the art will recognize from this invention that they may easily swap the dual SMAD inhibition method for the stacked single method proposed here. A person skilled in the art will be able to determine the progress of the differentiation and what stage the neural cells have developed into. Determining the progress of differentiation may be by analysis of certain expression markers or at certain stages this may be assessed visually.

In the present context, the term “dual SMAD inhibition” refers to simultaneously suppressing the BMP and TGFB pathways that typically activate SMAD proteins 1/5/8 and 2/3, respectively, whereas the term “stacked SMAD inhibition” refers to suppression of the BMP pathway alone and particularly at multiple levels in a stacked manner (i.e., interfering with both the pathway ligand and receptor). The method of the invention comprises a stacked single SMAD inhibition procedure. The method of the invention does not comprise dual SMAD inhibition. I.e., it does not comprise simultaneously suppressing the BMP and TGFB pathways that typically activate SMAD proteins 1/5/8 and 2/3, respectively. Instead, the method of the invention comprises stacked SMAD inhibition, i.e., suppression of the BMP pathway alone and particularly at multiple levels in a stacked manner (i.e., interfering with both the pathway ligand and receptor).

Thus, a first aspect of the present invention provides an in vitro method for differentiating a population of human pluripotent stem cells (hPSCs) into a population of neural cells, wherein neuronal differentiation in said hPSCs is induced by the inhibition of the Small Mothers Against Decapentaplegic (SMAD) signaling pathway, i.e., by stacked SMAD inhibition, by culturing the population of hPSCs in a culture medium comprising two or more inducing factors consisting of two or more inhibitors of the SMAD signaling pathway, wherein a. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP ligand and b. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP receptor, and c. none of the inhibitors of the SMAD signaling pathway is a direct inhibitor of the TGFB pathway.

As used herein, the term “inhibitor of BMP ligands” means an inhibitor of the BMP signaling pathway that prevents the BMP protein from binding to its receptor by sequestration, denaturation, competitive inhibition or other methods. Suitable examples of inhibitors of BMP ligands include crossveinless, USAG-1 , noggin, greml , chordin and follistatin, including three isoforms of follistatin: FST288, FST303, FST315. In some embodiments, noggin is preferred. The one or more inhibitor of a BMP receptor interferes with a protein selected from the group consisting of ALK1 , ALK2, ALK3 and ALK6

As used herein the term “inhibitor of BMP receptors” means an inhibitor of the BMP signaling pathway that blocks the BMP receptor protein activity, i.e., preventing a BMP ligand from producing a signal by e.g., binding to the target BMP receptor. Suitable examples of inhibitors of BMP receptor include LDN-193189, dorsomorphin, LDN-212854, LDN-214117, and DMH1. In some embodiments, the inhibitor of BMP receptor is LDN- 212854. In currently preferred embodiments, the inhibitor of BMP ligand is noggin, and the inhibitor of BMP receptor is LDN-212854.

As used herein, the term “effective amount”, when referring to one or more inhibitors, means a concentration of the one or more inhibitors, which facilitates the downregulation in the hPSCs of markers for pluripotency to the extent so that less than 5%, such as less than 3%, or less than 1% of the hPSCs remain which are positive for the markers POU5F1, aka OCT4, NANOG, and CD9.

Use of at least one inhibitor of BMP ligand and at least one inhibitor of BMP receptor in effective amounts facilitates the differentiation of the hPSCs into neural cells. Thus, in embodiments at least 90% of the differentiated cells express SOX2.

The effective amount depends on the choice of inhibitors. For inhibitors of BMP ligands, an effective amount for noggin is from 50 to 1000 ng/ml, or from 75 to 500 ng/ml such as 100 ng/ml. It is contemplated that a person skilled in the art will be able to determine which concentration of another inhibitor of BMP ligand that gives an effective amount. Regarding inhibitors of BMP receptors an effective amount for LDN-212854 is from 10 to 1000 nM, from 50 to 500 nM, or from 75 to 200 nM or 100 nM. It is contemplated that a person skilled in the art will be able to determine which concentration of another inhibitor of BMP receptor that gives an effective amount.

As used herein “follistatin” may be any one of the three isoforms FST288, FST303, and FST315.

In an embodiment, the hPSCs are differentiated in a two-dimensional culture. In a further embodiment, the hPSCs are initially plated on a substrate. In an embodiment, the substrate comprises an extracellular matrix. In a further embodiment, the substrate comprises Poly-L-Lysine, Poly-D-Lysine, Poly-Ornithine, laminin, fibronectin, and/or collagen, and/or fragments thereof. In a more specific embodiment, the laminin or fragment thereof is selected from the group comprising of laminin-111 , laminin-521 , and laminin-511.

In another embodiment, the hPSCs are differentiated in a suspension culture.

In an embodiment the hPSCs are differentiated for a period of time whereby at least 50%, 60%, 70%, 80%, or at least 90% of the neural cells are no longer pluripotent. The differentiation may proceed to ensure that at least 95%, 99%, or 100% of the neural cells are no longer pluripotent. In an embodiment, the PSCs are differentiated for a period of time whereby at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the neural cells do not express one or more of the markers POU5F1 , NANOG and CD9. The differentiation protocols described in the examples herein lead to forebrain neural cells, midbrain neural cells and hindbrain/spinal cord neural cells.

Dissociation into single cell suspension

The method of the invention may further comprise a step of dissociating the neural cells to a single cell suspension. A person skilled in the art will recognize suitable techniques for dissociating the neural cells in order to ensure viability. The neural cells may be dissociated enzymatically or by chelating. The neural cells may also be dissociated by contacting the neural cells with a dissociating agent. Non-limiting examples of dissociating agents include accutase, trypsin, trypleSelect, collagenase, disapse, versene, EDTA, and/or ReLeSR. It is well recognized that dissociation of cells may cause stress, and the neural cells may be contacted with a ROCK inhibitor prior to the step of dissociating the neural cells. If added, the concentration of ROCK inhibitor is from about 0.1 pM to about 30 pM, preferably from about 1 pM to about 10 pM. The ROCK inhibitor may be Y-27632. As used herein, “Y-27632” refers to trans-4-(1-Aminoethyl)-N-(4-Pyridyl)-cyclohexane- carboxamide dihydrochloride with CAS no. 129830-38-2.

Maturation of the neural cells

The neural cell may be allowed to further mature. As used herein, the term “maturing” means a further development of stem cells that have already undergone an initial differentiation towards a specific germ layer. The terms “maturation” and “allowing to mature” may also be considered a further differentiation of the cells. Maturation will typically be the further development of cells towards a definitive cell type. Likely depending on the cell type’s maturation may simply require maintenance of the cells, such as by replacing the cell culture medium and ensuring viable conditions. Maturation of the cells may also require exposing the cells to further compounds or factors in order to facilitate the further development towards the definitive cell type.

In an embodiment, the neural cells are allowed to further mature into neurons.

Examples of differentiation to ventral midbrain cells are found in the publications by Nolbrant et al., Kirkeby ” Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation” 2017, Kriks et al., Studer “Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson’s disease” 2011 and Doi et al., Takahashi “Isolation of Human Induced Pluripotent Stem Cell-Derived Dopaminergic Progenitors by Cell Sorting for Successful Transplantation” 2014. Examples of differentiation to dorsal forebrain/pall ial cells are found in the publications by Shi et al., Livesey “Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses” 2011 and Espuny-Camacho et al., Vanderhaegen “Pyramidal Neurons Derived from Human Pluripotent Stem Cells Integrate Efficiently into Mouse Brain Circuits In Vivo” 2012.

Examples of differentiation to hindbrain/spinal cord cells are found in the publications by Du et al., Zhang “Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells” 2014; Amaroso et al., Wichterle “Accelerated High-Yield Generation of Limb-Innervating Motor Neurons from Human Stem Cells” 2013; Butts et al., McDevitt “V2a interneuron differentiation from mouse and human pluripotent stem cells” 2019.

However, none of the differentiation methods described involves the use of at least one inhibitor of BMP ligand and at least one inhibitor of BMP receptor.

As seen from the examples herein, the hPSCs may be contacted with one or more of the compounds selected from the group consisting of Sonic Hedgehog (SHH) agonist such as SHH or SAG, CHIR99021, FGF8, FGF2, retinoic acid, BDNF, GDNF, dcAMP, DAPT, and ascorbic acid (AA), and/or hPSCs may be contacted with a Sonic Hedgehog (SHH) agonist, such as SHH or SAG. In an embodiment, PSCs are contacted with a WNT activator or GSK3 inhibitor, such as CHIR99021. In an embodiment, hPSCs are contacted with fibroblast growth factor (FGF) 8.

Composition and use

The invention also relates to a composition comprising a population of hPSCs and a culture medium comprising two or more inducing factors consisting of two or more inhibitors of the SMAD signaling pathway, wherein a. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP ligand and b. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP receptor, and c. none of the inhibitors of the SMAD signaling pathway is a direct inhibitor of the TGFB pathway.

In an embodiment, the two or more inhibitors of SMAD signaling pathway are noggin and LDN-193189, and the composition does not comprise an inhibitor of nitric oxide synthase (NOS). The neural cells differentiated according to the protocols described herein and, optionally, subjected to differentiation into neurons, may be used as medicament. In a further embodiment, the neural cells are used for the treatment of a neural condition selected from the group comprising of Parkinson’s disease, stroke, traumatic brain injury, spinal cord injury, Huntington’s disease, dementia, Alzheimer’s disease, and other neurological conditions wherein neurons are lost or dysfunctional. In one particular embodiment, the neural cells are neurons, specifically dopaminergic cells, for the treatment of Parkinson’s disease.

PARTICULAR EMBODIMENTS

1. An in vitro method for differentiating a population of human pluripotent stem cells (hPSCs) into a population of neural cells, wherein neuronal differentiation in said hPSCs is induced by the inhibition of the Small Mothers Against Decapentaplegic (SMAD) signaling pathway by culturing the population of hPSCs in a culture medium comprising two or more inducing factors consisting of two or more inhibitors of the SMAD signaling pathway, wherein a. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP ligand and b. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP receptor, and c. none of the inhibitors of the SMAD signaling pathway is a direct inhibitor of the TGFB pathway.

2. The method according to embodiment 1 , wherein the one or more inhibitor of a BMP ligands is selected from crossveinless, USAG-1, noggin, follistatin including three isoforms: FST288, FST303, FST315, chordin and greml.

3. The method according to embodiment 1 or 2, wherein the one or more inhibitor of a BMP receptor interferes with a protein selected from the group consisting of ALK1, ALK2, ALK3 and ALK6.

4. The method according to any one of the preceding embodiments, wherein the one or more inhibitor of a BMP receptor is selected from LDN-193189, dorsomorphin, LDN-212854, LDN-214117, and DMH1.

5. The method according any one of the preceding embodiments, wherein the inhibitor of a BMP ligand is noggin. The method according any one of the preceding embodiments, wherein the inhibitor of a BMP receptor is LDN-193189. The method according any one of the preceding embodiments, wherein the inhibitor of a BMP ligand is noggin and wherein the inhibitor of a BMP receptor is LDN-193189. The method according to any one of the preceding embodiments, wherein the culture medium does not comprise any other added inducing factors. The method according to any one of the preceding embodiments, wherein the culture medium does not comprise an inhibitor of nitric oxide synthase (NOS). The method according to any one of the preceding embodiments, wherein the hPSCs are contacted simultaneously with the two or more inhibitors of the SMAD signaling pathway. The method according to any one of the preceding embodiments, wherein the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway, one after another within a 24-hour period. The method according to any one of the preceding embodiments, wherein the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway for 5 to 11 days, such as 6, 8, 9 or 10 days. The method according to any one of the preceding embodiments, wherein the population of neural cells is of the forebrain, midbrain, hindbrain, and/or spinal cord. The method according to any one of the preceding embodiments, wherein the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway for 9 days as an example for midbrain differentiation. The method according to any one of the preceding embodiments, wherein the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway for 11 days as an example for forebrain differentiation. The method according to any one of the preceding embodiments, wherein the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway from day 0. The method according to any one of the preceding embodiments, wherein the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway in an effective amount. 18. The method according to any one of the preceding embodiments, wherein the concentration of an inhibitor of a BMP ligand, specifically Noggin, is from 50 to 1000 ng/ml, or from 75 to 500 ng/ml.

19. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of a BMP ligand, specifically Noggin, is 100 ng/ml.

20. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of a BMP receptor, specifically LDN-193189, is from 10 to 1000 nM, from 50 to 500 nM, or from 75 to 200 nM.

21. The method according to any one of the preceding embodiments, wherein the concentration of the inhibitor of a BMP receptor, specifically LDN-193189, is 100 nM.

22. The method according to any one of the preceding embodiments, wherein the method results in a population of neural cell, wherein at least 90% are positive for SOX2.

23. The method according to any one of the preceding embodiments, wherein the method results in a population of neural cell, wherein less than 5% or 3% of the population of neural cells are positive for POLI5F1, NANOG and CD9.

24. The method according to any one of the preceding embodiments, wherein the method results in a population of neural cell, wherein less than 1% of the population of neural cells are positive for POLI5F1, NANOG and CD9.

25. The method according to any one of the preceding embodiments, wherein method results in a population of neural cells wherein the neural cells are non-native.

26. The method according to any one of the preceding embodiments, wherein the method comprises further steps for differentiating the resulting population of neural cells into neurons.

27. The method according to any one of the preceding embodiments, wherein method results in a population of neural cells wherein the neuronal cells are suitable for further differentiation into neurons.

28. The method according to any one of the preceding embodiments, wherein the two or more inhibitors of the SMAD signaling pathway are added at least every 48 hours, preferably at least every 24 hours or less. 29. A composition comprising a population of hPSCs and a culture medium comprising two or more inducing factors consisting of two or more inhibitors of the SMAD signaling pathway, wherein a. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP ligand and b. at least one inhibitor of the SMAD signaling pathway is an inhibitor of a BMP receptor, and c. none of the inhibitors of the SMAD signaling pathway is a direct inhibitor of the TGFB pathway.

30. The composition according to embodiment 29, wherein the two or more inhibitors of SMAD signaling pathway are noggin and LDN-193189.

31. The composition according to embodiments 29 or 30, wherein the composition does not comprise an inhibitor of nitric oxide synthase (NOS).

EXAMPLES

The following are non-limiting examples for carrying out the invention.

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Example 1 : Method for culture of human pluripotent stem cells

Human embryonic stem cell (hESCs) lines RC17 (Roslin CT) and 3053 (Novo Nordisk A/S) were cultured in iPS Brew media (Miltenyi Biotec) supplemented with 60 U/rnL Penicillin-15Streptomycin (P-S; Thermo Fisher Scientific) on human laminin-521 matrix (0.7-1.2 pg/cm2; Biolamina) coated culture ware. Media was changed daily, and cells passaged with EDTA 0.5mM (Thermo Fisher Scientific) every 4-6 days. Cultures were maintained at 37°C, humidity 95% and a 5% CO2level.

The cultured hPSCs may be differentiated according to published protocols towards various subtypes of neural cells such as dorsal forebrain cells of the cortical glutamatergic neuronal lineage (Shi et al., 2012 (a); Shi et al., 2012 (b)), ventral midbrain cells of the dopaminergic neuronal lineage (Nolbrant et al., 2017; Kirkeby et al., 2016; Kriks et al., 2011) and ventral spinal cord cells of the motor neuron lineages (Amaroso et al. ,2013; Du et al. ,2015). Example 2: Differentiation of human pluripotent stem cells to forebrain neural cells in dual and stacked SMAD inhibition conditions

To determine the effect of the stacked SMAD inhibition method, hESCs cells were differentiated to a dorsal forebrain lineage (Figure 7). This was performed according to an established protocol that utilizes a dual SMAD inhibition method (Shi et al. 2011 ; Shi et al 2012) and both the dual SMAD inhibition and new stacked SMAD inhibition approaches were compared in their capacity to elicit dorsal forebrain neural specification. hESCs were seeded and cultured on laminin-521 (1.2 pg/cm2) (BioLamina) coated culture ware, and once forming a 95-100% confluent monolayer exposed to differentiation media. From DIV0-11, the cells were cultured in an N2/B27-based media: 50% DMEM/F12+Glutamax (Gibco) 50% Neurobasal (Gibco), 2% B27 supplement with vitamin A CTS (Thermo Fisher), 1% N2 supplement CTS (Thermo Fisher), 5% GlutaMAX (Thermo Fisher), 0.2% Penicillin streptomycin (P/S; Thermo Fisher), 1% NEAA (Gibco), 0,089% B-Mercaptoethanol (Gibco). Dual SMAD inhibition method involved media supplemented with a BMP pathway inhibitor (Noggin at 100ng/mL) and TGFb pathway inhibitor (SB431542 at 10uM) in Figure 7A. This was compared to the stacked SMAD inhibition method where media was supplemented with two BMP pathway inhibitors, one targeting the BMP receptors (LDN-193189 at 100 nM; Miltenyi Biotec) and one targeting the BMP protein ligand itself (Noggin at 100 ng/mL; Miltenyi Biotec) in Figure 7B.

This data shows that the method efficiently exits hESCs from the undifferentiated state by reducing expression of pluripotency markers CD9, OCT3/4 and maintaining the neural marker SOX2 in the stacked SMAD inhibition method compared to dual SMAD inhibition controls (Figure 8-9). Concomitant to this, an upregulation in dorsal forebrain neural markers OTX2 and PAX6 was observed in the stacked SMAD inhibition method compared to dual SMAD inhibition controls (Figure10-11).

Example 3: Differentiation of human pluripotent stem cells to ventral midbrain neural cells in dual and stacked SMAD inhibition conditions

To determine the effect of the stacked SMAD inhibition method, hESCs cells were differentiated to a ventral midbrain lineage (Figure 1). This was performed according to an established protocol that utilizes a dual SMAD inhibition method (Nolbrant et al., 2017; Kirkeby et al., 2016) and both the dual SMAD inhibition and new stacked SMAD inhibition approaches were compared in their capacity to elicit ventral midbrain neural specification. hESCs were differentiated to ventral midbrain neural cells based on an established protocol (Nolbrant et al., Kirkeby: ’’Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation” 2017). In brief, hESC were grown to 70-90% confluency, then disassociated with 0.5 mM EDTA. The cells were seeded at 10⁴cells/cm² in cell culture flasks or plates coated with human laminin-111 (1.2-1.5 pg/cm²; BioLamina) and immediately put into contact with differentiation media. The cells were exposed to N2-based media from days in vitro (DIV) 0-9 50% DMEM/F12+Glutamax (Gibco), 50% Neurobasal (Gibco), 1% N2 supplement CTS (Thermo Fisher Scientific), 5% GlutaMAX (Thermo Fisher Scientific), 0.2% P-S (Thermo Fisher Scientific), supplemented with Sonic Hedgehog C24II (SHH; 500 ng/mL; Miltenyi Biotec) for ventral fate, GSK3pinhibitor CHIR99021 (CHIR; 0.5-0.6 pM; Miltenyi Biotec) to promote caudalization.

Dual SMAD inhibition method involved media supplemented with a BMP pathway inhibitor (Noggin at 100ng/mL) and TGFb pathway inhibitor (SB431542 at 10uM) from day 0-9 in Figure 1A. This was compared to the stacked SMAD inhibition method where media was supplemented with two BMP pathway inhibitors from day 0-9, one targeting the BMP receptors (LDN-193189 at 100 nM; Miltenyi Biotec) and one targeting the BMP protein ligand itself (Noggin at 100 ng/mL; Miltenyi Biotec) in Figure 1B.

This data shows that the method efficiently exits hESCs from the undifferentiated state by reducing expression of pluripotency markers CD9, OCT3/4 and maintaining the neural marker SOX2 in the stacked SMAD inhibition method compared to dual SMAD inhibition controls (Figure 2-4). Concomitant to this, an upregulation in ventral midbrain neural markers OTX2 and PAX6 were observed in the stacked SMAD inhibition method compared to dual SMAD inhibition controls (Figure 5-6).

Example 4: Differentiation of human pluripotent stem cells to spinal cord neural cells in dual and stacked SMAD inhibition conditions

To determine the effect of the stacked SMAD inhibition method, hESCs cells were differentiated to a spinal cord lineage (Figure 12). This was performed according to an established protocol that utilizes a dual SMAD inhibition method (Du et al. 2014) and both the dual SMAD inhibition and new stacked SMAD inhibition approaches were compared in their capacity to elicit spinal cord neural specification. hPSCs were seeded into 10pg/mL Laminin-521 precoated 24w/plates or T25 flasks (Sarstedt) and distributed evenly. The first 24 hours, cells were maintained with iPS-Brew and supplemented with 10pM Rho-associated kinase inhibitor Y27632 (ROCKi; Sigma) to enhance cell survival. Media was changed every 24 hours and cells were grown to 90- 100% confluence at which point differentiation was initiated. For the first 6 days of differentiation, cells were maintenance in “Pan Neuronal Base Media” - 40% Neurobasal (Gibco), DMEM/F12 + GlutaMAX (Gibco), 5% B27 supplement (Thermo Fisher), 1% N2 supplement (Thermo Fisher), 1% MEM Non-essential amino acids (NEAA; Gibco), 0.5% GlutaMAX (Gibco), 0.2% Penicillin/streptomycin (P/S; Thermo Fisher) - supplemented with 3pM CHIR99021 (CHIR; Miltenyi) was added for caudalization. Media was changed daily. At day 6, cells were dissociated using Accutase (Innovative Cell Technologies) and passaged with 10pM ROCKi Y27632 by re-seeding to 1 :6 ratio cells per cm2 coated with 10pg/mL LN-521. From days 6-12, cells were grown in Pan Neuronal Base Media supplemented with BMP pathway inhibitors LDN-193189 (100 nM; Miltenyi Biotec) and Noggin (100 ng/mL; Miltenyi Biotec) and also 1pM CHIR, 40-100nM SAG (Tocris) and 500nM Retinoic acid (RA; Sigma). Dual SMAD inhibition method involved media supplemented with a BMP pathway inhibitor (Noggin at 100ng/mL) and TGFb pathway inhibitor (SB431542 at 10uM) from day 0-6 in Figure 12A. This was compared to the stacked SMAD inhibition method where media was supplemented with two BMP pathway inhibitors from day 0-6, one targeting the BMP receptors (LDN-193189 at 100 nM; Miltenyi Biotec) and one targeting the BMP protein ligand itself (Noggin at 100 ng/mL; Miltenyi Biotec) in Figure 12B.

This data shows that the method efficiently exits hESCs from the undifferentiated state by reducing expression of pluripotency marker CD9 from >90% in hESC (Figure 13A) to <1% in the stacked SMAD inhibition method (Figure 13B) similar to control dual SMAD inhibition (Figure 13C). Concomitant to this, an upregulation in spinal cord marker OLIG2 and absence of forebrain/midbrain marker OTX2 was observed in the stacked SMAD inhibition method similar to dual SMAD inhibition controls (Figure14).

Du et al 2014 reference :

Zhong-Wei Du, Hong Chen, Huisheng Liu, Jianfeng Lu, Kun Qian, CindyTzu-Ling Huang, Xiaofen Zhong, Frank Fan & Su-Chun Zhang

Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells

Nature Communications 2014

Claims

24 CLAIMS

2. The method according to claim 1 , wherein the one or more inhibitor of a BMP ligands is selected from crossveinless, USAG-1 , noggin, follistatin including three isoforms: FST288, FST303, FST315, chordin and greml.

3. The method according to claim 1 or 2, wherein the one or more inhibitor of a BMP receptor interferes with a protein selected from the group consisting of ALK1 , ALK2, ALK3 and ALK6.

4. The method according to any one of the preceding claims, wherein the one or more inhibitor of a BMP receptor is selected from LDN-193189, dorsomorphin, LDN-212854, LDN-214117, and DMH1.

5. The method according any one of the preceding claims, wherein the inhibitor of a BMP ligand is noggin.

6. The method according any one of the preceding claims, wherein the inhibitor of a BMP receptor is LDN-193189.

7. The method according any one of the preceding claims, wherein the inhibitor of a BMP ligand is noggin and wherein the inhibitor of a BMP receptor is LDN-193189.

8. The method according to any one of the preceding claims, wherein the culture medium does not comprise any other added inducing factors.

9. The method according to any one of the preceding claims, wherein the culture medium does not comprise an inhibitor of nitric oxide synthase (NOS).

10. The method according to any one of the preceding claims, wherein the hPSCs are contacted simultaneously with the two or more inhibitors of the SMAD signaling pathway.

11. The method according to any one of the preceding claims, wherein the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway for 5 to 11 days, such as 6, 8, 9 or 10 days.

12. The method according to any one of the preceding claims, wherein the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway for 9 days as an example for midbrain differentiation.

13. The method according to any one of the preceding claims, wherein the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway from day 0.

14. The method according to any one of the preceding claims, wherein the hPSCs are contacted with the two or more inhibitors of the SMAD signaling pathway in an effective amount.

15. The method according to any one of the preceding claims, wherein the method results in a population of neural cell, wherein at least 90% are positive for SOX2.