WO2007002210A2 - Compositions de culture de cellules souches embryonnaires et methodes d’utilisation de celles-ci - Google Patents

Compositions de culture de cellules souches embryonnaires et methodes d’utilisation de celles-ci Download PDF

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WO2007002210A2
WO2007002210A2 PCT/US2006/024168 US2006024168W WO2007002210A2 WO 2007002210 A2 WO2007002210 A2 WO 2007002210A2 US 2006024168 W US2006024168 W US 2006024168W WO 2007002210 A2 WO2007002210 A2 WO 2007002210A2
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composition
igf
cell
activator
stem cell
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PCT/US2006/024168
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WO2007002210A3 (fr
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Allan J. Robins
Thomas C. Schulz
Stephen Dalton
E. Edward Baetge
Melissa Carpenter
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Bresagen, Inc.
University Of Georgia Research Foundation, Inc.
Cythera, Inc.
Robarts Research Institute
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Publication of WO2007002210A2 publication Critical patent/WO2007002210A2/fr
Publication of WO2007002210A3 publication Critical patent/WO2007002210A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/105—Insulin-like growth factors [IGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)

Definitions

  • the present invention generally relates to compositions and methods for culturing pluripotent stem cells, the cells created by these methods, and their uses thereof. Particularly, the invention relates to a defined medium for culturing pluripotent stem cells in the absence of a feeder cell layer, and in the absence of serum or serum replacement.
  • Embryonic stem (ES) cells represent a powerful model system for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo, as well as providing opportunities for genetic manipulation of mammals and resultant commercial, medical and agricultural applications. Furthermore, appropriate proliferation and differentiation of ES cells can be used to generate an unlimited source of cells suited to transplantation for treatment of diseases that result from cell damage or dysfunction.
  • EPL early primitive ectoderm-like cells as described in International Patent Application WO 99/53021
  • ICM/epiblast in vivo or in vitro derived ICM/epiblast
  • EG cells primordial germ cells
  • EC cells teratocarcinoma cells
  • pluripotent cells derived by dedifferentiation or by nuclear transfer will share some or all of these properties and applications.
  • KSR contains high levels of insulin, as well as other possible sources of growth factors, such as potentially found in Albumax ® (Invitrogen Corp.).
  • the extracellular matrix may also have been a source of matrix-associated growth factors.
  • One embodiment of the present invention relates to a novel, defined composition
  • a basal salt nutrient solution an activator of the IGF-I receptor (IGF-IR), serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family, wherein the composition is essentially serum free.
  • IGF-IR IGF-I receptor
  • FGF receptor FGF receptor
  • transferrin optionally, a member of the TGF- ⁇ family
  • the invention also encompasses a composition for culturing a pluripotent mammalian embryonic stem cell, comprising an extracellular matrix, and a medium comprising a basal salt nutrient solution, an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family, wherein the medium is essentially serum free, wherein a pluripotent mammalian stem cell remains undifferentiated for greater than approximately 5 passages in culture.
  • a cell composition comprising a pluripotent mammalian embryonic stem cell proliferating on an extracellular matrix in the presence of a defined medium, wherein the cell composition is essentially free of feeder cells, and wherein the defined medium comprises a basal salt nutrient solution, an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family.
  • the invention is further directed to a method of culturing a pluripotent mammalian embryonic stem cell comprising (a) providing a pluripotent mammalian embryonic stem cell; (b) plating the cell on an extracellular matrix; and (c) contacting the cell with a defined medium that is essentially serum free comprising an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family, wherein the stem cell proliferates in culture and remains undifferentiated essentially in the absence of serum or serum replacement in the medium.
  • a defined medium that is essentially serum free comprising an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family
  • the pluripotent mammalian embryonic stem cell proliferates and remains undifferentiated for greater than 5 passages in culture. In another embodiment, the pluripotent mammalian embryonic stem cell proliferates and remains undifferentiated for greater than one month in culture. In another embodiment, the contact of the cell with the defined medium is in the absence of a feeder layer. In another embodiment, the pluripotent mammalian embryonic stem cell is cultured on feeder cells prior to plating on the extracellular matrix. [0011] In particular embodiments of the invention, the member of the TGF- ⁇ family is selected from the group consisting of Nodal, Activin A, Activin B, TGF- ⁇ , BMP2 and BMP4.
  • the member of the TGF- ⁇ family is Activin A. It is contemplated that the activator of IGF-IR is selected from the group consisting of insulin and an insulin-like growth factor. In one embodiment, the insulin-like growth factor is IGF-I or IGF-2. In one embodiment, the insulin-like growth factor is IGF-I, which can be LongR 3 IGF-l. In one embodiment, the activator of an FGF receptor is FGF2. It is also contemplated that the serum albumin is selected from the group consisting of bovine serum albumin and human serum albumin. In a further embodiment, the medium is a non-conditioned medium.
  • the mammalian cell population used in the invention is selected from the group consisting of an embryonic stem cell, an ICM/epiblast cell, a primitive ectoderm cell, a primordial germ cell, and a teratocarcinoma cell.
  • the mammalian cell population comprises a human embryonic stem cell.
  • Figures IA-D show photomicrographs of BGOIv colonies grown in conditioned medium (CM) comprising FGF2 and BSA (Panel A); FGF2, insulin, and Activin A (Panel B); FGF, insulin, and BSA (Panel C); and FGF, LongR3-IGF-l, and BSA (Panel D).
  • CM conditioned medium
  • FIGS 2A-G show photomicrographs of BGOIv colonies grown in medium comprising FGF2 and insulin (Panel A); FGF and LongR 3 -IGF-l (Panel B); FGF2 (Panel C); insulin (Panel D); LongR 3 -IGF-l (Panel E); FGF2, and Insulin (Panel F); and FGF2 and LongR 3 -IGF-l (Panel G).
  • Panels A and B are in the presence of CM
  • Panels C-G are in the presence of non-conditioned medium.
  • Figures 3A-D show photomicrographs of BGOIv colonies grown in non- conditioned medium comprising FGF2 and LongR 3 -IGF-l (Panels A and B); and FGF2, LongR 3 -IGF-l, and Activin A (Panels C and D).
  • Figures 4A-E show the characteristics of a p2 culture. Using RT-PCR, Panel A shows that the cultures expressed multiple markers of pluripotency, including ABCG2, DPPA5, REXl, UTFl, NANOG, OCT4, SOX2, CRIPTO, and TERT.
  • Panel B shows a photomicrograph of a DAPI stained colony.
  • Panels C and D show immunostainings of the same colony as Panel B, demonstrating the uniform expression of Oct4 (Panel C), and SSEA-4 (Panel D). The colonies were also uniformly positive for alkaline phosphatase activity.
  • One embodiment of the present invention relates to a novel, defined composition
  • a novel, defined composition comprising a basal salt nutrient solution, an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family, wherein the composition is essentially serum free.
  • the invention also encompasses a composition for culturing a pluripotent mammalian embryonic stem cell, comprising an extracellular matrix, and a medium comprising a basal salt nutrient solution, an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family, wherein the medium is essentially serum free, wherein a pluripotent mammalian stem cell remains undifferentiated for greater than approximately 5 passages in culture. In one embodiment, the pluripotent mammalian stem cell remains undifferentiated for greater than approximately 1 month in culture.
  • a cell composition comprising a pluripotent mammalian embryonic stem cell proliferating on an extracellular matrix in the presence of a defined medium, wherein the cell composition is essentially free of feeder cells, and wherein the defined medium comprises a basal salt nutrient solution, an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family.
  • the invention is further directed to a method of culturing a pluripotent mammalian embryonic stem cell comprising (a) providing a pluripotent mammalian embryonic stem cell; (b) plating the cell on an extracellular matrix; and (c) contacting the cell with a defined medium that is essentially serum free comprising an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family, wherein the stem cell proliferates in culture and remains undifferentiated essentially in the absence of serum or serum replacement in the medium.
  • a defined medium that is essentially serum free comprising an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family
  • the pluripotent mammalian embryonic stem cell proliferates and remains undifferentiated for greater than 5 passages in culture. In a further embodiment, the pluripotent mammalian embryonic stem cell proliferates and remains undifferentiated for greater than 1 month in culture. In another embodiment, the contact of the cell with the defined medium is in the absence of a feeder layer. In another embodiment, the pluripotent mammalian embryonic stem cell is cultured on feeder cells prior to plating on the extracellular matrix.
  • the member of the TGF- ⁇ family is selected from the group consisting of Nodal, Activin A, Activin B, TGF- ⁇ , BMP2, and BMP4.
  • the member of the TGF- ⁇ family is Activin A.
  • the activator of IGF-IR is selected from the group consisting of insulin and an insulin-like growth factor.
  • the insulin-like growth factor is IGF-I or IGF-2.
  • the insulin-like growth factor is IGF-I, which can be LongR 3 IGF-l.
  • the activator of an FGF receptor is FGF2.
  • FGF2 is initially present at a concentration of approximately 0.1 ng/ml to approximately 100 ng/ml, approximately 0.5 ng/ml to approximately 50 ng/ml, approximately 1 ng/ml to approximately 25 ng/ml, approximately 1 ng/ml to approximately 12 ng/ml, or is initially present at a concentration of approximately 8 ng/ml. °
  • the serum albumin is selected from the group consisting of bovine serum albumin and human serum albumin.
  • the medium is a non-conditioned medium.
  • the mammalian cell population used in the invention is selected from the group consisting of an embryonic stem cell, an ICM/epiblast cell, a primitive ectoderm cell, a primordial germ cell, and a teratocarcinoma cell.
  • the mammalian cell population comprises a human embryonic stem cell.
  • Human pluripotent cells offer unique opportunities for investigating early stages of human development as well as for therapeutic intervention in several disease states, such as diabetes mellitus and Parkinson's disease.
  • the use of insulin-producing ⁇ -cells derived from hESCs would offer a vast improvement over current cell therapy procedures that utilize cells from donor pancreases.
  • cell therapy treatments for diabetes mellitus, which utilize cells from donor pancreases are limited by the scarcity of high quality islet cells needed for transplant.
  • HESCs offer a source of starting material from which to develop substantial quantities of high quality differentiated cells for human cell therapies.
  • a basal salt nutrient solution refers to a mixture of salts that provide cells with water and certain bulk inorganic ions essential for normal cell metabolism, maintain intra- and extra-cellular osmotic balance, provide a carbohydrate as an energy source, and provide a buffering system to maintain the medium within the physiological pH range.
  • basal salt nutrient solutions include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMl 1640, Ham's F-10, Ham's F-12, ⁇ - Minimal Essential Medium ( ⁇ MEM), Glasgow's Minimal Essential Medium (G- MEM), and Iscove's Modified Dulbecco's Medium, and mixtures thereof.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basal Medium Eagle
  • RPMl 1640 Ham's F-10, Ham's F-12, ⁇ - Minimal Essential Medium ( ⁇ MEM), Glasgow's Minimal Essential Medium (G- MEM), and Iscove's Modified Dulbecco's Medium, and mixtures thereof.
  • the basal salt nutrient solution is an approximately 50:50 mixture of DMEM and Ham's F12.
  • the term "member of the TGF- ⁇ family” refers to growth factors that are generally characterized by one of skill in the art as belonging to the TGF- ⁇ family, either due to homology with known members of the TGF- ⁇ family, or due to similarity in function with known members of the TGF- ⁇ family.
  • the member of the TGF- ⁇ family is selected from the group consisting of Nodal, Activin A, Activin B, TGF- ⁇ , BMP2, and BMP4.
  • the member of the TGF- ⁇ family is Activin A.
  • the term "activator of an FGF receptor” refers to growth factors that are generally characterized by one of skill in the art as belonging to the FGF family, either due to homology with known members of the FGF family, or due to similarity in function with known members of the FGF family.
  • the activator of an FGF receptor is an FGF, such as, but not limited to ⁇ -FGF and FGF2.
  • the term "activator of IGF-IR” refers to mitogens that play a pivotal role in regulating cell proliferation, differentiation, and apoptosis.
  • the effects of an activator of IGF-IR are typically mediated through IGF-IR, although they can be mediated through other receptors.
  • the IGF-IR is also involved in cell transformation induced by tumor virus proteins and oncogene products, and the interaction is regulated by a group of specific binding proteins (IGFBPs).
  • IGFBPs specific binding proteins
  • a large group of IGFBP proteases hydrolyze IGFBPs, resulting in the release of bound IGFs that then resume their ability to interact with IGF-IR.
  • the ligands, the receptors, the binding proteins, and the proteases are all considered to be activators of IGF-IR.
  • the activator of IGF-IR is insulin.
  • the activator of IGF-IR is IGF-I, or IGF-2.
  • the IGF-I is an IGF-I analog.
  • IGF-I analogs include LongR 3 IGF-l, Des(l-3)IGF-1, [Arg 3 ]IGF-l, [Ala 31 ]IFG-l, Des(2,3)[Ala 31 ]IGF- 1, [Leu 24 ]IGFl, Des(2,3)[Leu 24 ]IGF-l, [Leu 60 ]IGF-l, [Ala 31 J[LeU 60 IIGF-I, [Leu 24 ][Ala 31 ]IGF-l, and combinations thereof.
  • the IFG-I analog is LongR 3 IGF-l, a recombinant analog of human insulin growth factor- 1 (JRH Biosciences; Yandell et al, 2004, BioProcess Intl., 56-64).
  • LongR 3 IGF-l has greatly decreased affinity for IGFBPs, and may therefore be more bioactive in cell culture.
  • the invention provides in certain embodiments that the compositions consist essentially of a basal salt nutrient solution, an activator of the PI3K pathway, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family, wherein the composition is essentially serum free.
  • the activator of the PI3K pathway is an activator of IGF-IR
  • the composition further comprises a second activator of the PI3K pathway that may act through a separate receptor.
  • insulin refers to a protein that binds to the insulin receptor and can induce signaling through the receptor. Alternatively, insulin can signal through other receptors, such as, but not limited to, IGF-IR.
  • insulin encompasses a protein having the polypeptide sequence of native human insulin, or of other mammalian insulin, or of any homologs to these sequences, and includes any zinc containing compound that may be used in place of insulin to give substantially the same results as insulin. Additionally, the term insulin encompasses polypeptide fragments that are capable of binding to the insulin or IGF-I receptor and inducing signaling through the receptor(s).
  • the invention further encompasses variants of naturally occurring members of the TGF- ⁇ family, and of naturally occurring activators of an FGF receptor and of IGF-IR.
  • variant includes chimeric or fusion polypeptides, homologs, analogs, orthologs, and paralogs.
  • the invention also provides chimeric or fusion polypeptides.
  • a "chimeric polypeptide" or “fusion polypeptide” comprises at least a portion of a member of the reference polypeptide operatively linked to a second, different polypeptide.
  • the second polypeptide has an amino acid sequence corresponding to a polypeptide which is not substantially identical to the reference polypeptide, and which is derived from the same or a different organism.
  • the term "operatively linked" is intended to indicate that the reference polypeptide and the second polypeptide are fused to each other so that both sequences fulfill the proposed function attributed to the sequence used.
  • the second polypeptide can be fused to the N-terminus or C-terminus of the reference polypeptide.
  • the fusion polypeptide is a GST-IGF-I fusion polypeptide in which an IGF-I sequence is fused to the C-terminus of the GST sequences.
  • fusion polypeptides can facilitate the purification of recombinant polypeptides.
  • the fusion polypeptide can contain a heterologous signal sequence at its N-terminus.
  • expression and/or secretion of a polypeptide can be increased through use of a heterologous signal sequence.
  • homologs are defined herein as two nucleic acids or polypeptides that have similar, or “identical,” nucleotide or amino acid sequences, respectively. Homologs include allelic variants, orthologs, paralogs, agonists, and antagonists as defined hereafter.
  • the term “homolog” further encompasses nucleic acid molecules that differ from a reference nucleotide sequence due to degeneracy of the genetic code and thus encode the same polypeptide as that encoded by the reference nucleotide sequence.
  • Naturally occurring refers to a nucleic or amino acid sequence that occurs in nature.
  • An agonist of a polypeptide can retain substantially the same, or a subset, of the biological activities of the polypeptide.
  • An antagonist of a polypeptide can inhibit one or more of the activities of the naturally occurring form of the polypeptide.
  • the pluripotent cells are contacted with an effective amount of an activator of an FGF receptor, an effective amount of an activator of IGF- IR, and optionally with an effective amount of a member of the TGF- ⁇ family.
  • the term "effective amount" of a compound refers to that concentration of the compound that is sufficient in the presence of the remaining components of the defined medium to effect the stabilization of the pluripotent cell in culture for greater than one month in the absence of a feeder cell and in the absence of serum or serum replacement. This concentration is readily determined by one of ordinary skill in the art.
  • the defined medium comprises a basal salt nutrient solution, and further comprises an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family.
  • other minor components such as L-glutamine, non-essential amino acids, amino acids, lipids, ascorbic acid, trace elements, antibiotics, ⁇ - Mercaptoethanol, and similar components may be present.
  • the defined medium can comprise a basal salt nutrient solution, an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family.
  • the member of the TGF- ⁇ family is selected from the group consisting of Nodal, Activin A, Activin B, TGF- ⁇ , BMP2, and BMP4.
  • the member of the TGF- ⁇ family is Activin A. It is contemplated that nodal is initially present at a concentration of approximately 0.1 ng/ml to approximately 100 ng/ml, more preferably approximately 0.5 ng/ml to approximately 75 ng/ml, more preferably approximately 1 ng/ml to approximately 50 ng/ml, or more preferably approximately 2.5 ng/ml to approximately 5 ng/ml.
  • Activin A is initially present at a concentration of approximately 0.01 ng/ml to approximately 1000 ng/ml, more preferably approximately 0.1 ng/ml to approximately 100 ng/ml, more preferably approximately 0.1 ng/ml to approximately 10 ng/ml, or most preferably at a concentration of approximately 1 ng/ml.
  • TGF- ⁇ is initially present at a concentration of approximately 0.01 ng/ml to approximately 100 ng/ml, more preferably approximately 0.1 ng/ml to approximately 50 ng/ml, or more preferably approximately 0.1 ng/ml to approximately 20 ng/ml.
  • BMP4 is initially present at a concentration of approximately 0.01 ng/ml to approximately 100 ng/ml, more preferably approximately 0.05 ng/ml to approximately 10 ng/ml, or more preferably approximately 0.05 ng/ml to approximately 0.01 ng/ml.
  • the activator of IGF-IR is selected from the group consisting of insulin and an insulin-like growth factor.
  • the activator of IGF-IR is insulin. It is contemplated that insulin is initially present at a concentration of approximately 1 ⁇ g/ml to approximately 1000 ⁇ g/ml, more preferably approximately 5 ⁇ g/ml to approximately 100 ⁇ g/ml, more preferably approximately 10 ⁇ g/ml to approximately 50 ⁇ g/ml, or most preferably at a concentration of approximately 20 ⁇ g/ml.
  • the insulin like growth factor is IGF- 1 or IGF-2.
  • the insulin-like growth factor is IGF-I, which can be LongR 3 IGF-l.
  • LongR 3 IGF-l is initially present at a concentration of approximately 1 ng/ml to approximately 1000 ng/ml, more preferably approximately 5 ng/ml to approximately 500 ng/ml, more preferably approximately 50 ng/ml to approximately 500 ng/ml, more preferably approximately 100 ng/ml to approximately 300 ng/ml, or at a concentration of approximately 100 ng/ml.
  • the activator of an FGF receptor is FGF2.
  • FGF2 is initially present at a concentration of approximately 0.1 ng/ml to approximately 100 ng/ml, more preferably approximately 0.5 ng/ml to approximately 50 ng/ml, more preferably approximately 1 ng/ml to approximately 25 ng/ml, more preferably approximately 1 ng/ml to approximately 12 ng/ml, or most preferably at a concentration of approximately 8 ng/ml.
  • the serum albumin is selected from bovine serum albumin and human serum albumin. It is contemplated that the serum albumin is initially present at a concentration of from approximately 0.02-5.0%, from approximately 0.05-2% or from approximately 0.1-0.5%.
  • the composition can further comprise trace elements.
  • Trace elements can be purchased commercially, for example, from Mediatech.
  • Non-limiting examples of trace elements include AlCl 3 , AgNO 3 , Ba(C 2 H 3 O 2 ) 2 , CdCl 2 , CdSO 4 , CoCl 2 , CrCl 3 , Cr 2 (SO 4 ) 3 , CuSO 4 , ferric citrate, GeO 2 , KI, KBr, LI, molybdic acid, MnSO 4 , MnCl 2 , NaF, Na 2 SiO 3 , NaVO 3 , NH 4 VO 3 , (NKU) 6 Mo 7 O 245 NiSO 4 , RbCl, selenium, Na 2 SeO 3 , H 2 SeO 3 , selenite-2Na, selenomethionone, SnCl 2 , ZnSO 4 , ZrOCl 2 , and mixtures and salts thereof. If selenium,
  • Non-limiting examples of such amino acids are Glycine, L-Alanine, L-Alanyl-L- Glutamine, L-Glutamine/Glutamax, L-Arginine hydrochloride, L-Asparagine-H 2 O, L- Aspartic acid, L-Cysteine hydrochloride-H 2 O, L-Cystine 2HCl, L-Glutamic Acid, L- Histidine hydrochloride-H 2 O, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L- Methionine, L-Phenylalanine, L-Proline, L-Hydroxyproline, L-Serine, L-Threonine, L- Tryptophan, L-Tyrosine disodium salt dihydrate, and L- Valine.
  • the amino acid is L-Isoleucine, L-Phenylalanine, L-Proline, L- Hydroxyproline, L- Valine, and mixtures thereof.
  • the defined medium can comprise ascorbic acid.
  • ascorbic acid is present at an initial concentration of approximately 1 mg/L to approximately 1000 mg/L, or from approximately 2 mg/L to approximately 500 mg/L, or from approximately 5 mg/L to approximately 100 mg/L, or from approximately 10 mg/L to approximately 100 mg/L or approximately at 50 mg/L.
  • the composition comprises an inactivator of Notch signaling.
  • an "inactivator of Notch signaling” refers to an agent that antagonizes the activity of one or more Notch proteins or any of their upstream or downstream signaling components through any of its possible signaling pathways.
  • the compound used to inactivate Notch signaling can be any compound known in the art, or later discovered.
  • Non-limiting examples of inactivators of Notch signaling include dominant-negative, truncated Delta or Serrate molecules that lack intracellular domains, and inhibitors of the gamma-secretase complex.
  • the defined medium can comprise a growth hormone.
  • the growth hormone present in the defined medium will be of the same species as the pluripotent mammalian cell that is cultured with the defined media.
  • the growth hormone is human growth hormone.
  • growth hormone is present at an initial concentration of approximately 0.001 ng/ml to approximately 1000 ng/ml, more preferably approximately 0.001 ng/ml to approximately 250 ng/ml, or more preferably approximately 0.01 ng/ml to approximately 150 ng/ml.
  • the defined media of the invention is essentially free of serum and serum replacement, and is essentially serum free.
  • essentially serum free refers to a medium that does not contain serum or serum replacement, or that contains essentially no serum or serum replacement.
  • "essentially” means that a de minimus or reduced amount of a component, such as serum or serum replacement, may be present that does not eliminate the improved bioactive culturing capacity of the medium or environment.
  • essentially serum free medium or environment can contain less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% serum wherein the presently improved bioactive maintenance capacity of the medium or environment is still observed.
  • the essentially serum free medium does not contain serum or serum replacement, or only contains trace amounts of serum or serum replacement from the isolation of components of the serum or serum replacement that are added to the defined media.
  • the pluripotent cells are contacted with the defined media of the invention in the absence of serum or serum replacement, and in the absence of a feeder cell layer, such that the cells are maintained in an undifferentiated state for at least one month.
  • Pluripotency can be determined through characterization of the cells with respect to surface markers, transcriptional markers, karyotype, and ability to differentiate to cells of the three germ layers. These characteristics are well known to those of ordinary skill in the art.
  • isolated refers to being substantially separated from the natural source of the cells such that the cell, cell line, cell culture, or population of cells are capable of being cultured in vitro.
  • the term "isolating" is used to refer to the physical selection of one or more cells out of a group of two or more cells, wherein the cells are selected based on cell morphology and/or the expression of various markers.
  • the term “express” refers to the transcription of a polynucleotide or translation of a polypeptide in a cell, such that levels of the molecule are measurably higher in a cell that expresses the molecule than they are in a cell that does not express the molecule.
  • Methods to measure the expression of a molecule are well known to those of ordinary skill in the art, and include without limitation, Northern blotting, RT-PCR, in situ hybridization, Western blotting, and immunostaining.
  • the term “contacting” i.e., contacting a cell e.g.
  • a pluripotent cell, with a compound is intended to include incubating the compound and the cell together in vitro (e.g., adding the compound to cells in culture).
  • the term "contacting" is not intended to include the in vivo exposure of cells to a defined cell medium comprising an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family, that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).
  • the step of contacting the cell with a defined cell medium comprising an activator of IGF-IR, serum albumin, an activator of an FGF receptor, transferrin, and optionally, a member of the TGF- ⁇ family, can be conducted in any suitable manner.
  • the cells may be treated in adherent culture, or in suspension culture. It is understood that the cells contacted with the defined medium can be further treated with a cell differentiation environments to stabilize the cells, or to differentiate the cells.
  • the compositions and methods described herein have several useful features. For example, the compositions and methods described herein are useful for modeling the early stages of human development.
  • compositions and methods described herein can also serve for therapeutic intervention in disease states, such as neurodegenerative disorders, diabetes mellitus or renal failure, such as by the development of pure tissue or cell types.
  • disease states such as neurodegenerative disorders, diabetes mellitus or renal failure, such as by the development of pure tissue or cell types.
  • the term “differentiate” refers to the production of a cell type that is more differentiated than the cell type from which it is derived. The term therefore encompasses cell types that are partially and terminally differentiated.
  • the term “enriched” refers to a cell culture that contains more than approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the desired cell lineage.
  • the cell types that differentiate from embryonic stem cells have several uses in various fields of research and development including but not limited to drug discovery, drug development and testing, toxicology, production of cells for therapeutic purposes as well as basic science research. These cell types express molecules that are of interest in a wide range of research fields. These include the molecules known to be required for the functioning of the various cell types as described in standard reference texts. These molecules include, but are not limited to, cytokines, growth factors, cytokine receptors, extracellular matrix, transcription factors, secreted polypeptides and other molecules, and growth factor receptors.
  • the pluripotent cell is a human cell.
  • pluripotent human cell encompasses pluripotent cells obtained from human embryos, fetuses, or adult tissues.
  • the pluripotent human cell is a human pluripotent embryonic stem cell.
  • the pluripotent human cell is a human pluripotent fetal stem cell, such as a primordial germ cell.
  • the pluripotent human cell is a human pluripotent adult stem cell.
  • the term “pluripotent” refers to a cell capable of at least developing into one of ectodermal, endodermal, and mesodermal cells.
  • pluripototent refers to cells that are totipotent and multipotent.
  • the term “totipotent cell” refers to a cell capable of developing into all lineages of cells.
  • multipotent refers to a cell that is not terminally differentiated.
  • multipotent refers to a cell that, without manipulation (i.e., nuclear transfer or dedifferentiation inducement), is incapable of forming differentiated cell types derived from all three germ layers (mesoderm, ectoderm, and endoderm), or in other words, is a cell that is partially differentiated.
  • the pluripotent human cell can be selected from the group consisting of a human embryonic stem (ES) cell; a human inner cell mass (ICM)/epiblast cell; a human primitive ectoderm cell, such as an early primitive ectoderm cell (EPL); a human primordial germ (EG) cell; and a human teratocarcinoma (EC) cell.
  • the human pluripotent cells of the present invention can be derived using any method known to those of skill in the art.
  • the human pluripotent cells can be produced using de-differentiation and nuclear transfer methods.
  • the human ICM/epiblast cell or the primitive ectoderm cell used in the present invention can be derived in vivo or in vitro.
  • EPL cells may be generated in adherent culture or as cell aggregates in suspension culture, as described in PCT Publication No. WO 99/53021.
  • the human pluripotent cells can be passaged using any method known to those of skill in the art, including, manual passaging methods, and bulk passaging methods such as antibody selection and protease passaging.
  • the embryonic stem cell of the invention has a normal karyotype, while in other embodiments, the embryonic stem cell has an abnormal karyotype. In one embodiment, a majority of the embryonic stem cells have an abnormal karyotype. It is contemplated that greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater than 95% of metaphases examined will display an abnormal karyotype. In certain embodiments, the abnormal karyotype is evident after the cells have been cultured for greater than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 passages.
  • the abnormal karyotype comprises a trisomy of at least one autosomal chromosome, wherein the autosomal chromosome is selected from the group consisting of chromosomes 1, 7, 8, 12, 14, and 17.
  • the abnormal karyotype comprises a trisomy of more than one autosomal chromosome, wherein at least one of the more than one autosomal chromosomes is selected from the group consisting of chromosomes 1, 7, 8, 12, 14, and 17.
  • the autosomal chromosome is chromosome 12 or 17.
  • the abnormal karyotype comprises an additional sex chromosome.
  • the karyotype comprises two X chromosomes and one Y chromosome. It is also contemplated that translocations of chromosomes may occur, and such translocations are encompassed within the term "abnormal karyotype.” Combinations of the foregoing chromosomal abnormalities and other chromosomal abnormalities are also encompassed by the invention.
  • the pluripotent cells of the invention can be differentiated through contact with a cell differentiation environment.
  • the term "cell differentiation environment” refers to a cell culture condition wherein the pluripotent cells are induced to differentiate, or are induced to become a human cell culture enriched in differentiated cells.
  • the differentiated cell lineage induced by the growth factor will be homogeneous in nature.
  • the term “homogeneous,” refers to a population that contains more than approximately 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the desired cell lineage.
  • a cell differentiating medium or environment may be utilized to partially, terminally, or reversibly differentiate the pluripotent cells of the present invention.
  • the medium of the cell differentiation environment may contain a variety of components including, for example, KODMEM medium (Knockout Dulbecco's Modified Eagle's Medium), DMEM, Ham's F12 medium, FBS (fetal bovine serum), FGF2 (fibroblast growth factor 2), KSR, or hLIF (human leukemia inhibitory factor).
  • the cell differentiation environment can also contain supplements such as L-Glutamine, NEAA (non-essential amino acids), P/S (penicillin/streptomycin), N2, and ⁇ -mercaptoethanol ( ⁇ -ME).
  • additional factors may be added to the cell differentiation environment, including, but not limited to, fibronectin, laminin, heparin, heparin sulfate, retinoic acid, members of the epidermal growth factor family (EGFs), members of the fibroblast growth factor family (FGFs) including FGF2 and/or FGF8, members of the platelet derived growth factor family (PDGFs), transforming growth factor (TGF)/ bone morphogenetic protein (BMP)/ growth and differentiation factor (GDF) factor family antagonists including but not limited to noggin, follistatin, chordin, gremlin, cerberus/DAN family proteins, ventropin, high dose activin, and amnionless.
  • EGFs epidermal growth factor family
  • FGFs fibroblast growth factor family
  • PDGFs platelet derived growth factor family
  • TGF transforming growth factor
  • BMP bone morphogenetic protein
  • GDF growth and differentiation factor
  • TGF/BMP/GDF antagonists could also be added in the form of TGF/BMP/GDF receptor-Fc chimeras.
  • Other factors that may be added include molecules that can activate or inactivate signaling through Notch receptor family, including but not limited to proteins of the Delta-like and Jagged families as well as inhibitors of Notch processing or cleavage.
  • Other growth factors may include members of the insulin like growth factor family (IGF), insulin, the wingless related (WNT) factor family, and the hedgehog factor family.
  • the cell culture environment comprises plating the cells in an adherent culture.
  • the terms "plated” and “plating” refer to any process that allows a cell to be grown in adherent culture.
  • the substrate for the adherent culture may comprise any one or combination of polyornithine, laminin, poly-lysine, purified collagen, gelatin, f ⁇ bronectin, tenascin, vitronectin, entactin, heparin sulfate proteoglycans, poly glycolytic acid (PGA), poly lactic acid (PLA), and poly lactic-gly colic acid (PLGA).
  • the substrate for the adherent culture may comprise the matrix laid down by a feeder layer, or laid down by the pluripotent human cell or cell culture.
  • extracellular matrix encompasses solid substrates such as but not limited to those described above, as well as the matrix laid down by a feeder cell layer or by the pluripotent human cell or cell culture.
  • the cells are plated on matrigel coated plates.
  • the cells are plated on f ⁇ bronectin coated plates.
  • the plates are prepared by coating with 10 ⁇ g/ml human plasma f ⁇ bronectin (Invitrogen, #33016-015), diluted in tissue grade water, for 2-3 hours at room temperature.
  • a feeder cell is a cell that grows in vitro, that is co-cultured with a target cell and stabilizes the target cell in its current state of differentiation.
  • a feeder cell layer can be used interchangeably with the term “feeder cell.”
  • the term "essentially free of a feeder cell” refers to tissue culture conditions that do not contain feeder cells, or that contain a de minimus number of feeder cells. By “de minimus”, it is meant that number of feeder cells that are carried over to the instant culture conditions from previous culture conditions where the pluripotent cells may have been cultured on feeder cells.
  • conditioned medium is obtained from a feeder cell that stabilizes the target cell in its current state of differentiation.
  • the defined medium is a non- conditioned medium, which is a medium that is not obtained from a feeder cell.
  • stabilize refers to the differentiation state of a cell. When a cell or cell population is stabilized, it will continue to proliferate over multiple passages in culture, and preferably indefinitely in culture; additionally, each cell in the culture is preferably of the same differentiation state, and when the cells divide, typically yield cells of the same cell type or yield cells of the same differentiation state.
  • a stabilized cell or cell population does not further differentiate or de-differentiate if the cell culture conditions are not altered, and the cells continue to be passaged and are not overgrown.
  • the cell that is stabilized is capable of proliferation in the stable state indefinitely, or for at least more than 2 passages.
  • it is stable for more than 5 passages, more than 10 passages, more than 15 passages, more than 20 passages, more than 25 passages, or most preferably, it is stable for more than 30 passages.
  • the cell is stable for greater than approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or 11 months of continuous passaging.
  • the cell is stable for greater than approximately 1 year of continuous passaging.
  • stem cells are maintained in culture in a pluripotent state by routine passage in the defined medium until it is desired that they be differentiated.
  • proliferate refers to an increase in the number cells in a cell culture.
  • the progression of the hESC culture to the desired cell lineage, or its maintenance in an undifferentiated state can be monitored by quantitating expression of marker genes characteristic of the desired cell lineage as well as the lack of expression of marker genes characteristic of hESCs and other cell types.
  • quantitating gene expression of such marker genes is through the use of quantitative PCR (Q-PCR). Methods of performing Q-PCR are well known in the art.
  • compositions comprising the desired cell lineage that are substantially free of other cell types can be produced.
  • compositions comprising mixtures of hESCs and the desired cell lineage can be produced.
  • cells of the desired cell lineage can be isolated by using an affinity tag that is specific for such cells.
  • an affinity tag specific for a target cell is an antibody that is specific to a marker polypeptide that is present on the cell surface of the target cell but which is not substantially present on other cell types that would be found in a cell culture produced by the methods described herein.
  • the pluripotent cells can be passaged using enzymatic, non-enzymatic, or manually dissociation methods prior to and/or after contact with the defined medium of the invention.
  • Non-limiting examples of enzymatic dissociation methods include the use of proteases such as trypsin, collagenase, dispase, and accutase. In one embodiment, accutase is used to passage the contacted cells.
  • the resultant culture can comprise a mixture of singlets, doublets, triplets, and clumps of cells that vary in size depending on the enzyme used.
  • a non-limiting example of a non-enzymatic dissociation method is a cell dispersal buffer. Manual passaging techniques have been well described in the art, such as in Schulz et al, 2004, Stem Cells, 22(7): 1218-38. The choice of passaging method is influenced by the choice of extracellular matrix, and is easily determined by one of ordinary skill in the art.
  • the BGOIv hESC line is a karyotypically variant cell line, which exhibits stable karyotype containing specific trisomies (karyotype: 49, XXY,+12,+17).
  • Parent cultures were maintained as described previously (Schulz et al, 2003, BMC Neurosci., 4:27; Schulz et al, 2004, Stem Cells, 22(7): 1218-38; Rosier et al, 2004, Dev. Dynamics, 229:259-274; Brimble et al, 2004, Stem Cells Dev., 13:585-596).
  • the cells were grown in dishes coated with Matrigel or fibronectin, in conditioned media from MEFs (MEF-CM) comprising DMEM:F12 with 20% KSR, 8 ng/ml FGF2, 2 mM L-Glutamine, Ix non-essential amino acids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 0.1 mM ⁇ -mercaptoethanol (Sigma) with collagenase passaging.
  • MEFs MEFs
  • the defined culture media tested herein comprised DMEM/F12, 2 mM
  • LongR 3 -IGFl is a modified version of IGFl that has reduced affinity for IGFl binding proteins, some of which are expressed in hESCs.
  • the media further comprises trace elements such as selenium, ascorbic acid, amino acids, and a source of lipids.
  • trace elements such as selenium, ascorbic acid, amino acids, and a source of lipids.
  • Matrigel coated dishes were prepared by diluting Growth Factor Reduced BD matrigel matrix (BD Biosciences) to a final concentration of 1:30 in cold DMEM/F-12. 1 ml/35 mm dish was used to coat dishes for 1-2 hours at room temperature or at least overnight at 4°C. Plates were stored up to one week at 4°C. Matrigel solution was removed immediately before use.
  • parent cultures were plated into 6-well dishes for comparison of multiple conditions.
  • Cultures were either plated directly into the test conditions, or were plated with MEF-CM for one day to promote adherence, prior to changing the media to the test conditions. The cultures were assessed every day and graded based on morphological criteria 4 to 5 days after plating.
  • the grading scale of 1 to 5 involved examining the whole culture and assessing overall proportion of undifferentiated colonies, their relative size, and proportion of colonies or parts of colonies exhibiting obvious differentiation. Grade 5 indicates "ideal" cultures, with large undifferentiated colonies and negligible differentiation. Grade 4 indicates a very good culture, but with some obvious differentiation. Grade 3 indicates acceptable cultures, but with around half the colonies exhibiting obvious differentiation. Grade 2 cultures are predominantly differentiated, with occasional putative undifferentiated cells. Grade 1 cultures contain differentiated colonies or the cultures did not adhere or did not survive. Cultures that exhibited good expansion of undifferentiated cells were passaged to assess the generation of stable sublines in these semi-defined media.
  • BGOIv cells were cultured as described above. Specifically, BGOIv cells were plated to 6-well dishes that had been coated with matrigel. Cells were plated at a concentration such that well-defined and isolated colonies are visible after 4-5 days. The cells were cultured for one day in 20% KSR MEF-CM to ensure effective plating. After one day, the media was changed to the experimental conditions shown in Table 1.
  • the base media (1:1 DMEM/F12, 2 mM L-glutamine, Ix non-essential amino acids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 0.1 mM ⁇ - mercaptoethanol, 4 ng/ml FGF2) had been conditioned on MEFs for 24 hours.
  • the components that were added to this media to generate the experimental conditions were an additional 4 ng/ml FGF2 (8ng/ml final), and 20 ⁇ g/ml insulin, 50 ng/ml LongR 3 -IGFl, 100 ng/ml Activin A, and/or 0.2% BSA in the indicated combinations.
  • BGOIv cells were cultured as described above. Specifically, BGOIv cells were plated to 6-well dishes that had been coated with matrigel. The cells were plated directly in the experimental conditions indicated in Table 2. The base media for conditions 1 and 2 (1:1 DMEM/F12, 2 mM L-glutamine, Ix non-essential amino acids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 0.1 mM ⁇ -mercaptoethanol, 4 ng/ml FGF2, 10 ⁇ g/ml transferrin) had been conditioned on 3VEEFs for 24 hours, and an additional 4 ng/ml FGF2 (8 ng/ml final concentration) and 0.2% BSA were added prior to use.
  • the base media for conditions 1 and 2 (1:1 DMEM/F12, 2 mM L-glutamine, Ix non-essential amino acids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 0.1 mM ⁇ -mercap
  • Condition 1 included 20 ⁇ g/ml insulin and condition 2 included 50 ng/ml LongR 3 -IGFl.
  • the base media was not conditioned.
  • the components that were added to the base media were an additional 4 ng/ml FGF2 (8 ng/ml final), and 20 ⁇ g/ml insulin, 50 ng/ml LongR 3 -IGFl, 100 ng/ml Activin A, and/or 0.2% BSA in the indicated combinations.
  • Passaged cultures are indicated by thicker border in grading column and grading of pi cultures.
  • Table 3 indicates the titrations that were analyzed.
  • the base media comprised 1:1 DMEM/F12, 2 mM L-glutamine, Ix non-essential amino acids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 0.1 mM 2-Mecaptoethanol, 8 ng/ml FGF2, and
  • BSA human serum albumin
  • BMP4 0.1 ng/ml BMP4, but were not successfully passaged. Higher concentrations of BMP4, 1—10 ng/ml, caused differentiation.
  • LongR 3 -IGFl was titrated from approximately 50 to 500 ng/ml.
  • TGF- ⁇ was titrated from approximately 0.1 - 20 ng/ml. Cultures grown in all the conditions containing TGF- ⁇ were graded at 3 to 4. Overall, there were large undifferentiated regions within colonies, but cells in the colonies were not as tightly packed as expected. Some of these cultures were passaged, but the cells differentiated. [0090] Nodal was titrated from approximately 1 - 50 ng/ml. Cultures containing 2.5 or 5 ng/ml nodal were given a grading of 4, with higher and lower concentrations receiving a lower grading. Some of these cultures were passaged, but the cells differentiated. [0091] BSA was titrated from approximately 0.02 - 1%.
  • LongR 3 -IGFl all contained some colonies of undifferentiated cells, and were graded at 3 to 4.
  • a culture containing 1 ng/ml activin and 500 ng/ml LongR 3 -IGFl contained a higher level of differentiation and was grade 3.
  • Tissue culture dishes were coated with 10 ⁇ g/ml human plasma fibronectin (Invitrogen, #33016-015), diluted in tissue grade water, for 2-3 hours at room temperature. The dishes were washed with tissue culture grade water before use.
  • parental BGOIv cells were passaged to pi cultures with Accutase (Innovative Cell Technologies) according to the manufacturer's instructions.
  • Accutase comprised Ix accutase enzymes in PBS, containing 0.5 mM EDTA. These p0 cultures were graded at 4-5, and were successfully passaged to pi and subsequently p2 cultures with Accutase passaging.
  • the pi and p2 cultures were grown on human fibronectin in the presence of defined medium comprising 1:1 DMEM/F12, 2 mM L-glutamine, Ix non-essential amino acids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 0.1 mM ⁇ -mercaptoethanol, 8 ng/ml FGF2, 10 ⁇ g/ml transferrin, 0.2% BSA, 100 or 200 ng/ml LongR 3 -IGFl, and 1 ng/ml Activin A. These cultures were grade 4 at pi and grade 3 at p2.
  • the p2 cultures consisted almost entirely of morphologically undifferentiated cells in colonies, but appeared to have slower growth and smaller colonies than observed in p0 cultures, which accounts for the lower grading. If the slower growth rate was not considered, these p2 cultures were grade 4-5. [0097] 0.01% defined lipids (Invitrogen), 50 ⁇ g/ml ascorbic acid, and 0.0017 ⁇ g/ml sodium selenite were added to the p2 culture containing 100 ⁇ g/ml LongR 3 - IGFl, two days after plating, to assess if this caused obvious improvements in the growth rate. No obvious effect was observed after 2 days, and cultures were harvested for analysis 4 days after plating at p2.
  • RNA was prepared from a p2 culture containing 100 ⁇ g/ml LongR 3 - IGFl and was analyzed by RT-PCR as described previously (Brimble et ah, 2004 Stem Cells Dev. 13:585-596). These p2 cultures expressed multiple markers of pluripotency, including ABCG2, DPPA5, REXl, UTFl, NANOG, OCT4, SOX2, CRIPTO, and TERT ( Figure 4A). [0099] A p2 culture containing 200 ⁇ g/ml LongR 3 -IGFl was fixed in 4% paraformaldehyde (PFA) (Fisher Scientific, Hampton, NH) and 4% sucrose (Sigma) in Ix PBS.
  • PFA paraformaldehyde
  • the samples was blocked with 3% goat serum (Invitrogen), 1% polyvinyl pyrolidone (Sigma), and 0.3% Triton X-100 (Sigma) in Ix PBS (block buffer) and then incubated with primary antibodies diluted in block buffer for 1 hour at room temperature.
  • the primary antibodies were mouse anti-SSEA-4 (Chemicon) and rabbit anti-OCT4 (Santa Cruz).
  • the sample was then washed and incubated for 1 hour in secondary antibodies diluted 1:1,000 in block buffer, followed by washing.
  • the secondary antibodies were goat anti-rabbit antibody conjugated with alexa594 (red), and goat anti-mouse antibody conjugated with Alexa-488 (green), (Molecular Probes).
  • BGOIv cells were cultured as described above, with minor changes.
  • BGOIv cells were plated to 6-well dishes that had been coated with fibronectin. Tissue culture dishes were coated with 10 ⁇ g/ml human plasma fibronectin (Invitrogen, #33016-015), diluted in tissue grade water, for 2-3 hours at room temperature. The dishes were washed with tissue culture grade water before use. [00103] The cells were cultured directly in the defined media conditions, or in
  • the defined media comprised approximately 1:1 DMEM/F12, 2 mM L-glutamine, Ix non-essential amino acids, 0.5 U/ml penicillin, 0.5 U/ml streptomycin, 0.1 mM ⁇ -Mercaptoethanol, 10 ⁇ g/ml transferrin, 8 ng/ml FGF2, 50 ng/ml LongR 3 -IGFl and 1 ng/ml Activin A.
  • the concentrations of different lipid sources were titrated within the defined media, in order to identify the optimal concentrations of lipids. Ex-Cyte lipids and Invitrogen defined lipids were titrated ranging from approximately 0.01% to approximately 1%.
  • BGOIv cells expanded effectively in defined media containing lipids, forming large colonies of undifferentiated cells, with low levels of differentiation. These cultures were grades 4 to 5. The pO cultures were successfully passaged using accutase to pi cultures as described above. The morphological gradings of the pi cultures are shown below in Tables 4-5.
  • Tables 4 and 5 indicate the titrations analyzed, and the graded morphology of the pi cultures.
  • the addition of lipids results in changes in the morphology of the colonies: they are more three-dimensional, although the cells still grow as a single layer within the colony.
  • the colonies are thicker in the presence of the lipids, and refract the light more than colonies cultured without lipids.
  • Pl cultures are successfully passaged to further cultures in the presence of defined media containing lipids at a concentration of approximately 0.0001% to approximately 1%, without losing the characteristics of pluripotent cells.

Abstract

La présente invention concerne des compositions et des méthodes pour la culture et la maintenance de cellules souches de mammifères pluripotentes indifférenciées. Plus particulièrement, la présente invention concerne un milieu défini utile en l’absence de couche nourricière et de sérum ou de sérum de remplacement, qui contient un activateur de récepteur IGF-IR, un activateur de récepteur FGF, de la sérumalbumine, de la transferrine et, éventuellement, un membre de la famille TGF-β.
PCT/US2006/024168 2005-06-20 2006-06-20 Compositions de culture de cellules souches embryonnaires et methodes d’utilisation de celles-ci WO2007002210A2 (fr)

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