Improved reprogramming methods and cell culture platforms

Abstract

To provide improved reprogramming methods and cell culture platforms.SOLUTION: The present invention provides compositions and methods for manufacturing pluripotent cells. Particularly, the invention provides improved culture platforms for manufacturing pluripotent cells with ground state pluripotency. In various embodiments, the invention contemplates, in part, a composition comprising: (a) a Wnt pathway agonist; (b) a MEK inhibitor; and (c) a ROCK inhibitor. In certain embodiments, the composition further comprises bFGF or LIF.SELECTED DRAWING: Figure 1E

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 61 / 947,979, filed March 4, 2014, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Description The sequence listing associated with this application is provided in text form in lieu of paper and is incorporated herein by reference. The name of the text file containing the sequence listing is FATE_122_01WO_ST25.txt. The text file is 8 KB, was created on March 4, 2015, and was submitted electronically via EFS-Web simultaneously with the filing of this application. background

[0003] The present invention relates generally to compositions and methods for producing pluripotent cells. In particular, the present invention relates to improved culture substrates for producing pluripotent cells with ground state pluripotency. [Background technology]

[0004] Today's pluripotent stem cell-based disease and toxicology screening studies, as well as future autologous and allogeneic pluripotent stem cell therapies, require robust and reproducible methods for cell line generation and expansion of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). hiPSCs have been generated by ectopic expression of pluripotency factors introduced via genomically integrated retroviral and lentiviral expression systems. Efforts to eliminate as many integration events as possible have included replacing some reprogramming factors with small molecule inhibitors. Furthermore, non-integrative methods have proven inefficient and labor-intensive, require additional reprogramming factors, or are not effective at reprogramming all somatic cells (Lee et al., 2013).

[0005] Several challenges associated with pluripotent stem cell culture remain to be addressed if the cells are to be suitable for future industrial and clinical applications. In the most commonly used conventional culture systems, hESCs and hiPSCs are maintained on feeder cells and passaged as clumps to prevent extensive cell death and genomic aberrations (Thomson et al., 1998). The inability to culture hiPSCs in a feeder-free (FF) environment significantly limits their potential industrial-scale screening or cell therapy applications (Skottman et al., 2007; Valamehr et al., 2011). Furthermore, recent studies on improving hiPSCs have focused on lentivirus-induced hiPSCs, which are not transgene-free, limiting the therapeutic relevance of such studies.

[0006] Another challenge that remains to be successfully addressed besides genome modification is the tendency of human pluripotent stem cells to spontaneously differentiate in culture ( Pera and Trounson, 2004 ; Sathananthan and Trounson, 2005 ; Valamehr et al., 2011 ).

[0007] Although work in hESCs and hiPSCs has been described, maintaining the ground state resulting in genome-modified human pluripotent stem cells requires continuous ectopic expression of pluripotency genes ( Hanna et al., 2010a ), which is inappropriate for industrial and clinical grade pluripotent cells.

[0008] Thus, the lack of compositions and methods for high-throughput, transgene- or footprint-free generation of human pluripotent cell products has proven to be a major obstacle to the development and commercialization of future pluripotent stem cell therapies. Summary of the Invention [Means for solving the problem]

[0009] The present invention generally provides an improved cell culture substrate.

[0010] In various embodiments, the present invention contemplates, in part, a composition comprising: (a) a Wnt pathway agonist; (b) a MEK inhibitor; and (c) a ROCK inhibitor, but not a TGFβR inhibitor.

[0011] In a specific embodiment, the Wnt pathway agonist is a GSK3 inhibitor.

[0012] In certain embodiments, the GSK3 inhibitor is CHIR99021 or BIO.

[0013] In additional embodiments, the MEK inhibitor is PD98059 or PD032901.

[0014] In a further embodiment, the ROCK inhibitor is thiazovivin or Y27632.

[0015] In some embodiments, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD032901, and the ROCK inhibitor is thiazovivin.

[0016] In certain embodiments, any of the above compositions further comprises bFGF or LIF.

[0017] In further embodiments, any of the above compositions further comprise bFGF and LIF.

[0018] In various embodiments, a culture medium comprising any of the above compositions is provided that does not contain a TGFβR inhibitor.

[0019] In some embodiments, a method of culturing one or more pluripotent cells, comprising culturing the one or more pluripotent cells in a cell culture medium according to any of the culture media described above.

[0020] In additional embodiments, the one or more pluripotent cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0021] In a specific embodiment, the one or more pluripotent cells are iPSCs.

[0022] In certain embodiments, the composition comprises a population of pluripotent cells.

[0023] In a further embodiment, the population of pluripotent cells is a homogenous population of pluripotent cells.

[0024] In a specific embodiment, at least 95% of the population of pluripotent cells express SSEA4-FITC and TRA1-81 or TRA1-60.

[0025] In some embodiments, up to 5% of the population of pluripotent cells express alpha-smooth muscle actin (SMA), TUJ1, or FoxA2.

[0026] In a specific embodiment, the pluripotent cells have been pre-cultured in a cell culture medium containing a TGFβR inhibitor.

[0027] In additional embodiments, culturing the pluripotent cells in cell culture medium reduces spontaneous differentiation of the cultured cells.

[0028] In one embodiment, the expression of one or more differentiation marker genes in the cultured cells is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the expression of one or more differentiation marker genes in pluripotent cells cultured in medium containing a TGFβR inhibitor, and the differentiation marker genes include FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3 , GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury) and ZIC1.

[0029] In another embodiment, the one or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1.

[0030] In yet another embodiment, in the cultured cells, expression of two or more differentiation marker genes is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to expression of two or more differentiation marker genes in pluripotent cells cultured in a medium containing a TGFβR inhibitor, and the differentiation marker genes are selected from the group consisting of FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GA TA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury) and ZIC1.

[0031] In yet another embodiment, the two or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1.

[0032] In a specific embodiment, in the cultured cells, expression of three or more differentiation marker genes is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to expression of three or more differentiation marker genes in pluripotent cells cultured in medium containing a TGFβR inhibitor, and the differentiation marker genes are selected from the group consisting of FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GAT A3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC , PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D It is selected from the group consisting of FOXG1, LEFTY1, TUJ1, T gene (Brachyury) and ZIC1.

[0033] In certain embodiments, the three or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1.

[0034] In additional embodiments, the expression of five or more differentiation marker genes in the cultured cells is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the expression of the five or more differentiation marker genes in pluripotent cells cultured in medium containing a TGFβR inhibitor, and the differentiation marker genes are FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GAT A3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC , PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D It is selected from the group consisting of FOXG1, LEFTY1, TUJ1, T gene (Brachyury) and ZIC1.

[0035] In a further embodiment, the five or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1.

[0036] In certain embodiments, culturing pluripotent cells in cell culture medium maintains or induces a ground state of pluripotency.

[0037] In a specific embodiment, the ground state of pluripotency of one or more pluripotent cells is maintained for at least five passages.

[0038] In certain specific embodiments, the ground state of pluripotency of one or more pluripotent cells is maintained for at least 10 passages.

[0039] In a further specific embodiment, the ground state of pluripotency of one or more pluripotent cells is maintained for at least 50 passages.

[0040] In additional specific embodiments, the ground state of pluripotency of one or more pluripotent cells is maintained for at least 100 passages.

[0041] In various embodiments, the method further comprises dissociating one or more pluripotent cells during passaging.

[0042] In certain embodiments, the viability of one or more pluripotent cells is maintained during passaging.

[0043] In certain specific embodiments, the one or more pluripotent cells comprise a normal karyotype.

[0044] In certain additional embodiments, the one or more pluripotent cells are cultured in a feeder-free environment.

[0045] In certain further embodiments, the genomic stability of the one or more pluripotent cells is maintained for at least 10 passages.

[0046] In certain related embodiments, the genomic stability of the one or more pluripotent cells is maintained for at least 50 passages.

[0047] In certain other embodiments, the genomic stability of the one or more pluripotent cells is maintained for at least 100 passages.

[0048] In various embodiments, the present invention contemplates, in part, a method of adapting pluripotent cells to feeder-free culture, the method comprising: (a) isolating one or more pluripotent cells that are cultured in the presence of feeder cells; and (b) culturing the one or more pluripotent cells in a chemically defined cell culture medium comprising a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a ROCK inhibitor, and no TGFβR inhibitor.

[0049] In various specific embodiments, the present invention contemplates, in part, a method of culturing pluripotent cells that have been enzymatically passaged as single cells, the method comprising: (a) enzymatically treating one or more pluripotent cells to passage the single pluripotent cells; (b) culturing the single pluripotent cells in a feeder-free environment; and (c) culturing the single pluripotent cells in a chemically defined cell culture medium comprising a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a ROCK inhibitor, and no TGFβR inhibitor.

[0050] In various certain embodiments, the present invention contemplates, in part, a method of reducing spontaneous differentiation of one or more pluripotent cells, the method comprising: (a) culturing the one or more pluripotent cells in a feeder-free environment; and (b) culturing the one or more pluripotent cells in a chemically defined cell culture medium comprising a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a ROCK inhibitor, and no TGFβR inhibitor.

[0051] In various additional embodiments, the invention contemplates, in part, a method of producing induced pluripotent stem cells (iPSCs), the method comprising: (a) obtaining one or more non-pluripotent cells; (b) reprogramming the one or more non-pluripotent cells to a pluripotent state; and (c) culturing the pluripotent cells in cell culture medium that does not contain a TGFβR inhibitor, thereby generating iPSCs.

[0052] In a specific embodiment, the one or more non-pluripotent cells comprise somatic cells.

[0053] In some embodiments, the one or more non-pluripotent cells comprise adult stem cells.

[0054] In certain embodiments, one or more non-pluripotent cells are reprogrammed to a pluripotent state by increasing the expression of endogenous OCT4 in the cells.

[0055] In a further embodiment, reprogramming one or more non-pluripotent cells to a pluripotent state comprises introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more reprogramming factors selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, and SV40LT.

[0056] In additional embodiments, reprogramming one or more non-pluripotent cells to a pluripotent state comprises introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, and SV40LT.

[0057] In certain embodiments, reprogramming one or more non-pluripotent cells to a pluripotent state comprises introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, SOX2, and NANOG.

[0058] In certain embodiments, reprogramming one or more non-pluripotent cells to a pluripotent state comprises introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, NANOG, ECAT1, UTF1, and ESRRB.

[0059] In certain embodiments, reprogramming one or more non-pluripotent cells to a pluripotent state comprises introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, ECAT1, and UTF1.

[0060] In a specific embodiment, the one or more polynucleotides is a lentiviral vector.

[0061] In some embodiments, the one or more polynucleotides are episomal vectors.

[0062] In a related specific embodiment, the cell culture medium comprises a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a ROCK inhibitor.

[0063] In further specific embodiments, reprogramming the one or more non-pluripotent cells comprises contacting the one or more non-pluripotent cells with a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a TGFβR inhibitor, and optionally a ROCK inhibitor.

[0064] In additional embodiments, the iPSCs comprise a population of iPSCs.

[0065] In a specific embodiment, the population of iPSCs is a homogenous population of iPSCs.

[0066] In a specific embodiment, at least 95% of the population of iPSCs express SSEA4 and TRA1-81 or TRA1-60.

[0067] In certain embodiments, up to 5% of the population of pluripotent cells express alpha-smooth muscle actin (SMA), TUJ1, or FoxA2.

[0068] In certain embodiments, culturing pluripotent cells in cell culture medium reduces spontaneous differentiation or maintains or induces a ground state of pluripotency.

[0069] In additional embodiments, the expression of one or more, two or more, three or more, four or more, or five or more differentiation marker genes is reduced in the iPSCs by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the expression of one or more differentiation marker genes in iPSCs cultured in medium comprising a TGFβR inhibitor, the differentiation marker genes being selected from the group consisting of FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, C The gene is selected from the group consisting of YP2B6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury), and ZIC1.

[0070] In specific embodiments, the one or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1.

[0071] In certain embodiments, the reduction in spontaneous differentiation is maintained for at least 5 passages.

[0072] In other embodiments, the reduction in spontaneous differentiation is maintained for at least 10 passages.

[0073] In a specific embodiment, the reduction in spontaneous differentiation is maintained for at least 50 passages.

[0074] In additional embodiments, the reduction in spontaneous differentiation is maintained for at least 100 passages.

[0075] In a further embodiment, the method comprises dissociating the iPSCs during passaging.

[0076] In additional embodiments, the viability of the iPSCs is maintained during passaging.

[0077] In certain embodiments, the iPSCs comprise a normal karyotype.

[0078] In other embodiments, the iPSCs are cultured in a feeder-free environment.

[0079] In a specific embodiment, the genomic stability of the iPSCs is maintained for at least 10 passages.

[0080] In additional embodiments, the genomic stability of the iPSCs is maintained for at least 50 passages.

[0081] In additional specific embodiments, the genomic stability of the iPSCs is maintained for at least 100 passages.

[0082] In various embodiments, the invention provides, in part, induced pluripotent stem cells (iPSCs) comprising ground state pluripotency produced according to any one of the above embodiments.

[0083] In various certain embodiments, the present invention provides, in part, induced pluripotent stem cells (iPSCs) that comprise ground state pluripotency, wherein the iPSCs do not contain exogenously introduced polynucleotides encoding reprogramming factor polypeptides.

[0084] In various specific embodiments, the invention provides, in part, methods for producing induced pluripotent stem cells (iPSCs), the methods comprising: (a) obtaining one or more pluripotent stem cells; and (b) culturing the one or more pluripotent stem cells in a cell culture medium that does not contain a TGFβR inhibitor, thereby generating ground state iPSCs.

[0085] In certain embodiments, the one or more iPSCs comprise reprogrammed somatic cells.

[0086] In additional embodiments, the one or more iPSCs comprise reprogrammed adult stem cells.

[0087] In other embodiments, one or more iPSCs are reprogrammed to a pluripotent state by increasing the expression of endogenous OCT4 in the one or more iPSCs.

[0088] In a specific embodiment, the one or more iPSCs have been reprogrammed by introducing one or more polynucleotides encoding one or more reprogramming factors selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, and SV40LT into one or more non-pluripotent cells.

[0089] In certain specific embodiments, one or more iPSCs have been reprogrammed by introducing one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, and SV40LT.

[0090] In additional embodiments, the one or more iPSCs have been reprogrammed by introducing one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, SOX2, and NANOG into one or more non-pluripotent cells.

[0091] In additional embodiments, the one or more iPSCs have been reprogrammed by introducing one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, NANOG, ECAT1, UTF1, and ESRRB into one or more non-pluripotent cells.

[0092] In another embodiment, one or more iPSCs have been reprogrammed by introducing one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, ECAT1, and UTF1 into one or more non-pluripotent cells.

[0093] In various embodiments, the lentiviral vector comprises one or more polynucleotides.

[0094] In a specific embodiment, the episomal vector comprises one or more polynucleotides.

[0095] In other embodiments, the cell culture medium comprises a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a ROCK inhibitor.

[0096] In certain embodiments, obtaining one or more iPSCs comprises contacting one or more non-pluripotent or partially pluripotent cells with a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a TGFβR inhibitor, and optionally a ROCK inhibitor, to generate one or more iPSCs.

[0097] In additional embodiments, the iPSCs comprise a population of iPSCs.

[0098] In additional embodiments, the population of iPSCs is a homogenous population of iPSCs.

[0099] In a specific embodiment, at least 95% of the population of iPSCs express SSEA4 and TRA1-81 or TRA1-60.

[0100] In a specific embodiment, the one or more iPSCs are obtained by reprogramming a population of pluripotent cells, wherein up to 5% of the population of pluripotent cells express alpha-smooth muscle actin (SMA), TUJ1, or FoxA2.

[0101] In a further embodiment, the method comprises culturing one or more iPSCs in a cell culture medium that reduces spontaneous differentiation or maintains or induces a ground state of pluripotency.

[0102] In certain embodiments, iPSCs with reduced spontaneous differentiation include those in which the expression of one or more, two or more, three or more, four or more, or five or more differentiation marker genes is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the expression of one or more differentiation marker genes in iPSCs cultured in medium containing a TGFβR inhibitor, and the differentiation marker genes include FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0 B1, CXCR4, CYP2B6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury) and ZIC1.

[0103] In additional embodiments, the one or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1.

[0104] In other embodiments, the reduced spontaneous differentiation is maintained for at least 5 passages.

[0105] In certain embodiments, the reduced spontaneous differentiation is maintained for at least 10 passages.

[0106] In additional embodiments, the reduced spontaneous differentiation is maintained for at least 50 passages.

[0107] In a specific embodiment, the reduced spontaneous differentiation is maintained for at least 100 passages.

[0108] In a further embodiment, the method comprises dissociating one or more iPSCs during passaging.

[0109] In a specific embodiment, the viability of one or more iPSCs is maintained during passaging.

[0110] In other embodiments, the one or more iPSCs comprise a normal karyotype.

[0111] In additional embodiments, one or more iPSCs are cultured in a feeder-free environment.

[0112] In certain embodiments, the genomic stability of one or more iPSCs is maintained for at least 10 passages.

[0113] In additional embodiments, the genomic stability of the one or more iPSCs is maintained for at least 50 passages.

[0114] In a specific embodiment, the genomic stability of one or more iPSCs is maintained for at least 100 passages.

[0115] In various specific embodiments, the present invention provides, in part, induced pluripotent stem cells (iPSCs) comprising ground state pluripotency produced according to any one of the above embodiments.

[0116] In various embodiments, the present invention provides, in part, induced pluripotent stem cells (iPSCs) that comprise ground state pluripotency, wherein the iPSCs do not contain exogenously introduced polynucleotides encoding reprogramming factor polypeptides.

[0117] In some embodiments, there is provided a method for reprogramming a non-pluripotent cell into a pluripotent cell, the method comprising introducing into the non-pluripotent cell one or more polynucleotides encoding (i) at least one polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) at least one polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, thereby reprogramming the non-pluripotent cell into a pluripotent cell.

[0118] In specific embodiments, the introducing includes (i) introducing one or more polynucleotides encoding an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) introducing an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide.

[0119] In another embodiment, the introducing comprises (i) introducing one or more polynucleotides encoding an OCT4 polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide, or (ii) introducing an OCT-4 polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide.

[0120] In some embodiments, the one or more polynucleotides are introduced by retrovirus, Sendai virus, adenovirus, episome, minicircle, vector system with an expression cassette, or mRNA.

[0121] In a specific embodiment, the retrovirus is a lentivirus.

[0122] In another embodiment, the pluripotent cells do not comprise an exogenous polynucleotide.

[0123] In another embodiment, the one or more polynucleotides are cleaved by CRE-mediated cleavage.

[0124] In some embodiments, the method further comprises introducing into the non-pluripotent cell (i) a polynucleotide encoding an SV40LT polypeptide, or (ii) an SV40LT polypeptide.

[0125] In one embodiment, the method comprises contacting the non-pluripotent cell with at least one of a TGFβR inhibitor, a Wnt pathway agonist, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway agonist is optionally a GSK3 inhibitor.

[0126] In another embodiment, the non-pluripotent cells are contacted with a TGFβR inhibitor, a Wnt pathway agonist, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway agonist is optionally a GSK3 inhibitor.

[0127] In a specific embodiment, the method further comprises culturing the pluripotent cells in a culture medium comprising a Wnt pathway agonist, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway agonist is optionally a GSK3 inhibitor, and the culture medium does not contain a TGFβR inhibitor.

[0128] In some embodiments, the Rock inhibitor is thiazovivin or Y27632, the TGFβR inhibitor is A-83-01 or SB431542, the GSK3 inhibitor is CHIR99021 or BIO, or the MEK inhibitor is PD98059 or PD032901.

[0129] In another embodiment, the pluripotency of the pluripotent cells is maintained for at least 5 cell divisions or at least 10 cell divisions.

[0130] In a specific embodiment, the one or more polynucleotides are introduced as a polycistronic vector comprising multiple polynucleotides separated by at least one 2A peptide.

[0131] In one embodiment, the polycistronic vector comprises multiple polynucleotides encoding OCT4 polypeptides.

[0132] In some embodiments, the methods include identifying pluripotent cells by selecting for OCT4 expression in the pluripotent cells.

[0133] In one embodiment, selecting for OCT4 expression comprises selecting for ectopic Oct-4 expression.

[0134] In another embodiment, the culturing generates a population of pluripotent stem cells.

[0135] In another embodiment, the population of pluripotent stem cells is at least 70% homogeneous, at least 80% homogeneous, or at least 90% homogeneous.

[0136] In yet another embodiment, at least 70%, at least 80%, or at least 90% of the population of pluripotent cells express SSEA and Tra-181.

[0137] In certain embodiments, the pluripotent cells or population of pluripotent cells are capable of single cell passage.

[0138] In one embodiment, the cells generated by single cell passaging have a normal karyotype.

[0139] In one embodiment, the invention provides a pluripotent cell produced according to any one of the above methods.

[0140] In another embodiment, the present invention provides a composition comprising an isolated non-pluripotent cell comprising (i) one or more exogenous polynucleotides encoding at least one polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) at least one exogenous polypeptide selected from an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide.

[0141] In one embodiment, the cell comprises (i) one or more exogenous polynucleotides encoding at least two of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) at least two exogenous polypeptides selected from an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide.

[0142] In another embodiment, the cell comprises (i) one or more exogenous polynucleotides encoding at least three of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) at least three exogenous polypeptides selected from an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide.

[0143] In one specific embodiment, the cell comprises (i) one or more exogenous polynucleotides encoding an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) an exogenous OCT4 polypeptide, an exogenous ECAT1 polypeptide, an exogenous UTF1 polypeptide, an exogenous NANOG polypeptide, and an exogenous ESRRB polypeptide.

[0144] In another embodiment, the cell comprises one or more exogenous polynucleotides encoding an OCT4 polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide, or an exogenous OCT4 polypeptide, an exogenous ECAT1 polypeptide, and an exogenous UTF1 polypeptide.

[0145] In one embodiment, the cells are contacted with at least one of a TGFβR inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor.

[0146] In another embodiment, the cells are contacted with a TGFβR inhibitor, a Wnt pathway activator, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway activator is optionally a GSK3 inhibitor.

[0147] In specific embodiments, the Rock inhibitor is thiazovivin or Y27632, the TGFβR inhibitor is A-83-01 or SB431542, the GSK3 inhibitor is CHIR99021 or BIO, or the MEK inhibitor is PD98059 or PD032901.

[0148] In another embodiment, the one or more exogenous polynucleotides are introduced into the non-pluripotent cells via a retrovirus, Sendai virus, adenovirus, episome, minicircle, vector system carrying an expression cassette, or mRNA.

[0149] In another embodiment, the retrovirus is a lentivirus.

[0150] In a specific embodiment, the cells do not comprise an exogenous polynucleotide.

[0151] In certain embodiments, the one or more exogenous polynucleotides are removed by CRE-mediated cleavage.

[0152] In another embodiment, the cell comprises an exogenous polynucleotide encoding an SV40LT antigen polypeptide or an exogenous SV40LT antigen polypeptide.

[0153] In one embodiment, the one or more exogenous polynucleotides are introduced as a polycistronic vector comprising multiple polynucleotides separated by at least one 2A peptide.

[0154] In another embodiment, the polycistronic vector comprises multiple polynucleotides encoding OCT4 polypeptides.

[0155] In certain embodiments, the exogenous polynucleotide encoding an OCT4 polypeptide is linked to a selectable marker.

[0156] In one specific embodiment, the present invention provides a composition consisting of at least one, at least two, or at least three of: (i) a cDNA encoding an OCT4 polypeptide, (ii) a cDNA encoding an ECAT1 polypeptide, (iii) a cDNA encoding a UTF1 polypeptide, (iv) a cDNA encoding a NANOG polypeptide, and (v) a cDNA encoding an ESRRB polypeptide.

[0157] In one embodiment, the composition consists of a cDNA encoding an OCT4 polypeptide, a cDNA encoding an ECAT1 polypeptide, a cDNA encoding a UTF1 polypeptide, a cDNA encoding a NANOG polypeptide, and a cDNA encoding an ESRRB polypeptide.

[0158] In another embodiment, the composition consists of a cDNA encoding an OCT4 polypeptide, a cDNA encoding an ECAT1 polypeptide, and a cDNA encoding a UTF1 polypeptide.

[0159] In specific embodiments, each cDNA is encoded by a retrovirus, Sendai virus, adenovirus, episome, minicircle, vector system with an expression cassette, or mRNA.

[0160] In one embodiment, the present invention provides a vector comprising one or more polynucleotides encoding at least one reprogramming factor polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide.

[0161] In certain embodiments, the one or more polynucleotides encode an OCT polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide.

[0162] In another embodiment, the one or more polynucleotides encode an OCT polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide.

[0163] In another specific embodiment, the vector further comprises a polynucleotide encoding an SV40LT antigen polypeptide.

[0164] In one embodiment, the vector is a retrovirus, Sendai virus, adenovirus, episome, minicircle, vector system with an expression cassette, or mRNA.

[0165] In one embodiment, the retrovirus is a lentivirus.

[0166] In one specific embodiment, the present invention provides a kit for reprogramming non-pluripotent cells into pluripotent cells, comprising one or more polynucleotides encoding at least one polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, or an ESRRB polypeptide; or at least one polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, or an ESRRB polypeptide; and at least one of a TGFβR inhibitor, a Wnt pathway activator, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway activator is optionally a GSK3 inhibitor.

[0167] In one embodiment, the one or more polynucleotides encode an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or at least one polypeptide comprises an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide.

[0168] In another embodiment, one or more of the one or more polynucleotides encode an OCT4 polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide, or at least one polypeptide comprises an OCT4 polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide.

[0169] In one embodiment, the kit comprises a polynucleotide encoding an SV40LT antigen polypeptide or an SV40LT antigen polypeptide.

[0170] In another embodiment, the kit comprises a TGFβR inhibitor, a Wnt pathway activator, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway activator is optionally a GSK3 inhibitor.

[0171] In one embodiment, the Rock inhibitor is thiazovivin or Y27632, the TGFβR inhibitor is A-83-01 or SB431542, the GSK3 inhibitor is CHIR99021 or BIO, or the MEK inhibitor is PD98059 or PD032901.

[0172] In another embodiment, the at least one polynucleotide is encoded by a retrovirus, Sendai virus, adenovirus, episome, minicircle, vector system comprising an expression cassette, or mRNA.

[0173] In another embodiment, the retrovirus is a lentivirus.

[0174] In one embodiment, at least one polynucleotide is encoded by a polycistronic vector, with each polynucleotide separated by a 2A peptide.

[0175] In yet another embodiment, the polycistronic vector comprises two or more polynucleotides encoding an OCT4 polypeptide.

[0176] In a specific embodiment, at least one polynucleotide encoding an OCT4 polypeptide is linked to a selectable marker.

[0177] In another specific embodiment, the present invention provides a method for generating a population of pluripotent stem cells, the method comprising: providing a population of non-pluripotent cells; introducing into the population of non-pluripotent cells a polynucleotide encoding an OCT4 polypeptide linked to a selectable marker; incubating the population of non-pluripotent cells with the polynucleotide under conditions sufficient to reprogram at least a portion of the population of non-pluripotent cells into pluripotent cells; and selecting for cells that express the selectable marker, thereby providing a population of pluripotent stem cells.

[0178] In another embodiment, the polynucleotide is introduced as a polycistronic vector comprising multiple polynucleotides encoding OCT4 polypeptides.

[0179] In one embodiment, the multiple polynucleotides are separated by at least one 2A peptide.

[0180] In yet another embodiment, at least 10% of the cells in the population of cells express SSEA and TRA-181. In certain embodiments, for example, the following are provided: (Item 1) (a) Wnt pathway agonists and; (b) MEK inhibitor and; (c) a ROCK inhibitor, The composition does not contain a TGFβR inhibitor. (Item 2) 2. The composition of claim 1, wherein the Wnt pathway agonist is a GSK3 inhibitor. (Item 3) 3. The composition of claim 2, wherein the GSK3 inhibitor is CHIR99021 or BIO. (Item 4) 2. The composition of claim 1, wherein the MEK inhibitor is PD98059 or PD032901. (Item 5) 2. The composition of claim 1, wherein the ROCK inhibitor is thiazovivin or Y27632. (Item 6) 2. The composition of claim 1, wherein the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD032901, and the ROCK inhibitor is thiazovivin. (Item 7) 7. The composition of any one of items 1 to 6, further comprising bFGF or LIF. (Item 8) The composition according to any one of items 1 to 6, further comprising bFGF and LIF. The composition according to item 1, Culture medium without TGFβR inhibitors. (Item 10) 10. A method for culturing one or more pluripotent cells, comprising culturing the one or more pluripotent cells in the cell culture medium of item 9. (Item 11) 11. The method of claim 10, wherein the one or more pluripotent cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). (Item 12) 11. The method of claim 10, wherein the one or more pluripotent cells are iPSCs. (Item 13) 11. The method of claim 10, wherein the composition comprises a population of pluripotent cells. (Item 14) Item 15. The method of item 13, wherein the population of pluripotent cells is a homogenous population of pluripotent cells. 14. The method of claim 13, wherein at least 95% of the population of pluripotent cells expresses SSEA4-FITC and TRA1-81 or TRA1-60. (Item 16) 14. The method of claim 13, wherein up to 5% of the population of pluripotent cells express alpha-smooth muscle actin (SMA), TUJ1, or FoxA2. (Item 17) 11. The method of claim 10, wherein the pluripotent cells are pre-cultured in a cell culture medium containing a TGFβR inhibitor. (Item 18) 14. The method of claim 13, wherein culturing the pluripotent cells in the cell culture medium reduces spontaneous differentiation of the cultured cells. (Item 19) The expression of one or more differentiation marker genes in the cultured cells is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the expression of one or more differentiation marker genes in pluripotent cells cultured in a medium containing a TGFβR inhibitor, and the differentiation marker genes include FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3, GATA4, GATA5, GATA6, GATA7, GATA8, GATA9, GATA10, GATA11, GATA12, GATA13, GATA14, GATA15, GATA16, GATA17, GATA18, GATA19, GATA19, GATA16, GATA19 ... 19. The method of item 18, wherein the gene is selected from the group consisting of TA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury), and ZIC1. (Item 20) 20. The method of claim 19, wherein the one or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1. (Item 21) In the cultured cells, expression of two or more differentiation marker genes is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to expression of two or more differentiation marker genes in pluripotent cells cultured in a medium containing a TGFβR inhibitor, and the differentiation marker genes are selected from the group consisting of FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3, and G 19. The method of item 18, wherein the gene is selected from the group consisting of ATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury), and ZIC1. (Item 22) 22. The method of claim 21, wherein the two or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1. (Item 23) In the cultured cells, expression of three or more differentiation marker genes is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to expression of three or more differentiation marker genes in pluripotent cells cultured in a medium containing a TGFβR inhibitor, and the differentiation marker genes are FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3, G 19. The method of item 18, wherein the gene is selected from the group consisting of ATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury), and ZIC1. (Item 24) 24. The method of claim 23, wherein the three or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1. (Item 25) In the cultured cells, expression of five or more differentiation marker genes is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to expression of five or more differentiation marker genes in pluripotent cells cultured in a medium containing a TGFβR inhibitor, and the differentiation marker genes are FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3, G 19. The method of item 18, wherein the gene is selected from the group consisting of ATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury), and ZIC1. (Item 26) 26. The method of item 25, wherein the five or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1. (Item 27) 20. The method of claim 18, wherein culturing the pluripotent cells in the cell culture medium maintains or induces a ground state of pluripotency. (Item 28) 28. The method of claim 27, wherein the ground state of pluripotency of the one or more pluripotent cells is maintained for at least five passages. (Item 29) 28. The method of claim 27, wherein the ground state of pluripotency of the one or more pluripotent cells is maintained for at least 10 passages. (Item 30) 28. The method of claim 27, wherein the ground state of pluripotency of the one or more pluripotent cells is maintained for at least 50 passages. (Item 31) 28. The method of claim 27, wherein the ground state of pluripotency of the one or more pluripotent cells is maintained for at least 100 passages. (Item 32) 32. The method of any one of paragraphs 10 to 31, further comprising dissociating the one or more pluripotent cells during passaging. (Item 33) 33. The method of claim 32, wherein the viability of the one or more pluripotent cells is maintained during passaging. (Item 34) 34. The method of any one of paragraphs 10 to 33, wherein the one or more pluripotent cells comprise a normal karyotype. (Item 35) 35. The method of any one of paragraphs 10 to 34, wherein the one or more pluripotent cells are cultured in a feeder-free environment. (Item 36) 36. The method of any one of paragraphs 10 to 35, wherein the genomic stability of the one or more pluripotent cells is maintained for at least 10 passages. (Item 37) 36. The method of any one of paragraphs 10 to 35, wherein the genomic stability of the one or more pluripotent cells is maintained for at least 50 passages. (Item 38) 36. The method of any one of paragraphs 10 to 35, wherein the genomic stability of the one or more pluripotent cells is maintained for at least 100 passages. (Item 39) 1. A method for adapting pluripotent cells to feeder-free culture, comprising: (a) isolating one or more pluripotent cells that are cultured in the presence of feeder cells; (b) culturing the one or more pluripotent cells in a chemically defined cell culture medium containing a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a ROCK inhibitor, but not containing a TGFβR inhibitor. 1. A method of culturing pluripotent cells that have been enzymatically passaged as single cells, comprising: (a) enzymatically treating one or more pluripotent cells to passage a single pluripotent cell; (b) culturing said single pluripotent cell in a feeder-free environment; (c) culturing the single pluripotent cell in a chemically defined cell culture medium comprising a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a ROCK inhibitor, in the absence of a TGFβR inhibitor. (Item 41) 1. A method for reducing spontaneous differentiation of one or more pluripotent cells, comprising: (a) culturing the one or more pluripotent cells in a feeder-free environment; (b) culturing the one or more pluripotent cells in a chemically defined cell culture medium containing a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a ROCK inhibitor, but not containing a TGFβR inhibitor. 1. A method for producing induced pluripotent stem cells (iPSCs), comprising: (a) obtaining one or more non-pluripotent cells; (b) reprogramming the one or more non-pluripotent cells to a pluripotent state; (c) culturing the pluripotent cells in a cell culture medium that does not contain a TGFβR inhibitor, thereby generating iPSCs. (Item 43) 43. The method of claim 42, wherein the one or more non-pluripotent cells comprise somatic cells. (Item 44) 43. The method of claim 42, wherein the one or more non-pluripotent cells comprise adult stem cells. (Item 45) 43. The method of claim 42, wherein the one or more non-pluripotent cells are reprogrammed to a pluripotent state by increasing expression of endogenous OCT4 in the cells. (Item 46) 43. The method of claim 42, wherein reprogramming the one or more non-pluripotent cells to the pluripotent state comprises introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more reprogramming factors selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, and SV40LT. (Item 47) 43. The method of claim 42, wherein reprogramming the one or more non-pluripotent cells to the pluripotent state comprises introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, and SV40LT. (Item 48) 43. The method of claim 42, wherein reprogramming the one or more non-pluripotent cells to the pluripotent state comprises introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, SOX2, and NANOG. (Item 49) 43. The method of claim 42, wherein reprogramming the one or more non-pluripotent cells to the pluripotent state comprises introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, NANOG, ECAT1, UTF1, and ESRRB. (Item 50) 43. The method of claim 42, wherein reprogramming the one or more non-pluripotent cells to the pluripotent state comprises introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, ECAT1, and UTF1. (Item 51) 51. The method of any one of items 46 to 50, wherein the one or more polynucleotides is a lentiviral vector. (Item 52) 51. The method of any one of items 46 to 50, wherein the one or more polynucleotides are episomal vectors. (Item 53) 53. The method of any one of items 42 to 52, wherein the cell culture medium comprises a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a ROCK inhibitor. (Item 54) 54. The method of any one of items 42 to 53, wherein reprogramming the one or more non-pluripotent cells comprises contacting the one or more non-pluripotent cells with a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a TGFβR inhibitor, and optionally a ROCK inhibitor. (Item 55) 43. The method of claim 42, wherein the iPSCs comprise a population of iPSCs. (Item 56) 56. The method of claim 55, wherein the population of iPSCs is a homogenous population of iPSCs. (Item 57) 56. The method of claim 55, wherein at least 95% of the population of iPSCs express SSEA4 and TRA1-81 or TRA1-60. (Item 58) 56. The method of claim 55, wherein up to 5% of the population of pluripotent cells express alpha-smooth muscle actin (SMA), TUJ1, or FoxA2. (Item 59) 59. The method of any one of paragraphs 42 to 58, wherein culturing the pluripotent cells in the cell culture medium reduces spontaneous differentiation or maintains or induces a ground state of pluripotency. (Item 60) The expression of one or more, two or more, three or more, four or more, or five or more differentiation marker genes is reduced in the iPSCs by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to the expression of one or more differentiation marker genes in iPSCs cultured in a medium containing a TGFβR inhibitor, and the differentiation marker genes include FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B 6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury), and ZIC1. (Item 61) 61. The method of claim 60, wherein the one or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1. (Item 62) 61. The method of paragraph 60, wherein the reduction in spontaneous differentiation is maintained for at least 5 passages. (Item 63) 61. The method of paragraph 60, wherein the reduction in spontaneous differentiation is maintained for at least 10 passages. (Item 64) 61. The method of paragraph 60, wherein the reduction in spontaneous differentiation is maintained for at least 50 passages. (Item 65) 61. The method of paragraph 60, wherein the reduction in spontaneous differentiation is maintained for at least 100 passages. (Item 66) 66. The method of any one of paragraphs 42 to 65, comprising dissociating the iPSCs during passaging. (Item 67) 67. The method of claim 66, wherein the viability of the iPSCs is maintained during passaging. (Item 68) 68. The method of any one of items 42 to 67, wherein the iPSCs comprise a normal karyotype. (Item 69) 69. The method of any one of paragraphs 42 to 68, wherein the iPSCs are cultured in a feeder-free environment. (Item 70) 70. The method of any one of paragraphs 42 to 69, wherein the genomic stability of the iPSCs is maintained for at least 10 passages. (Item 71) 70. The method of any one of paragraphs 42 to 69, wherein the genomic stability of the iPSCs is maintained for at least 50 passages. (Item 72) 70. The method of any one of paragraphs 42 to 69, wherein the genomic stability of the iPSCs is maintained for at least 100 passages. (Item 73) 73. Induced pluripotent stem cells (iPSCs) containing ground state pluripotency generated according to any one of items 42 to 72. (Item 74) An induced pluripotent stem cell (iPSC) that comprises ground state pluripotency, wherein the iPSC does not contain an exogenously introduced polynucleotide encoding a reprogramming factor polypeptide. (Item 75) 1. A method for producing induced pluripotent stem cells (iPSCs), comprising: (a) obtaining one or more pluripotent stem cells; (b) culturing the one or more pluripotent stem cells in a cell culture medium that does not contain a TGFβR inhibitor, thereby generating ground state iPSCs. (Item 76) 76. The method of claim 75, wherein the one or more iPSCs comprise reprogrammed somatic cells. (Item 77) 76. The method of claim 75, wherein the one or more iPSCs comprise reprogrammed adult stem cells. (Item 78) 79. The method of claim 75, wherein the one or more iPSCs are reprogrammed to a pluripotent state by increasing expression of endogenous OCT4 in the one or more iPSCs. 76. The method of claim 75, wherein the one or more iPSCs have been reprogrammed by introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more reprogramming factors selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, and SV40LT. (Item 80) 76. The method of paragraph 75, wherein the one or more iPSCs have been reprogrammed by introducing one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, ECAT1, UTF1, ESRRB, and SV40LT. (Item 81) 76. The method of claim 75, wherein the one or more iPSCs have been reprogrammed by introducing one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, SOX2, and NANOG into the one or more non-pluripotent cells. (Item 82) 76. The method of claim 75, wherein the one or more iPSCs have been reprogrammed by introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, NANOG, ECAT1, UTF1, and ESRRB. (Item 83) 76. The method of claim 75, wherein the one or more iPSCs have been reprogrammed by introducing one or more polynucleotides encoding one or more copies of a reprogramming factor selected from the group consisting of OCT4, ECAT1, and UTF1 into the one or more non-pluripotent cells. (Item 84) 84. The method of any one of items 79 to 83, wherein a lentiviral vector comprises the one or more polynucleotides. (Item 85) 84. The method of any one of items 79 to 83, wherein an episomal vector comprises the one or more polynucleotides. (Item 86) 76. The method of claim 75, wherein the cell culture medium comprises a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a ROCK inhibitor. (Item 87) 87. The method of any one of items 75 to 86, wherein obtaining one or more iPSCs comprises contacting the one or more non-pluripotent or partially pluripotent cells with a Wnt pathway agonist, optionally a GSK3 inhibitor; a MEK inhibitor; and a TGFβR inhibitor, and optionally a ROCK inhibitor, to generate the one or more iPSCs. (Item 88) 76. The method of claim 75, wherein the iPSCs comprise a population of iPSCs. (Item 89) 90. The method of claim 88, wherein the population of iPSCs is a homogenous population of iPSCs. (Item 90) 89. The method of paragraph 88, wherein at least 95% of the population of iPSCs express SSEA4 and TRA1-81 or TRA1-60. (Item 91) 89. The method of claim 88, wherein the one or more iPSCs are obtained by reprogramming a population of pluripotent cells, and wherein up to 5% of the population of pluripotent cells express alpha-smooth muscle actin (SMA), TUJ1, or FoxA2. (Item 92) 76. The method of claim 75, wherein culturing the one or more iPSCs in the cell culture medium reduces spontaneous differentiation or maintains or induces a ground state of pluripotency. (Item 93) The iPSCs with reduced spontaneous differentiation include those having gene expression, wherein expression of one or more, two or more, three or more, four or more, or five or more differentiation marker genes is reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to expression of one or more differentiation marker genes in iPSCs cultured in a medium containing a TGFβR inhibitor, and the differentiation marker genes include FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4 ... 93. The method of item 92, wherein the gene is selected from the group consisting of CR4, CYP2B6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury), and ZIC1. (Item 94) 94. The method of item 93, wherein the one or more differentiation marker genes are selected from the group consisting of T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1. (Item 95) 94. The method of paragraph 93, wherein the reduced spontaneous differentiation is maintained for at least 5 passages. (Item 96) 94. The method of paragraph 93, wherein the reduced spontaneous differentiation is maintained for at least 10 passages. (Item 97) 94. The method of paragraph 93, wherein the reduced spontaneous differentiation is maintained for at least 50 passages. (Item 98) 94. The method of paragraph 93, wherein the reduced spontaneous differentiation is maintained for at least 100 passages. (Item 99) 99. The method of any one of paragraphs 75 to 98, comprising dissociating the one or more iPSCs during passaging. (Item 100) 91. The method of claim 90, wherein the viability of the one or more iPSCs is maintained during passaging. (Item 101) 101. The method of any one of paragraphs 75 to 100, wherein the one or more iPSCs comprise a normal karyotype. (Item 102) 102. The method of any one of paragraphs 75 to 101, wherein the one or more iPSCs are cultured in a feeder-free environment. (Item 103) 103. The method of any one of paragraphs 75 to 102, wherein the genomic stability of the one or more iPSCs is maintained for at least 10 passages. (Item 104) 103. The method of any one of paragraphs 75 to 102, wherein the genomic stability of the one or more iPSCs is maintained for at least 50 passages. (Item 105) 103. The method of any one of paragraphs 75 to 102, wherein the genomic stability of the one or more iPSCs is maintained for at least 100 passages. (Item 106) 106. Induced pluripotent stem cells (iPSCs) containing ground state pluripotency produced according to any one of items 75 to 105. (Item 107) An induced pluripotent stem cell (iPSC) that comprises ground state pluripotency, wherein the iPSC does not contain an exogenously introduced polynucleotide encoding a reprogramming factor polypeptide. (Item 108) A method for reprogramming a non-pluripotent cell into a pluripotent cell, the method comprising introducing into the non-pluripotent cell one or more polynucleotides encoding (i) at least one polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) at least one polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, thereby reprogramming the non-pluripotent cell into a pluripotent cell. (Item 109) Item 109. The method of Item 108, wherein the introducing comprises (i) introducing one or more polynucleotides encoding an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) introducing an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide. (Item 110) Item 109. The method of Item 108, wherein the introducing comprises (i) introducing one or more polynucleotides encoding an OCT4 polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide, or (ii) introducing an OCT-4 polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide. (Item 111) 111. The method of any one of items 108 to 110, wherein the one or more polynucleotides are introduced by retrovirus, Sendai virus, adenovirus, episome, minicircle, vector system with an expression cassette, or mRNA. (Item 112) 112. The method of claim 111, wherein the retrovirus is a lentivirus. (Item 113) 112. The method of claim 111, wherein the pluripotent cells do not contain an exogenous polynucleotide. (Item 114) 114. The method of claim 113, wherein the one or more polynucleotides are cleaved by CRE-mediated cleavage. (Item 115) 115. The method of any one of items 108 to 114, further comprising introducing into the non-pluripotent cell (i) a polynucleotide encoding an SV40LT polypeptide, or (ii) an SV40LT polypeptide. (Item 116) 116. The method of any one of paragraphs 108 to 115, further comprising contacting the non-pluripotent cells with at least one of a TGFβR inhibitor, a Wnt pathway agonist, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway agonist is optionally a GSK3 inhibitor. (Item 117) 117. The method of claim 116, wherein the non-pluripotent cells are contacted with a TGFβR inhibitor, a Wnt pathway agonist, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway agonist is optionally a GSK3 inhibitor. (Item 118) 118. The method of any one of paragraphs 108 to 117, further comprising culturing the pluripotent cells in a culture medium comprising a Wnt pathway agonist, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway agonist is optionally a GSK3 inhibitor, and the culture medium does not contain a TGFβR inhibitor. (Item 119) 119. The method of any one of items 116 to 118, wherein (a) the Rock inhibitor is thiazovivin or Y27632, (b) the TGFβR inhibitor is A-83-01 or SB431542, (c) the GSK3 inhibitor is CHIR99021 or BIO, or (d) the MEK inhibitor is PD98059 or PD032901. (Item 120) 120. The method of paragraph 118 or 119, wherein the pluripotency of the pluripotent cells is maintained for at least 5 cell divisions or at least 10 cell divisions. (Item 121) 121. The method of any one of items 108 to 120, wherein the one or more polynucleotides are introduced as a polycistronic vector comprising multiple polynucleotides separated by at least one 2A peptide. (Item 122) 122. The method of claim 121, wherein the polycistronic vector comprises multiple polynucleotides encoding Oct4 polypeptides. (Item 123) 123. The method of any one of paragraphs 108 to 122, further comprising identifying the pluripotent cells by selecting for Oct34 expression in the pluripotent cells. (Item 124) 124. The method of claim 123, wherein selecting for Oct4 expression comprises selecting for ectopic Oct4 expression. (Item 125) 125. The method of any one of paragraphs 118 to 124, wherein the culturing generates a population of pluripotent stem cells. (Item 126) 126. The method of paragraph 125, wherein the population of pluripotent stem cells is at least 70% homogeneous, at least 80% homogeneous, or at least 90% homogeneous. (Item 127) 127. The method of paragraph 125 or 126, wherein at least 70%, at least 80%, or at least 90% of the population of pluripotent cells express SSEA and Tra-181. (Item 128) 128. The method of any one of paragraphs 108 to 127, wherein the pluripotent cell or population of pluripotent cells is capable of single cell passaging. (Item 129) 129. The method of claim 128, wherein the cells generated by single cell passaging have a normal karyotype. (Item 130) 130. A pluripotent cell produced according to the method of any one of items 108 to 129. (Item 131) A composition comprising an isolated non-pluripotent cell comprising (i) one or more exogenous polynucleotides encoding at least one polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) at least one exogenous polypeptide selected from an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide. (Item 132) 132. The composition of claim 131, wherein the cells comprise (i) one or more exogenous polynucleotides encoding at least two of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) at least two exogenous polypeptides selected from an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide. (Item 133) Item 132. The composition of item 131, wherein the cells comprise (i) one or more exogenous polynucleotides encoding at least three of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) at least three exogenous polypeptides selected from an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide. (Item 134) Item 132. The composition of item 131, wherein the cells comprise (i) one or more exogenous polynucleotides encoding an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) an exogenous OCT4 polypeptide, an exogenous ECAT1 polypeptide, an exogenous UTF1 polypeptide, an exogenous NANOG polypeptide, and an exogenous ESRRB polypeptide. (Item 135) Item 132. The composition of item 131, wherein the cells comprise (i) one or more exogenous polynucleotides encoding an OCT4 polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide, or (ii) an exogenous OCT4 polypeptide, an exogenous ECAT1 polypeptide, and an exogenous UTF1 polypeptide. (Item 136) 136. The composition of any one of items 121 to 135, wherein the cells are contacted with at least one of a TGFβR inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor. (Item 137) 137. The composition of claim 136, wherein the cells are contacted with a TGFβR inhibitor, a Wnt pathway activator, a MEK inhibitor, and a ROCK inhibitor, and the Wnt pathway activator is optionally a GSK3 inhibitor. (Item 138) 138. The composition of claim 136 or 137, wherein (a) the Rock inhibitor is thiazovivin or Y27632, (b) the TGFβR inhibitor is A-83-01 or SB431542, (c) the GSK3 inhibitor is CHIR99021 or BIO, or (d) the MEK inhibitor is PD98059 or PD032901. (Item 139) 139. The composition of any one of items 131 to 138, wherein the one or more exogenous polynucleotides are introduced into the non-pluripotent cell by retrovirus, Sendai virus, adenovirus, episome, minicircle, vector system carrying an expression cassette, or mRNA. (Item 140) 140. The method of claim 139, wherein the retrovirus is a lentivirus. (Item 141) 140. The composition of claim 139, wherein the cells do not contain an exogenous polynucleotide. (Item 142) 142. The method of claim 141, wherein the one or more exogenous polynucleotides are removed by CRE-mediated cleavage. (Item 143) 143. The composition of any one of items 131 to 142, wherein the cell further comprises (i) an exogenous polynucleotide encoding an SV40LT antigen polypeptide, or (ii) an exogenous SV40LT antigen polypeptide. (Item 144) 144. The composition of any one of items 131 to 143, wherein the one or more exogenous polynucleotides are introduced as a polycistronic vector comprising multiple polynucleotides separated by at least one 2A peptide. (Item 145) 145. The composition of claim 144, wherein the polycistronic vector comprises multiple polynucleotides encoding OCT4 polypeptides. (Item 146) 146. The composition of any one of items 131 to 145, wherein the exogenous polynucleotide encoding an OCT4 polypeptide is linked to a selectable marker. (Item 147) A composition consisting of at least one, at least two, or at least three of: (i) a cDNA encoding an OCT4 polypeptide, (ii) a cDNA encoding an ECAT1 polypeptide, (iii) a cDNA encoding a UTF1 polypeptide, (iv) a cDNA encoding a NANOG polypeptide, and (v) a cDNA encoding an ESRRB polypeptide. (Item 148) Item 148. The composition according to Item 147, comprising a cDNA encoding an OCT4 polypeptide, a cDNA encoding an ECAT1 polypeptide, a cDNA encoding a UTF1 polypeptide, a cDNA encoding a NANOG polypeptide, and a cDNA encoding an ESRRB polypeptide. (Item 149) Item 149. The composition according to item 148, consisting of a cDNA encoding an OCT4 polypeptide, a cDNA encoding an ECAT1 polypeptide, and a cDNA encoding a UTF1 polypeptide. (Item 150) 150. The composition of any one of items 147 to 149, wherein each cDNA is encoded by a retrovirus, a Sendai virus, an adenovirus, an episome, a minicircle, a vector system with an expression cassette, or by mRNA. (Item 151) A vector comprising one or more polynucleotides encoding at least one reprogramming factor polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide. (Item 152) Item 152. The vector of Item 151, wherein the one or more polynucleotides encode an OCT polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide. (Item 153) Item 153. The vector of Item 152, wherein the one or more polynucleotides encode an OCT polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide. (Item 154) 154. The vector of any one of items 151 to 153, wherein the vector further comprises a polynucleotide encoding an SV40LT antigen polypeptide. (Item 155) 155. The vector of any one of items 151 to 154, which is a retrovirus, a Sendai virus, an adenovirus, an episome, a minicircle, a vector system with an expression cassette, or an mRNA. (Item 156) Item 156. The vector of item 155, wherein the retrovirus is a lentivirus. (Item 157) 1. A kit for reprogramming a non-pluripotent cell into a pluripotent cell, comprising: (a) one or more polynucleotides encoding at least one polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, or an ESRRB polypeptide; or (b) at least one polypeptide selected from the group consisting of an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, or an ESRRB polypeptide; and (c) The kit comprises at least one of a TGFβR inhibitor, a Wnt pathway activator, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway activator is optionally a GSK3 inhibitor. (Item 158) Item 158. The kit of Item 157, wherein (i) the one or more polynucleotides encode an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide, or (ii) the at least one polypeptide comprises an OCT4 polypeptide, an ECAT1 polypeptide, a UTF1 polypeptide, a NANOG polypeptide, and an ESRRB polypeptide. (Item 159) Item 158. The kit of Item 157, wherein (i) the one or more polynucleotides encode an OCT4 polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide, or (ii) the at least one polypeptide comprises an OCT4 polypeptide, an ECAT1 polypeptide, and a UTF1 polypeptide. (Item 160) 160. The kit of any one of items 157 to 159, further comprising: (i) a polynucleotide encoding an SV40LT antigen polypeptide; or (ii) an SV40LT antigen polypeptide. (Item 161) 161. The kit of any one of items 157 to 160, comprising a TGFβR inhibitor, a Wnt pathway activator, a MEK inhibitor, and a ROCK inhibitor, wherein the Wnt pathway activator is optionally a GSK3 inhibitor. (Item 162) 162. The kit of any one of items 157 to 161, wherein (a) the Rock inhibitor is thiazovivin or Y27632, (b) the TGFβR inhibitor is A-83-01 or SB431542, (c) the GSK3 inhibitor is CHIR99021 or BIO, or (d) the MEK inhibitor is PD98059 or PD032901. (Item 163) 163. The kit of any one of items 157 to 162, wherein the at least one polynucleotide is encoded by a lentivirus, a Sendai virus, an adenovirus, an episome, a minicircle, a vector system with an expression cassette, or an mRNA. (Item 164) Item 164. The kit of item 163, wherein the retrovirus is a lentivirus. (Item 165) 165. The kit of any one of items 157 to 164, wherein the at least one polynucleotide is encoded by a polycistronic vector and each polynucleotide is separated by a 2A peptide. (Item 166) 166. The kit of claim 165, wherein the polycistronic vector comprises two or more polynucleotides encoding OCT4 polypeptides. (Item 167) 167. The kit of any one of items 157 to 166, wherein the at least one polynucleotide encoding an OCT4 polypeptide is linked to a selectable marker. (Item 168) 1. A method for generating a population of pluripotent stem cells, comprising: a) providing a population of non-pluripotent cells; b) introducing into said population of non-pluripotent cells a polynucleotide encoding an OCT4 polypeptide linked to a selectable marker; c) incubating said population of non-pluripotent cells with said polynucleotide under conditions sufficient to reprogram at least a portion of said population of non-pluripotent cells into pluripotent cells; d) selecting for cells that express the selectable marker, thereby providing a population of pluripotent stem cells. (Item 169) 169. The method of claim 168, wherein the polynucleotide is introduced as a polycistronic vector comprising multiple polynucleotides encoding OCT4 polypeptides. (Item 170) 170. The method of claim 169, wherein the multiple polynucleotides are separated by 2A peptides. (Item 171) 170. The method of claim 169, wherein at least 10% of the cells in the population of cells express SSEA and TRA-181. [Brief explanation of the drawings]

[0181] [Figure 1A]Figures 1A-1E show results from a multi-stage culture platform for improved reprogramming and hiPSC maintenance. (1A) Lentivirus-generated hiPSC clone FTi088 maintained a homogenous population of undifferentiated cells in SMC4, while spontaneous differentiation was observed in lentivirus-generated hiPSC line FTi096 cultured in SMC4. Spontaneous differentiation was minimized when FTi096 was transferred to FMM for three passages, as shown by morphology (upper panel) and flow cytometry of SSEA4 and TRA1-81 (lower panel). (1B) qRT-PCR of transgene expression of the viral-like sequence WPRE. Expression was normalized to GAPDH and compared to WPRE expression in the parental fibroblast cell line 4 days after lentivirus infection (Day 4 PI). An uninfected fibroblast cell line (Fibroblast) and the human ESC line HUES9 were used as negative controls. Values ​​for each set are indicated above the bars. (1C) Screening of the effects of various media components on the SSEA4 and TRA1-81 populations of transgene-free lentivirus-induced hiPSCs after 10 passages: removal of SB431542 (-TGFβRi), increase of bFGF from 10 to 100 ng / mL, and addition of 10 ng / mL LIF. (1D) Fibroblast cell lines were transfected with a lentiviral construct containing the gene set OCT4 / KLF4 / SOX2, split into various media (conventional media; Conv.), cultured for 17 days, and sorted into SSEA4 and TRA1-81 double-positive populations on day 17. The sort gates are highlighted in blue. Each set was cultured for an additional 10 days in its respective media, except for the SMC4 set, which was split into FMM and SMC4. On day 27, the cultures were again sorted into SSEA4 and TRA1-81 double-positive populations, seeded at the normalized density of the sorting event, and maintained in the respective media for an additional 9 days. Conventional culture set gating was expanded to achieve normalized cell numbers. On day 36, each culture was stained for OCT4 and NANOG expression. Representative immunocytochemistry images are shown in the right panel of each set. (1E) Colony counts for day 36 staining are discussed in (1D).Error bars indicate three for FRM to FMM and FRM, and two for FMM and hESC. [Figure 1B]Figures 1A-1E show results from a multi-stage culture platform for improved reprogramming and hiPSC maintenance. (1A) Lentivirus-generated hiPSC clone FTi088 maintained a homogenous population of undifferentiated cells in SMC4, while spontaneous differentiation was observed in lentivirus-generated hiPSC line FTi096 cultured in SMC4. Spontaneous differentiation was minimized when FTi096 was transferred to FMM for three passages, as shown by morphology (upper panel) and flow cytometry of SSEA4 and TRA1-81 (lower panel). (1B) qRT-PCR of transgene expression of the viral-like sequence WPRE. Expression was normalized to GAPDH and compared to WPRE expression in the parental fibroblast cell line 4 days after lentivirus infection (Day 4 PI). An uninfected fibroblast cell line (Fibroblast) and the human ESC line HUES9 were used as negative controls. Values ​​for each set are indicated above the bars. (1C) Screening of the effects of various media components on the SSEA4 and TRA1-81 populations of transgene-free lentivirus-induced hiPSCs after 10 passages: removal of SB431542 (-TGFβRi), increase of bFGF from 10 to 100 ng / mL, and addition of 10 ng / mL LIF. (1D) Fibroblast cell lines were transfected with a lentiviral construct containing the gene set OCT4 / KLF4 / SOX2, split into various media (conventional media; Conv.), cultured for 17 days, and sorted into SSEA4 and TRA1-81 double-positive populations on day 17. The sort gates are highlighted in blue. Each set was cultured for an additional 10 days in its respective media, except for the SMC4 set, which was split into FMM and SMC4. On day 27, the cultures were again sorted into SSEA4 and TRA1-81 double-positive populations, seeded at the normalized density of the sorting event, and maintained in the respective media for an additional 9 days. Conventional culture set gating was expanded to achieve normalized cell numbers. On day 36, each culture was stained for OCT4 and NANOG expression. Representative immunocytochemistry images are shown in the right panel of each set. (1E) Colony counts for day 36 staining are discussed in (1D).Error bars indicate three for FRM to FMM and FRM, and two for FMM and hESC. [Figure 1C]Figures 1A-1E show results from a multi-stage culture platform for improved reprogramming and hiPSC maintenance. (1A) Lentivirus-generated hiPSC clone FTi088 maintained a homogenous population of undifferentiated cells in SMC4, while spontaneous differentiation was observed in lentivirus-generated hiPSC line FTi096 cultured in SMC4. Spontaneous differentiation was minimized when FTi096 was transferred to FMM for three passages, as shown by morphology (upper panel) and flow cytometry of SSEA4 and TRA1-81 (lower panel). (1B) qRT-PCR of transgene expression of the viral-like sequence WPRE. Expression was normalized to GAPDH and compared to WPRE expression in the parental fibroblast cell line 4 days after lentivirus infection (Day 4 PI). An uninfected fibroblast cell line (Fibroblast) and the human ESC line HUES9 were used as negative controls. Values ​​for each set are indicated above the bars. (1C) Screening of the effects of various media components on the SSEA4 and TRA1-81 populations of transgene-free lentivirus-induced hiPSCs after 10 passages: removal of SB431542 (-TGFβRi), increase of bFGF from 10 to 100 ng / mL, and addition of 10 ng / mL LIF. (1D) Fibroblast cell lines were transfected with a lentiviral construct containing the gene set OCT4 / KLF4 / SOX2, split into various media (conventional media; Conv.), cultured for 17 days, and sorted into SSEA4 and TRA1-81 double-positive populations on day 17. The sort gates are highlighted in blue. Each set was cultured for an additional 10 days in its respective media, except for the SMC4 set, which was split into FMM and SMC4. On day 27, the cultures were again sorted into SSEA4 and TRA1-81 double-positive populations, seeded at the normalized density of the sorting event, and maintained in the respective media for an additional 9 days. Conventional culture set gating was expanded to achieve normalized cell numbers. On day 36, each culture was stained for OCT4 and NANOG expression. Representative immunocytochemistry images are shown in the right panel of each set. (1E) Colony counts for day 36 staining are discussed in (1D).Error bars indicate three for FRM to FMM and FRM, and two for FMM and hESC. [Figure 1D]Figures 1A-1E show results from a multi-stage culture platform for improved reprogramming and hiPSC maintenance. (1A) Lentivirus-generated hiPSC clone FTi088 maintained a homogenous population of undifferentiated cells in SMC4, while spontaneous differentiation was observed in lentivirus-generated hiPSC line FTi096 cultured in SMC4. Spontaneous differentiation was minimized when FTi096 was transferred to FMM for three passages, as shown by morphology (upper panel) and flow cytometry of SSEA4 and TRA1-81 (lower panel). (1B) qRT-PCR of transgene expression of the viral-like sequence WPRE. Expression was normalized to GAPDH and compared to WPRE expression in the parental fibroblast cell line 4 days after lentivirus infection (Day 4 PI). An uninfected fibroblast cell line (Fibroblast) and the human ESC line HUES9 were used as negative controls. Values ​​for each set are indicated above the bars. (1C) Screening of the effects of various media components on the SSEA4 and TRA1-81 populations of transgene-free lentivirus-induced hiPSCs after 10 passages: removal of SB431542 (-TGFβRi), increase of bFGF from 10 to 100 ng / mL, and addition of 10 ng / mL LIF. (1D) Fibroblast cell lines were transfected with a lentiviral construct containing the gene set OCT4 / KLF4 / SOX2, split into various media (conventional media; Conv.), cultured for 17 days, and sorted into SSEA4 and TRA1-81 double-positive populations on day 17. The sort gates are highlighted in blue. Each set was cultured for an additional 10 days in its respective media, except for the SMC4 set, which was split into FMM and SMC4. On day 27, the cultures were again sorted into SSEA4 and TRA1-81 double-positive populations, seeded at the normalized density of the sorting event, and maintained in the respective media for an additional 9 days. Conventional culture set gating was expanded to achieve normalized cell numbers. On day 36, each culture was stained for OCT4 and NANOG expression. Representative immunocytochemistry images are shown in the right panel of each set. (1E) Colony counts for day 36 staining are discussed in (1D).Error bars indicate three for FRM to FMM and FRM, and two for FMM and hESC. [Figure 1E]Figures 1A-1E show results from a multi-stage culture platform for improved reprogramming and hiPSC maintenance. (1A) Lentivirus-generated hiPSC clone FTi088 maintained a homogenous population of undifferentiated cells in SMC4, while spontaneous differentiation was observed in lentivirus-generated hiPSC line FTi096 cultured in SMC4. Spontaneous differentiation was minimized when FTi096 was transferred to FMM for three passages, as shown by morphology (upper panel) and flow cytometry of SSEA4 and TRA1-81 (lower panel). (1B) qRT-PCR of transgene expression of the viral-like sequence WPRE. Expression was normalized to GAPDH and compared to WPRE expression in the parental fibroblast cell line 4 days after lentivirus infection (Day 4 PI). An uninfected fibroblast cell line (Fibroblast) and the human ESC line HUES9 were used as negative controls. Values ​​for each set are indicated above the bars. (1C) Screening of the effects of various media components on the SSEA4 and TRA1-81 populations of transgene-free lentivirus-induced hiPSCs after 10 passages: removal of SB431542 (-TGFβRi), increase of bFGF from 10 to 100 ng / mL, and addition of 10 ng / mL LIF. (1D) Fibroblast cell lines were transfected with a lentiviral construct containing the gene set OCT4 / KLF4 / SOX2, split into various media (conventional media; Conv.), cultured for 17 days, and sorted into SSEA4 and TRA1-81 double-positive populations on day 17. The sort gates are highlighted in blue. Each set was cultured for an additional 10 days in its respective media, except for the SMC4 set, which was split into FMM and SMC4. On day 27, the cultures were again sorted into SSEA4 and TRA1-81 double-positive populations, seeded at the normalized density of the sorting event, and maintained in the respective media for an additional 9 days. Conventional culture set gating was expanded to achieve normalized cell numbers. On day 36, each culture was stained for OCT4 and NANOG expression. Representative immunocytochemistry images are shown in the right panel of each set. (1E) Colony counts for day 36 staining are discussed in (1D).Error bars indicate three for FRM to FMM and FRM, and two for FMM and hESC. [Figure 2A] Figures 2A-2E show that individual episome-reprogrammed hiPSCs were efficiently selected and seeded into 96-well plates for clonal expansion. (2A) Schematic timing diagram of episome-reprogramming, multi-step culture platform, flow cytometry sorting, and clonal expansion. (2B) Flow cytometry profile of episome-reprogrammed cells maintained at the FRM-FMM transition in FF cultures (outlined in 2A) at the indicated days post-transfection. The sorting gating strategy used for each parental line (SSEA4+ / TRA1-81+ / CD30+ population) is indicated by its corresponding color in the histogram panel below, representing the percentage of wells in the 96-well plate containing individual hiPSC clones. Wells containing multiple clones or differentiated clones were not scored. The solid line represents the average percentage across all derivatives, and the dotted line represents the standard deviation. (2C) Flow profile of FTC007 cells induced to reprogram 19 days after transfection, maintained in conventional medium in the presence of MEF cells. The induced population was taken from the same population of FTC007 cells in (2B), but then processed in a different culture medium. (2D) Immunocytochemical analysis of various pluripotency markers of sorted colonies in a 96-well plate. The right corner panel represents DAPI staining. (2E) qRT-PCR of NANOG expression in each well of a SSEA4 / TRA1-81 / CD30 direct sorting (FACS) 96-well plate at three cells per well. The expression range is between zero and four times that of H1 human ESCs, as explained in the legend, and is normalized to GAPDH. [Figure 2B]Figures 2A-2E show that individual episome-reprogrammed hiPSCs were efficiently selected and seeded into 96-well plates for clonal expansion. (2A) Schematic timing diagram of episome-reprogramming, multi-step culture platform, flow cytometry sorting, and clonal expansion. (2B) Flow cytometry profile of episome-reprogrammed cells maintained at the FRM-FMM transition in FF cultures (outlined in 2A) at the indicated days post-transfection. The sorting gating strategy used for each parental line (SSEA4+ / TRA1-81+ / CD30+ population) is indicated by its corresponding color in the histogram panel below, representing the percentage of wells in the 96-well plate containing individual hiPSC clones. Wells containing multiple clones or differentiated clones were not scored. The solid line represents the average percentage across all derivatives, and the dotted line represents the standard deviation. (2C) Flow profile of FTC007 cells induced to reprogram 19 days after transfection, maintained in conventional medium in the presence of MEF cells. The induced population was taken from the same population of FTC007 cells in (2B), but then processed in a different culture medium. (2D) Immunocytochemical analysis of various pluripotency markers of sorted colonies in a 96-well plate. The right corner panel represents DAPI staining. (2E) qRT-PCR of NANOG expression in each well of a SSEA4 / TRA1-81 / CD30 direct sorting (FACS) 96-well plate at three cells per well. The expression range is between zero and four times that of H1 human ESCs, as explained in the legend, and is normalized to GAPDH. [Figure 2C]Figures 2A-2E show that individual episome-reprogrammed hiPSCs were efficiently selected and seeded into 96-well plates for clonal expansion. (2A) Schematic timing diagram of episome-reprogramming, multi-step culture platform, flow cytometry sorting, and clonal expansion. (2B) Flow cytometry profile of episome-reprogrammed cells maintained at the FRM-FMM transition in FF cultures (outlined in 2A) at the indicated days post-transfection. The sorting gating strategy used for each parental line (SSEA4+ / TRA1-81+ / CD30+ population) is indicated by its corresponding color in the histogram panel below, representing the percentage of wells in the 96-well plate containing individual hiPSC clones. Wells containing multiple clones or differentiated clones were not scored. The solid line represents the average percentage across all derivatives, and the dotted line represents the standard deviation. (2C) Flow profile of FTC007 cells induced to reprogram 19 days after transfection, maintained in conventional medium in the presence of MEF cells. The induced population was taken from the same population of FTC007 cells in (2B), but then processed in a different culture medium. (2D) Immunocytochemical analysis of various pluripotency markers of sorted colonies in a 96-well plate. The right corner panel represents DAPI staining. (2E) qRT-PCR of NANOG expression in each well of a SSEA4 / TRA1-81 / CD30 direct sorting (FACS) 96-well plate at three cells per well. The expression range is between zero and four times that of H1 human ESCs, as explained in the legend, and is normalized to GAPDH. [Figure 2D]Figures 2A-2E show that individual episome-reprogrammed hiPSCs were efficiently selected and seeded into 96-well plates for clonal expansion. (2A) Schematic timing diagram of episome-reprogramming, multi-step culture platform, flow cytometry sorting, and clonal expansion. (2B) Flow cytometry profile of episome-reprogrammed cells maintained at the FRM-FMM transition in FF cultures (outlined in 2A) at the indicated days post-transfection. The sorting gating strategy used for each parental line (SSEA4+ / TRA1-81+ / CD30+ population) is indicated by its corresponding color in the histogram panel below, representing the percentage of wells in the 96-well plate containing individual hiPSC clones. Wells containing multiple clones or differentiated clones were not scored. The solid line represents the average percentage across all derivatives, and the dotted line represents the standard deviation. (2C) Flow profile of FTC007 cells induced to reprogram 19 days after transfection, maintained in conventional medium in the presence of MEF cells. The induced population was taken from the same population of FTC007 cells in (2B), but then processed in a different culture medium. (2D) Immunocytochemical analysis of various pluripotency markers of sorted colonies in a 96-well plate. The right corner panel represents DAPI staining. (2E) qRT-PCR of NANOG expression in each well of a SSEA4 / TRA1-81 / CD30 direct sorting (FACS) 96-well plate at three cells per well. The expression range is between zero and four times that of H1 human ESCs, as explained in the legend, and is normalized to GAPDH. [Figure 2E]Figures 2A-2E show that individual episome-reprogrammed hiPSCs were efficiently selected and seeded into 96-well plates for clonal expansion. (2A) Schematic timing diagram of episome-reprogramming, multi-step culture platform, flow cytometry sorting, and clonal expansion. (2B) Flow cytometry profile of episome-reprogrammed cells maintained at the FRM-FMM transition in FF cultures (outlined in 2A) at the indicated days post-transfection. The sorting gating strategy used for each parental line (SSEA4+ / TRA1-81+ / CD30+ population) is indicated by its corresponding color in the histogram panel below, representing the percentage of wells in the 96-well plate containing individual hiPSC clones. Wells containing multiple clones or differentiated clones were not scored. The solid line represents the average percentage across all derivatives, and the dotted line represents the standard deviation. (2C) Flow profile of FTC007 cells induced to reprogram 19 days after transfection, maintained in conventional medium in the presence of MEF cells. The induced population was taken from the same population of FTC007 cells in (2B), but then processed in a different culture medium. (2D) Immunocytochemical analysis of various pluripotency markers of sorted colonies in a 96-well plate. The right corner panel represents DAPI staining. (2E) qRT-PCR of NANOG expression in each well of a SSEA4 / TRA1-81 / CD30 direct sorting (FACS) 96-well plate at three cells per well. The expression range is between zero and four times that of H1 human ESCs, as explained in the legend, and is normalized to GAPDH. [Figure 3A]Figures 3A-3F. Episomal reprogrammed hiPSC clones maintain their undifferentiated state and do not contain transgene sequences. (3A) Typical morphology of hiPSC clones 24 hours after single-cell passaging. (3B) Representative images of hiPSC clones in culture. (3C) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1, FTC007-c1 p4; Lane 2, FTC007-c21 p4; Lane 3, FTC016-c25 p5; Lane 4, FTC016-c36 p5; Lane 5, FTC017-c11 p7; Lane 6, FTC017-c14 p7; Lane 7, FTC017-c17 p6 (a line maintaining the episomal construct served as a positive control); Lane 8, untransfected FTC007; Lane 9, hiPSCs generated using a lentiviral construct (serving as a control for cross-contamination); Lane 10, episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (3D) Pluripotency markers detected by immunofluorescence for the expression of OCT4, NANOG, TRA1-81, and TRA160. (3E) Flow cytometry profiles of selected hiPSC clones from various parental lines. The top row profile is SSEA4 / TRA1-81 surface expression. The bottom row profile is OCT4 / NANOG intracellular expression. (3F) qRT-PCR analysis of endogenous pluripotency gene expression. Data were normalized to GAPDH and compared to HUES9 hESCs. For KLF4 expression, two data points were greater than 15-fold higher than HUES9 and were recorded on the graph. Error bars represent the standard deviation of replicates. [Figure 3B]Figures 3A-3F. Episomal reprogrammed hiPSC clones maintain their undifferentiated state and do not contain transgene sequences. (3A) Typical morphology of hiPSC clones 24 hours after single-cell passaging. (3B) Representative images of hiPSC clones in culture. (3C) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1, FTC007-c1 p4; Lane 2, FTC007-c21 p4; Lane 3, FTC016-c25 p5; Lane 4, FTC016-c36 p5; Lane 5, FTC017-c11 p7; Lane 6, FTC017-c14 p7; Lane 7, FTC017-c17 p6 (a line maintaining the episomal construct served as a positive control); Lane 8, untransfected FTC007; Lane 9, hiPSCs generated using a lentiviral construct (serving as a control for cross-contamination); Lane 10, episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (3D) Pluripotency markers detected by immunofluorescence for the expression of OCT4, NANOG, TRA1-81, and TRA160. (3E) Flow cytometry profiles of selected hiPSC clones from various parental lines. The top row profile is SSEA4 / TRA1-81 surface expression. The bottom row profile is OCT4 / NANOG intracellular expression. (3F) qRT-PCR analysis of endogenous pluripotency gene expression. Data were normalized to GAPDH and compared to HUES9 hESCs. For KLF4 expression, two data points were greater than 15-fold higher than HUES9 and were recorded on the graph. Error bars represent the standard deviation of replicates. [Figure 3C]Figures 3A-3F. Episomal reprogrammed hiPSC clones maintain their undifferentiated state and do not contain transgene sequences. (3A) Typical morphology of hiPSC clones 24 hours after single-cell passaging. (3B) Representative images of hiPSC clones in culture. (3C) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1, FTC007-c1 p4; Lane 2, FTC007-c21 p4; Lane 3, FTC016-c25 p5; Lane 4, FTC016-c36 p5; Lane 5, FTC017-c11 p7; Lane 6, FTC017-c14 p7; Lane 7, FTC017-c17 p6 (a line maintaining the episomal construct served as a positive control); Lane 8, untransfected FTC007; Lane 9, hiPSCs generated using a lentiviral construct (serving as a control for cross-contamination); Lane 10, episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (3D) Pluripotency markers detected by immunofluorescence for the expression of OCT4, NANOG, TRA1-81, and TRA160. (3E) Flow cytometry profiles of selected hiPSC clones from various parental lines. The top row profile is SSEA4 / TRA1-81 surface expression. The bottom row profile is OCT4 / NANOG intracellular expression. (3F) qRT-PCR analysis of endogenous pluripotency gene expression. Data were normalized to GAPDH and compared to HUES9 hESCs. For KLF4 expression, two data points were greater than 15-fold higher than HUES9 and were recorded on the graph. Error bars represent the standard deviation of replicates. [Figure 3D]Figures 3A-3F. Episomal reprogrammed hiPSC clones maintain their undifferentiated state and do not contain transgene sequences. (3A) Typical morphology of hiPSC clones 24 hours after single-cell passaging. (3B) Representative images of hiPSC clones in culture. (3C) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1, FTC007-c1 p4; Lane 2, FTC007-c21 p4; Lane 3, FTC016-c25 p5; Lane 4, FTC016-c36 p5; Lane 5, FTC017-c11 p7; Lane 6, FTC017-c14 p7; Lane 7, FTC017-c17 p6 (a line maintaining the episomal construct served as a positive control); Lane 8, untransfected FTC007; Lane 9, hiPSCs generated using a lentiviral construct (serving as a control for cross-contamination); Lane 10, episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (3D) Pluripotency markers detected by immunofluorescence for the expression of OCT4, NANOG, TRA1-81, and TRA160. (3E) Flow cytometry profiles of selected hiPSC clones from various parental lines. The top row profile is SSEA4 / TRA1-81 surface expression. The bottom row profile is OCT4 / NANOG intracellular expression. (3F) qRT-PCR analysis of endogenous pluripotency gene expression. Data were normalized to GAPDH and compared to HUES9 hESCs. For KLF4 expression, two data points were greater than 15-fold higher than HUES9 and were recorded on the graph. Error bars represent the standard deviation of replicates. [Figure 3E]Figures 3A-3F. Episomal reprogrammed hiPSC clones maintain their undifferentiated state and do not contain transgene sequences. (3A) Typical morphology of hiPSC clones 24 hours after single-cell passaging. (3B) Representative images of hiPSC clones in culture. (3C) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1, FTC007-c1 p4; Lane 2, FTC007-c21 p4; Lane 3, FTC016-c25 p5; Lane 4, FTC016-c36 p5; Lane 5, FTC017-c11 p7; Lane 6, FTC017-c14 p7; Lane 7, FTC017-c17 p6 (a line maintaining the episomal construct served as a positive control); Lane 8, untransfected FTC007; Lane 9, hiPSCs generated using a lentiviral construct (serving as a control for cross-contamination); Lane 10, episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (3D) Pluripotency markers detected by immunofluorescence for the expression of OCT4, NANOG, TRA1-81, and TRA160. (3E) Flow cytometry profiles of selected hiPSC clones from various parental lines. The top row profile is SSEA4 / TRA1-81 surface expression. The bottom row profile is OCT4 / NANOG intracellular expression. (3F) qRT-PCR analysis of endogenous pluripotency gene expression. Data were normalized to GAPDH and compared to HUES9 hESCs. For KLF4 expression, two data points were greater than 15-fold higher than HUES9 and were recorded on the graph. Error bars represent the standard deviation of replicates. [Figure 3F]Figures 3A-3F. Episomal reprogrammed hiPSC clones maintain their undifferentiated state and do not contain transgene sequences. (3A) Typical morphology of hiPSC clones 24 hours after single-cell passaging. (3B) Representative images of hiPSC clones in culture. (3C) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1, FTC007-c1 p4; Lane 2, FTC007-c21 p4; Lane 3, FTC016-c25 p5; Lane 4, FTC016-c36 p5; Lane 5, FTC017-c11 p7; Lane 6, FTC017-c14 p7; Lane 7, FTC017-c17 p6 (a line maintaining the episomal construct served as a positive control); Lane 8, untransfected FTC007; Lane 9, hiPSCs generated using a lentiviral construct (serving as a control for cross-contamination); Lane 10, episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (3D) Pluripotency markers detected by immunofluorescence for the expression of OCT4, NANOG, TRA1-81, and TRA160. (3E) Flow cytometry profiles of selected hiPSC clones from various parental lines. The top row profile is SSEA4 / TRA1-81 surface expression. The bottom row profile is OCT4 / NANOG intracellular expression. (3F) qRT-PCR analysis of endogenous pluripotency gene expression. Data were normalized to GAPDH and compared to HUES9 hESCs. For KLF4 expression, two data points were greater than 15-fold higher than HUES9 and were recorded on the graph. Error bars represent the standard deviation of replicates. [Figure 4A]Figures 4A-4E. Genomic stability and pluripotency are maintained during continuous single-cell and FF culture. (4A) Cytogenetic analysis of 20-40 G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (4B) Flow cytometry profiles and cytogenetic analysis of long-term passage (p25-30) hiPSC clones in FF and single-cell culture. (4C) Directed differentiation of FTC017-c11 on days 3-4. (4D) Embryoid body formation and differentiation of hiPSC clones showing three germ layer differentiation. Immunocytochemical analysis performed 28 days after differentiation: ectoderm, TUJ1; mesoderm, alpha-smooth muscle actin (aSMA); endoderm, AFP. (4E) Histological sections of teratomas derived from FTC007-c21 and FTC016-c25, representing each somatic cell lineage. Black arrow, endoderm; white arrow, ectoderm; grey arrow, mesoderm. [Figure 4B] Figures 4A-4E. Genomic stability and pluripotency are maintained during continuous single-cell and FF culture. (4A) Cytogenetic analysis of 20-40 G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (4B) Flow cytometry profiles and cytogenetic analysis of long-term passage (p25-30) hiPSC clones in FF and single-cell culture. (4C) Directed differentiation of FTC017-c11 on days 3-4. (4D) Embryoid body formation and differentiation of hiPSC clones showing three germ layer differentiation. Immunocytochemical analysis performed 28 days after differentiation: ectoderm, TUJ1; mesoderm, alpha-smooth muscle actin (aSMA); endoderm, AFP. (4E) Histological sections of teratomas derived from FTC007-c21 and FTC016-c25, representing each somatic cell lineage. Black arrow, endoderm; white arrow, ectoderm; grey arrow, mesoderm. [Figure 4C]Figures 4A-4E. Genomic stability and pluripotency are maintained during continuous single-cell and FF culture. (4A) Cytogenetic analysis of 20-40 G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (4B) Flow cytometry profiles and cytogenetic analysis of long-term passage (p25-30) hiPSC clones in FF and single-cell culture. (4C) Directed differentiation of FTC017-c11 on days 3-4. (4D) Embryoid body formation and differentiation of hiPSC clones showing three germ layer differentiation. Immunocytochemical analysis performed 28 days after differentiation: ectoderm, TUJ1; mesoderm, alpha-smooth muscle actin (aSMA); endoderm, AFP. (4E) Histological sections of teratomas derived from FTC007-c21 and FTC016-c25, representing each somatic cell lineage. Black arrow, endoderm; white arrow, ectoderm; grey arrow, mesoderm. [Figure 4D] Figures 4A-4E. Genomic stability and pluripotency are maintained during continuous single-cell and FF culture. (4A) Cytogenetic analysis of 20-40 G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (4B) Flow cytometry profiles and cytogenetic analysis of long-term passage (p25-30) hiPSC clones in FF and single-cell culture. (4C) Directed differentiation of FTC017-c11 on days 3-4. (4D) Embryoid body formation and differentiation of hiPSC clones showing three germ layer differentiation. Immunocytochemical analysis performed 28 days after differentiation: ectoderm, TUJ1; mesoderm, alpha-smooth muscle actin (aSMA); endoderm, AFP. (4E) Histological sections of teratomas derived from FTC007-c21 and FTC016-c25, representing each somatic cell lineage. Black arrow, endoderm; white arrow, ectoderm; grey arrow, mesoderm. [Figure 4E]Figures 4A-4E. Genomic stability and pluripotency are maintained during continuous single-cell and FF culture. (4A) Cytogenetic analysis of 20-40 G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (4B) Flow cytometry profiles and cytogenetic analysis of long-term passage (p25-30) hiPSC clones in FF and single-cell culture. (4C) Directed differentiation of FTC017-c11 on days 3-4. (4D) Embryoid body formation and differentiation of hiPSC clones showing three germ layer differentiation. Immunocytochemical analysis performed 28 days after differentiation: ectoderm, TUJ1; mesoderm, alpha-smooth muscle actin (aSMA); endoderm, AFP. (4E) Histological sections of teratomas derived from FTC007-c21 and FTC016-c25, representing each somatic cell lineage. Black arrow, endoderm; white arrow, ectoderm; grey arrow, mesoderm. [Figure 5A]Figures 5A-5J. Derivation of hiPSC clones with a minimal number of reprogramming factors. (5A) OCT4, SOX2, and NANOG were cloned into pCEP4 in various formats. The table shows the vector systems and abbreviations. (5B) SSEA4 and TRA1-81 flow cytometry profiles of reprogramming kinetics induced by various gene combinations 13 days after induction. (5C) Efficiency histograms showing the presence of TRA1-81-positive hiPSC clones in wells of a 96-well plate at three and nine cells per well. (5D) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1: 2xO+OS+ONS+T-c7 p6; Lane 2: 2xO+OS+ONS+T-c10 p6; Lane 3: 2xO+ONS+T-c5 p5; Lane 4: 2xO+ONS+T-c9 p5; Lane 5: 2xO+OS+T-c7 p7; Lane 6: 2xO+OS+T-c9 p6; Lane 7: untransfected FTC007; Lane 8: hiPSCs generated using a lentiviral construct; Lane 9: episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (5E) Morphology of clone 9 derived from 2xO+OS+T. (5F) Pluripotency markers detected by immunofluorescence for expression of OCT4, NANOG, TRA1-81, and TRA160. Images were taken at 10x magnification. (5G) Flow profiles of hiPSC clones derived from selected gene sets. The top row of profiles represents SSEA4 / TRA1-81 surface expression. The bottom row of profiles represents OCT4 / NANOG intracellular expression. (5H) Directed differentiation of selected hiPSC clones approximately 72-96 hours after induction. (5I) Cytogenetic analysis of G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (5J) Histological sections of teratomas derived from hiPSC clone 2xO+OS+ONS+T-c10, representing each somatic cell lineage. Left panel, endoderm; middle panel, mesoderm; right panel, ectoderm. [Figure 5B]Figures 5A-5J. Derivation of hiPSC clones with a minimal number of reprogramming factors. (5A) OCT4, SOX2, and NANOG were cloned into pCEP4 in various formats. The table shows the vector systems and abbreviations. (5B) SSEA4 and TRA1-81 flow cytometry profiles of reprogramming kinetics induced by various gene combinations 13 days after induction. (5C) Efficiency histograms showing the presence of TRA1-81-positive hiPSC clones in wells of a 96-well plate at three and nine cells per well. (5D) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1: 2xO+OS+ONS+T-c7 p6; Lane 2: 2xO+OS+ONS+T-c10 p6; Lane 3: 2xO+ONS+T-c5 p5; Lane 4: 2xO+ONS+T-c9 p5; Lane 5: 2xO+OS+T-c7 p7; Lane 6: 2xO+OS+T-c9 p6; Lane 7: untransfected FTC007; Lane 8: hiPSCs generated using a lentiviral construct; Lane 9: episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (5E) Morphology of clone 9 derived from 2xO+OS+T. (5F) Pluripotency markers detected by immunofluorescence for expression of OCT4, NANOG, TRA1-81, and TRA160. Images were taken at 10x magnification. (5G) Flow profiles of hiPSC clones derived from selected gene sets. The top row of profiles represents SSEA4 / TRA1-81 surface expression. The bottom row of profiles represents OCT4 / NANOG intracellular expression. (5H) Directed differentiation of selected hiPSC clones approximately 72-96 hours after induction. (5I) Cytogenetic analysis of G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (5J) Histological sections of teratomas derived from hiPSC clone 2xO+OS+ONS+T-c10, representing each somatic cell lineage. Left panel, endoderm; middle panel, mesoderm; right panel, ectoderm. [Figure 5C]Figures 5A-5J. Derivation of hiPSC clones with a minimal number of reprogramming factors. (5A) OCT4, SOX2, and NANOG were cloned into pCEP4 in various formats. The table shows the vector systems and abbreviations. (5B) SSEA4 and TRA1-81 flow cytometry profiles of reprogramming kinetics induced by various gene combinations 13 days after induction. (5C) Efficiency histograms showing the presence of TRA1-81-positive hiPSC clones in wells of a 96-well plate at three and nine cells per well. (5D) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1: 2xO+OS+ONS+T-c7 p6; Lane 2: 2xO+OS+ONS+T-c10 p6; Lane 3: 2xO+ONS+T-c5 p5; Lane 4: 2xO+ONS+T-c9 p5; Lane 5: 2xO+OS+T-c7 p7; Lane 6: 2xO+OS+T-c9 p6; Lane 7: untransfected FTC007; Lane 8: hiPSCs generated using a lentiviral construct; Lane 9: episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (5E) Morphology of clone 9 derived from 2xO+OS+T. (5F) Pluripotency markers detected by immunofluorescence for expression of OCT4, NANOG, TRA1-81, and TRA160. Images were taken at 10x magnification. (5G) Flow profiles of hiPSC clones derived from selected gene sets. The top row of profiles represents SSEA4 / TRA1-81 surface expression. The bottom row of profiles represents OCT4 / NANOG intracellular expression. (5H) Directed differentiation of selected hiPSC clones approximately 72-96 hours after induction. (5I) Cytogenetic analysis of G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (5J) Histological sections of teratomas derived from hiPSC clone 2xO+OS+ONS+T-c10, representing each somatic cell lineage. Left panel, endoderm; middle panel, mesoderm; right panel, ectoderm. [Figure 5D]Figures 5A-5J. Derivation of hiPSC clones with a minimal number of reprogramming factors. (5A) OCT4, SOX2, and NANOG were cloned into pCEP4 in various formats. The table shows the vector systems and abbreviations. (5B) SSEA4 and TRA1-81 flow cytometry profiles of reprogramming kinetics induced by various gene combinations 13 days after induction. (5C) Efficiency histograms showing the presence of TRA1-81-positive hiPSC clones in wells of a 96-well plate at three and nine cells per well. (5D) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1: 2xO+OS+ONS+T-c7 p6; Lane 2: 2xO+OS+ONS+T-c10 p6; Lane 3: 2xO+ONS+T-c5 p5; Lane 4: 2xO+ONS+T-c9 p5; Lane 5: 2xO+OS+T-c7 p7; Lane 6: 2xO+OS+T-c9 p6; Lane 7: untransfected FTC007; Lane 8: hiPSCs generated using a lentiviral construct; Lane 9: episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (5E) Morphology of clone 9 derived from 2xO+OS+T. (5F) Pluripotency markers detected by immunofluorescence for expression of OCT4, NANOG, TRA1-81, and TRA160. Images were taken at 10x magnification. (5G) Flow profiles of hiPSC clones derived from selected gene sets. The top row of profiles represents SSEA4 / TRA1-81 surface expression. The bottom row of profiles represents OCT4 / NANOG intracellular expression. (5H) Directed differentiation of selected hiPSC clones approximately 72-96 hours after induction. (5I) Cytogenetic analysis of G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (5J) Histological sections of teratomas derived from hiPSC clone 2xO+OS+ONS+T-c10, representing each somatic cell lineage. Left panel, endoderm; middle panel, mesoderm; right panel, ectoderm. [Figure 5E]Figures 5A-5J. Derivation of hiPSC clones with a minimal number of reprogramming factors. (5A) OCT4, SOX2, and NANOG were cloned into pCEP4 in various formats. The table shows the vector systems and abbreviations. (5B) SSEA4 and TRA1-81 flow cytometry profiles of reprogramming kinetics induced by various gene combinations 13 days after induction. (5C) Efficiency histograms showing the presence of TRA1-81-positive hiPSC clones in wells of a 96-well plate at three and nine cells per well. (5D) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1: 2xO+OS+ONS+T-c7 p6; Lane 2: 2xO+OS+ONS+T-c10 p6; Lane 3: 2xO+ONS+T-c5 p5; Lane 4: 2xO+ONS+T-c9 p5; Lane 5: 2xO+OS+T-c7 p7; Lane 6: 2xO+OS+T-c9 p6; Lane 7: untransfected FTC007; Lane 8: hiPSCs generated using a lentiviral construct; Lane 9: episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (5E) Morphology of clone 9 derived from 2xO+OS+T. (5F) Pluripotency markers detected by immunofluorescence for expression of OCT4, NANOG, TRA1-81, and TRA160. Images were taken at 10x magnification. (5G) Flow profiles of hiPSC clones derived from selected gene sets. The top row of profiles represents SSEA4 / TRA1-81 surface expression. The bottom row of profiles represents OCT4 / NANOG intracellular expression. (5H) Directed differentiation of selected hiPSC clones approximately 72-96 hours after induction. (5I) Cytogenetic analysis of G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (5J) Histological sections of teratomas derived from hiPSC clone 2xO+OS+ONS+T-c10, representing each somatic cell lineage. Left panel, endoderm; middle panel, mesoderm; right panel, ectoderm. [Figure 5F]Figures 5A-5J. Derivation of hiPSC clones with a minimal number of reprogramming factors. (5A) OCT4, SOX2, and NANOG were cloned into pCEP4 in various formats. The table shows the vector systems and abbreviations. (5B) SSEA4 and TRA1-81 flow cytometry profiles of reprogramming kinetics induced by various gene combinations 13 days after induction. (5C) Efficiency histograms showing the presence of TRA1-81-positive hiPSC clones in wells of a 96-well plate at three and nine cells per well. (5D) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1: 2xO+OS+ONS+T-c7 p6; Lane 2: 2xO+OS+ONS+T-c10 p6; Lane 3: 2xO+ONS+T-c5 p5; Lane 4: 2xO+ONS+T-c9 p5; Lane 5: 2xO+OS+T-c7 p7; Lane 6: 2xO+OS+T-c9 p6; Lane 7: untransfected FTC007; Lane 8: hiPSCs generated using a lentiviral construct; Lane 9: episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (5E) Morphology of clone 9 derived from 2xO+OS+T. (5F) Pluripotency markers detected by immunofluorescence for expression of OCT4, NANOG, TRA1-81, and TRA160. Images were taken at 10x magnification. (5G) Flow profiles of hiPSC clones derived from selected gene sets. The top row of profiles represents SSEA4 / TRA1-81 surface expression. The bottom row of profiles represents OCT4 / NANOG intracellular expression. (5H) Directed differentiation of selected hiPSC clones approximately 72-96 hours after induction. (5I) Cytogenetic analysis of G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (5J) Histological sections of teratomas derived from hiPSC clone 2xO+OS+ONS+T-c10, representing each somatic cell lineage. Left panel, endoderm; middle panel, mesoderm; right panel, ectoderm. [Figure 5G]Figures 5A-5J. Derivation of hiPSC clones with a minimal number of reprogramming factors. (5A) OCT4, SOX2, and NANOG were cloned into pCEP4 in various formats. The table shows the vector systems and abbreviations. (5B) SSEA4 and TRA1-81 flow cytometry profiles of reprogramming kinetics induced by various gene combinations 13 days after induction. (5C) Efficiency histograms showing the presence of TRA1-81-positive hiPSC clones in wells of a 96-well plate at three and nine cells per well. (5D) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1: 2xO+OS+ONS+T-c7 p6; Lane 2: 2xO+OS+ONS+T-c10 p6; Lane 3: 2xO+ONS+T-c5 p5; Lane 4: 2xO+ONS+T-c9 p5; Lane 5: 2xO+OS+T-c7 p7; Lane 6: 2xO+OS+T-c9 p6; Lane 7: untransfected FTC007; Lane 8: hiPSCs generated using a lentiviral construct; Lane 9: episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (5E) Morphology of clone 9 derived from 2xO+OS+T. (5F) Pluripotency markers detected by immunofluorescence for expression of OCT4, NANOG, TRA1-81, and TRA160. Images were taken at 10x magnification. (5G) Flow profiles of hiPSC clones derived from selected gene sets. The top row of profiles represents SSEA4 / TRA1-81 surface expression. The bottom row of profiles represents OCT4 / NANOG intracellular expression. (5H) Directed differentiation of selected hiPSC clones approximately 72-96 hours after induction. (5I) Cytogenetic analysis of G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (5J) Histological sections of teratomas derived from hiPSC clone 2xO+OS+ONS+T-c10, representing each somatic cell lineage. Left panel, endoderm; middle panel, mesoderm; right panel, ectoderm. [Figure 5H]Figures 5A-5J. Derivation of hiPSC clones with a minimal number of reprogramming factors. (5A) OCT4, SOX2, and NANOG were cloned into pCEP4 in various formats. The table shows the vector systems and abbreviations. (5B) SSEA4 and TRA1-81 flow cytometry profiles of reprogramming kinetics induced by various gene combinations 13 days after induction. (5C) Efficiency histograms showing the presence of TRA1-81-positive hiPSC clones in wells of a 96-well plate at three and nine cells per well. (5D) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1: 2xO+OS+ONS+T-c7 p6; Lane 2: 2xO+OS+ONS+T-c10 p6; Lane 3: 2xO+ONS+T-c5 p5; Lane 4: 2xO+ONS+T-c9 p5; Lane 5: 2xO+OS+T-c7 p7; Lane 6: 2xO+OS+T-c9 p6; Lane 7: untransfected FTC007; Lane 8: hiPSCs generated using a lentiviral construct; Lane 9: episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (5E) Morphology of clone 9 derived from 2xO+OS+T. (5F) Pluripotency markers detected by immunofluorescence for expression of OCT4, NANOG, TRA1-81, and TRA160. Images were taken at 10x magnification. (5G) Flow profiles of hiPSC clones derived from selected gene sets. The top row of profiles represents SSEA4 / TRA1-81 surface expression. The bottom row of profiles represents OCT4 / NANOG intracellular expression. (5H) Directed differentiation of selected hiPSC clones approximately 72-96 hours after induction. (5I) Cytogenetic analysis of G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (5J) Histological sections of teratomas derived from hiPSC clone 2xO+OS+ONS+T-c10, representing each somatic cell lineage. Left panel, endoderm; middle panel, mesoderm; right panel, ectoderm. [Figure 5I]Figures 5A-5J. Derivation of hiPSC clones with a minimal number of reprogramming factors. (5A) OCT4, SOX2, and NANOG were cloned into pCEP4 in various formats. The table shows the vector systems and abbreviations. (5B) SSEA4 and TRA1-81 flow cytometry profiles of reprogramming kinetics induced by various gene combinations 13 days after induction. (5C) Efficiency histograms showing the presence of TRA1-81-positive hiPSC clones in wells of a 96-well plate at three and nine cells per well. (5D) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1: 2xO+OS+ONS+T-c7 p6; Lane 2: 2xO+OS+ONS+T-c10 p6; Lane 3: 2xO+ONS+T-c5 p5; Lane 4: 2xO+ONS+T-c9 p5; Lane 5: 2xO+OS+T-c7 p7; Lane 6: 2xO+OS+T-c9 p6; Lane 7: untransfected FTC007; Lane 8: hiPSCs generated using a lentiviral construct; Lane 9: episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (5E) Morphology of clone 9 derived from 2xO+OS+T. (5F) Pluripotency markers detected by immunofluorescence for expression of OCT4, NANOG, TRA1-81, and TRA160. Images were taken at 10x magnification. (5G) Flow profiles of hiPSC clones derived from selected gene sets. The top row of profiles represents SSEA4 / TRA1-81 surface expression. The bottom row of profiles represents OCT4 / NANOG intracellular expression. (5H) Directed differentiation of selected hiPSC clones approximately 72-96 hours after induction. (5I) Cytogenetic analysis of G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (5J) Histological sections of teratomas derived from hiPSC clone 2xO+OS+ONS+T-c10, representing each somatic cell lineage. Left panel, endoderm; middle panel, mesoderm; right panel, ectoderm. [Figure 5J]Figures 5A-5J. Derivation of hiPSC clones with a minimal number of reprogramming factors. (5A) OCT4, SOX2, and NANOG were cloned into pCEP4 in various formats. The table shows the vector systems and abbreviations. (5B) SSEA4 and TRA1-81 flow cytometry profiles of reprogramming kinetics induced by various gene combinations 13 days after induction. (5C) Efficiency histograms showing the presence of TRA1-81-positive hiPSC clones in wells of a 96-well plate at three and nine cells per well. (5D) PCR analysis of episomal DNA derived from various hiPSC clones. Lane 1: 2xO+OS+ONS+T-c7 p6; Lane 2: 2xO+OS+ONS+T-c10 p6; Lane 3: 2xO+ONS+T-c5 p5; Lane 4: 2xO+ONS+T-c9 p5; Lane 5: 2xO+OS+T-c7 p7; Lane 6: 2xO+OS+T-c9 p6; Lane 7: untransfected FTC007; Lane 8: hiPSCs generated using a lentiviral construct; Lane 9: episomal vector used as a positive control. An input of 100 ng of genomic DNA and 35 PCR cycles were used for all sets. (5E) Morphology of clone 9 derived from 2xO+OS+T. (5F) Pluripotency markers detected by immunofluorescence for expression of OCT4, NANOG, TRA1-81, and TRA160. Images were taken at 10x magnification. (5G) Flow profiles of hiPSC clones derived from selected gene sets. The top row of profiles represents SSEA4 / TRA1-81 surface expression. The bottom row of profiles represents OCT4 / NANOG intracellular expression. (5H) Directed differentiation of selected hiPSC clones approximately 72-96 hours after induction. (5I) Cytogenetic analysis of G-banded metaphase cells from various hiPSC clones maintained in FF and single-cell culture. (5J) Histological sections of teratomas derived from hiPSC clone 2xO+OS+ONS+T-c10, representing each somatic cell lineage. Left panel, endoderm; middle panel, mesoderm; right panel, ectoderm. [Figure 6A]Figures 6A-6B. Relative gene expression profiles of episomal-induced hiPSCs in minimal-factor FMM. Heatmap results obtained from Fluidigm dynamic sequencing show the relative gene expression levels (RQ) of pluripotency (6A) and differentiation (6B) genes for conventionally maintained hiPSC lines, conventionally maintained H1 hESCs, and episomal hiPSC lines derived using various gene combinations maintained in FMM. Relative gene expression for each line is color-coded based on the three expression levels noted within each box and summarized in the legend (bottom right). All sets were performed in duplicate and normalized to the mean expression of two housekeeping genes (GAPDH and HPRT1), with reference to the median expression levels of six control conventional lines (OSK hiPSCs and H1 hESCs on MEFs), representing 1× values. [Figure 6B] Figures 6A-6B. Relative gene expression profiles of episomal-induced hiPSCs in minimal-factor FMM. Heatmap results obtained from Fluidigm dynamic sequencing show the relative gene expression levels (RQ) of pluripotency (6A) and differentiation (6B) genes for conventionally maintained hiPSC lines, conventionally maintained H1 hESCs, and episomal hiPSC lines derived using various gene combinations maintained in FMM. Relative gene expression for each line is color-coded based on the three expression levels noted within each box and summarized in the legend (bottom right). All sets were performed in duplicate and normalized to the mean expression of two housekeeping genes (GAPDH and HPRT1), with reference to the median expression levels of six control conventional lines (OSK hiPSCs and H1 hESCs on MEFs), representing 1× values. [Figure 7A]Figures 7A-7G show that FMM-maintained hiPSCs have reduced expression of differentiation genes and represent a basal state. (7A) A total of 339 probe sets were differentially expressed by more than or less than 2.5-fold between conventional and FMM cultures. Hierarchical clustering analysis of the 339 probe sets using the complete linkage method based on the Euclidean distance measure. (7B) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 213 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to FMM culture). (7C) List of genes representative of the basal or metastable pluripotent state. The list was obtained from references mentioned in the text. (7D) Hierarchical clustering analysis of the 231 probe sets corresponding to the genes in (7C) using the complete linkage method based on the Euclidean distance measure. (7E) RMA (log2) intensity of the probe sets corresponding to the genes in (7C). The left panel represents 39 probe sets for the basal state, and the right panel represents 188 probe sets for the metastable state. The average conventional culture intensity level is plotted on the X-axis, and the average FMM / SMC4 intensity is plotted on the Y-axis; black lines indicate equal expression. (7F) Gene expression comparison of genes located on the X chromosome between hiPSC clones induced and cultured in conventional culture medium and their counterparts adapted to SMC4 using Affymetrix probe sets. The probe set associated with XIST gene expression is highlighted. (7G) Representative images of HEK27me3 on hiPSC clones maintained in FMM or adapted to conventional culture for five passages. The dotted arrow in the left panel points to a representative nucleus lacking H3K27me3 staining, while the solid arrow in the right panel points to a nucleus positive for H3K27me3 staining. The percentage of staining-positive nuclei is shown in the lower left corner of each panel. FMM-cultured cells have larger nuclei. Scale bar = 50 μm. [Figure 7B]Figures 7A-7G show that FMM-maintained hiPSCs have reduced expression of differentiation genes and represent a basal state. (7A) A total of 339 probe sets were differentially expressed by more than or less than 2.5-fold between conventional and FMM cultures. Hierarchical clustering analysis of the 339 probe sets using the complete linkage method based on the Euclidean distance measure. (7B) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 213 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to FMM culture). (7C) List of genes representative of the basal or metastable pluripotent state. The list was obtained from references mentioned in the text. (7D) Hierarchical clustering analysis of the 231 probe sets corresponding to the genes in (7C) using the complete linkage method based on the Euclidean distance measure. (7E) RMA (log2) intensity of the probe sets corresponding to the genes in (7C). The left panel represents 39 probe sets for the basal state, and the right panel represents 188 probe sets for the metastable state. The average conventional culture intensity level is plotted on the X-axis, and the average FMM / SMC4 intensity is plotted on the Y-axis; black lines indicate equal expression. (7F) Gene expression comparison of genes located on the X chromosome between hiPSC clones induced and cultured in conventional culture medium and their counterparts adapted to SMC4 using Affymetrix probe sets. The probe set associated with XIST gene expression is highlighted. (7G) Representative images of HEK27me3 on hiPSC clones maintained in FMM or adapted to conventional culture for five passages. The dotted arrow in the left panel points to a representative nucleus lacking H3K27me3 staining, while the solid arrow in the right panel points to a nucleus positive for H3K27me3 staining. The percentage of staining-positive nuclei is shown in the lower left corner of each panel. FMM-cultured cells have larger nuclei. Scale bar = 50 μm. [Figure 7C]Figures 7A-7G show that FMM-maintained hiPSCs have reduced expression of differentiation genes and represent a basal state. (7A) A total of 339 probe sets were differentially expressed by more than or less than 2.5-fold between conventional and FMM cultures. Hierarchical clustering analysis of the 339 probe sets using the complete linkage method based on the Euclidean distance measure. (7B) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 213 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to FMM culture). (7C) List of genes representative of the basal or metastable pluripotent state. The list was obtained from references mentioned in the text. (7D) Hierarchical clustering analysis of the 231 probe sets corresponding to the genes in (7C) using the complete linkage method based on the Euclidean distance measure. (7E) RMA (log2) intensity of the probe sets corresponding to the genes in (7C). The left panel represents 39 probe sets for the basal state, and the right panel represents 188 probe sets for the metastable state. The average conventional culture intensity level is plotted on the X-axis, and the average FMM / SMC4 intensity is plotted on the Y-axis; black lines indicate equal expression. (7F) Gene expression comparison of genes located on the X chromosome between hiPSC clones induced and cultured in conventional culture medium and their counterparts adapted to SMC4 using Affymetrix probe sets. The probe set associated with XIST gene expression is highlighted. (7G) Representative images of HEK27me3 on hiPSC clones maintained in FMM or adapted to conventional culture for five passages. The dotted arrow in the left panel points to a representative nucleus lacking H3K27me3 staining, while the solid arrow in the right panel points to a nucleus positive for H3K27me3 staining. The percentage of staining-positive nuclei is shown in the lower left corner of each panel. FMM-cultured cells have larger nuclei. Scale bar = 50 μm. [Figure 7D]Figures 7A-7G show that FMM-maintained hiPSCs have reduced expression of differentiation genes and represent a basal state. (7A) A total of 339 probe sets were differentially expressed by more than or less than 2.5-fold between conventional and FMM cultures. Hierarchical clustering analysis of the 339 probe sets using the complete linkage method based on the Euclidean distance measure. (7B) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 213 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to FMM culture). (7C) List of genes representative of the basal or metastable pluripotent state. The list was obtained from references mentioned in the text. (7D) Hierarchical clustering analysis of the 231 probe sets corresponding to the genes in (7C) using the complete linkage method based on the Euclidean distance measure. (7E) RMA (log2) intensity of the probe sets corresponding to the genes in (7C). The left panel represents 39 probe sets for the basal state, and the right panel represents 188 probe sets for the metastable state. The average conventional culture intensity level is plotted on the X-axis, and the average FMM / SMC4 intensity is plotted on the Y-axis; black lines indicate equal expression. (7F) Gene expression comparison of genes located on the X chromosome between hiPSC clones induced and cultured in conventional culture medium and their counterparts adapted to SMC4 using Affymetrix probe sets. The probe set associated with XIST gene expression is highlighted. (7G) Representative images of HEK27me3 on hiPSC clones maintained in FMM or adapted to conventional culture for five passages. The dotted arrow in the left panel points to a representative nucleus lacking H3K27me3 staining, while the solid arrow in the right panel points to a nucleus positive for H3K27me3 staining. The percentage of staining-positive nuclei is shown in the lower left corner of each panel. FMM-cultured cells have larger nuclei. Scale bar = 50 μm. [Figure 7E]Figures 7A-7G show that FMM-maintained hiPSCs have reduced expression of differentiation genes and represent a basal state. (7A) A total of 339 probe sets were differentially expressed by more than or less than 2.5-fold between conventional and FMM cultures. Hierarchical clustering analysis of the 339 probe sets using the complete linkage method based on the Euclidean distance measure. (7B) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 213 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to FMM culture). (7C) List of genes representative of the basal or metastable pluripotent state. The list was obtained from references mentioned in the text. (7D) Hierarchical clustering analysis of the 231 probe sets corresponding to the genes in (7C) using the complete linkage method based on the Euclidean distance measure. (7E) RMA (log2) intensity of the probe sets corresponding to the genes in (7C). The left panel represents 39 probe sets for the basal state, and the right panel represents 188 probe sets for the metastable state. The average conventional culture intensity level is plotted on the X-axis, and the average FMM / SMC4 intensity is plotted on the Y-axis; black lines indicate equal expression. (7F) Gene expression comparison of genes located on the X chromosome between hiPSC clones induced and cultured in conventional culture medium and their counterparts adapted to SMC4 using Affymetrix probe sets. The probe set associated with XIST gene expression is highlighted. (7G) Representative images of HEK27me3 on hiPSC clones maintained in FMM or adapted to conventional culture for five passages. The dotted arrow in the left panel points to a representative nucleus lacking H3K27me3 staining, while the solid arrow in the right panel points to a nucleus positive for H3K27me3 staining. The percentage of staining-positive nuclei is shown in the lower left corner of each panel. FMM-cultured cells have larger nuclei. Scale bar = 50 μm. [Figure 7F]Figures 7A-7G show that FMM-maintained hiPSCs have reduced expression of differentiation genes and represent a basal state. (7A) A total of 339 probe sets were differentially expressed by more than or less than 2.5-fold between conventional and FMM cultures. Hierarchical clustering analysis of the 339 probe sets using the complete linkage method based on the Euclidean distance measure. (7B) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 213 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to FMM culture). (7C) List of genes representative of the basal or metastable pluripotent state. The list was obtained from references mentioned in the text. (7D) Hierarchical clustering analysis of the 231 probe sets corresponding to the genes in (7C) using the complete linkage method based on the Euclidean distance measure. (7E) RMA (log2) intensity of the probe sets corresponding to the genes in (7C). The left panel represents 39 probe sets for the basal state, and the right panel represents 188 probe sets for the metastable state. The average conventional culture intensity level is plotted on the X-axis, and the average FMM / SMC4 intensity is plotted on the Y-axis; black lines indicate equal expression. (7F) Gene expression comparison of genes located on the X chromosome between hiPSC clones induced and cultured in conventional culture medium and their counterparts adapted to SMC4 using Affymetrix probe sets. The probe set associated with XIST gene expression is highlighted. (7G) Representative images of HEK27me3 on hiPSC clones maintained in FMM or adapted to conventional culture for five passages. The dotted arrow in the left panel points to a representative nucleus lacking H3K27me3 staining, while the solid arrow in the right panel points to a nucleus positive for H3K27me3 staining. The percentage of staining-positive nuclei is shown in the lower left corner of each panel. FMM-cultured cells have larger nuclei. Scale bar = 50 μm. [Figure 7G]Figures 7A-7G show that FMM-maintained hiPSCs have reduced expression of differentiation genes and represent a basal state. (7A) A total of 339 probe sets were differentially expressed by more than or less than 2.5-fold between conventional and FMM cultures. Hierarchical clustering analysis of the 339 probe sets using the complete linkage method based on the Euclidean distance measure. (7B) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 213 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to FMM culture). (7C) List of genes representative of the basal or metastable pluripotent state. The list was obtained from references mentioned in the text. (7D) Hierarchical clustering analysis of the 231 probe sets corresponding to the genes in (7C) using the complete linkage method based on the Euclidean distance measure. (7E) RMA (log2) intensity of the probe sets corresponding to the genes in (7C). The left panel represents 39 probe sets for the basal state, and the right panel represents 188 probe sets for the metastable state. The average conventional culture intensity level is plotted on the X-axis, and the average FMM / SMC4 intensity is plotted on the Y-axis; black lines indicate equal expression. (7F) Gene expression comparison of genes located on the X chromosome between hiPSC clones induced and cultured in conventional culture medium and their counterparts adapted to SMC4 using Affymetrix probe sets. The probe set associated with XIST gene expression is highlighted. (7G) Representative images of HEK27me3 on hiPSC clones maintained in FMM or adapted to conventional culture for five passages. The dotted arrow in the left panel points to a representative nucleus lacking H3K27me3 staining, while the solid arrow in the right panel points to a nucleus positive for H3K27me3 staining. The percentage of staining-positive nuclei is shown in the lower left corner of each panel. FMM-cultured cells have larger nuclei. Scale bar = 50 μm. [Figure 8A]Figures 8A-8D. Episomal-induced reprogramming by FRM and FMM. (8A) SSEA4 and TRA1-81 flow profiles of a reprogrammed pool at day 10. (8B) Representative morphology of a typical colony observed during reprogramming. Images were taken 13 days after transfection. (8C) Episomal-reprogrammed fibroblasts maintained in FRM for the first 14 days were split and either maintained in FRM or switched to FMM. Then, 21 days after transfection, reprogrammed cultures were sorted to SSEA4 / TRA1-81 / CD30 and maintained in FRM or FMM for an additional 10 days before analysis. (8D) Morphology and flow profiles of representative cultures in FRM or FMM. White arrows point to regions of differentiated cells in cultures consisting of a mixture of undifferentiated and differentiated populations. Black arrows point to the distinct edge of the mostly undifferentiated population. The bottom panel shows a representative flow profile. FSC; forward / side scatter. [Figure 8B] Figures 8A-8D. Episomal-induced reprogramming by FRM and FMM. (8A) SSEA4 and TRA1-81 flow profiles of a reprogrammed pool at day 10. (8B) Representative morphology of a typical colony observed during reprogramming. Images were taken 13 days after transfection. (8C) Episomal-reprogrammed fibroblasts maintained in FRM for the first 14 days were split and either maintained in FRM or switched to FMM. Then, 21 days after transfection, reprogrammed cultures were sorted to SSEA4 / TRA1-81 / CD30 and maintained in FRM or FMM for an additional 10 days before analysis. (8D) Morphology and flow profiles of representative cultures in FRM or FMM. White arrows point to regions of differentiated cells in cultures consisting of a mixture of undifferentiated and differentiated populations. Black arrows point to the distinct edge of the mostly undifferentiated population. The bottom panel shows a representative flow profile. FSC; forward / side scatter. [Figure 8C]Figures 8A-8D. Episomal-induced reprogramming by FRM and FMM. (8A) SSEA4 and TRA1-81 flow profiles of a reprogrammed pool at day 10. (8B) Representative morphology of a typical colony observed during reprogramming. Images were taken 13 days after transfection. (8C) Episomal-reprogrammed fibroblasts maintained in FRM for the first 14 days were split and either maintained in FRM or switched to FMM. Then, 21 days after transfection, reprogrammed cultures were sorted to SSEA4 / TRA1-81 / CD30 and maintained in FRM or FMM for an additional 10 days before analysis. (8D) Morphology and flow profiles of representative cultures in FRM or FMM. White arrows point to regions of differentiated cells in cultures consisting of a mixture of undifferentiated and differentiated populations. Black arrows point to the distinct edge of the mostly undifferentiated population. The bottom panel shows a representative flow profile. FSC; forward / side scatter. [Figure 8D] Figures 8A-8D. Episomal-induced reprogramming by FRM and FMM. (8A) SSEA4 and TRA1-81 flow profiles of a reprogrammed pool at day 10. (8B) Representative morphology of a typical colony observed during reprogramming. Images were taken 13 days after transfection. (8C) Episomal-reprogrammed fibroblasts maintained in FRM for the first 14 days were split and either maintained in FRM or switched to FMM. Then, 21 days after transfection, reprogrammed cultures were sorted to SSEA4 / TRA1-81 / CD30 and maintained in FRM or FMM for an additional 10 days before analysis. (8D) Morphology and flow profiles of representative cultures in FRM or FMM. White arrows point to regions of differentiated cells in cultures consisting of a mixture of undifferentiated and differentiated populations. Black arrows point to the distinct edge of the mostly undifferentiated population. The bottom panel shows a representative flow profile. FSC; forward / side scatter. [Figure 9A]Figures 9A-9B. Reprogramming of various parental lines. (9A) Summary table of starting cell lines used in this study. Specific information associated with each line is shown, along with the percentage of the positive SSEA4 / TRA1-81 / CD30 population at the time of sorting after episomal transfection. (9B) Sorting and culture of CD34-rich cord blood cells. A volume of 0.5 ml of previously banked cord blood was used to extract 65,000 CD34+CD45+Lin− cells, which were cultured in suspension for 6 days before episomal transfection. [Figure 9B] Figures 9A-9B. Reprogramming of various parental lines. (9A) Summary table of starting cell lines used in this study. Specific information associated with each line is shown, along with the percentage of the positive SSEA4 / TRA1-81 / CD30 population at the time of sorting after episomal transfection. (9B) Sorting and culture of CD34-rich cord blood cells. A volume of 0.5 ml of previously banked cord blood was used to extract 65,000 CD34+CD45+Lin− cells, which were cultured in suspension for 6 days before episomal transfection. [Figure 10A] Figures 10A-10G. Characterization of hiPSCs during the reprogramming and maintenance process. (10A) Typical colony morphology 3 days after single-cell 96-well plate sorting. Scale bar represents 400 μm. (10B) Representative morphology of single-cell-derived hiPSC-like colonies 7-9 days after sorting from various starting cells. Scale bar represents 1000 μm. (10C) Immunocytochemical analysis of NANOG expression in hiPSC-like colonies in 96-well plates. (10D) Flow profile analysis of FTC007, induced to reprogram and maintained on Matrigel- or vitronectin-coated culture plates, on day 16. (10E) Brightfield image, (10F) immunofluorescence of OCT4 and NANOG, or (10G) flow cytometry analysis of SSEA4 and TRA1-81 in FTC016-c28, maintained continuously on Matrigel or in FMM for 5 passages on vitronectin. [Figure 10B] Figures 10A-10G. Characterization of hiPSCs during the reprogramming and maintenance process. (10A) Typical colony morphology 3 days after single-cell 96-well plate sorting. Scale bar represents 400 μm. (10B) Representative morphology of single-cell-derived hiPSC-like colonies 7-9 days after sorting from various starting cells. Scale bar represents 1000 μm. (10C) Immunocytochemical analysis of NANOG expression in hiPSC-like colonies in 96-well plates. (10D) Flow profile analysis of FTC007, induced to reprogram and maintained on Matrigel- or vitronectin-coated culture plates, on day 16. (10E) Brightfield image, (10F) immunofluorescence of OCT4 and NANOG, or (10G) flow cytometry analysis of SSEA4 and TRA1-81 in FTC016-c28, maintained continuously on Matrigel or in FMM for 5 passages on vitronectin. [Figure 10C] Figures 10A-10G. Characterization of hiPSCs during the reprogramming and maintenance process. (10A) Typical colony morphology 3 days after single-cell 96-well plate sorting. Scale bar represents 400 μm. (10B) Representative morphology of single-cell-derived hiPSC-like colonies 7-9 days after sorting from various starting cells. Scale bar represents 1000 μm. (10C) Immunocytochemical analysis of NANOG expression in hiPSC-like colonies in 96-well plates. (10D) Flow profile analysis of FTC007, induced to reprogram and maintained on Matrigel- or vitronectin-coated culture plates, on day 16. (10E) Brightfield image, (10F) immunofluorescence of OCT4 and NANOG, or (10G) flow cytometry analysis of SSEA4 and TRA1-81 in FTC016-c28, maintained continuously on Matrigel or in FMM for 5 passages on vitronectin. [Figure 10D]Figures 10A-10G. Characterization of hiPSCs during the reprogramming and maintenance process. (10A) Typical colony morphology 3 days after single-cell 96-well plate sorting. Scale bar represents 400 μm. (10B) Representative morphology of single-cell-derived hiPSC-like colonies 7-9 days after sorting from various starting cells. Scale bar represents 1000 μm. (10C) Immunocytochemical analysis of NANOG expression in hiPSC-like colonies in 96-well plates. (10D) Flow profile analysis of FTC007, induced to reprogram and maintained on Matrigel- or vitronectin-coated culture plates, on day 16. (10E) Brightfield image, (10F) immunofluorescence of OCT4 and NANOG, or (10G) flow cytometry analysis of SSEA4 and TRA1-81 in FTC016-c28, maintained continuously on Matrigel or in FMM for 5 passages on vitronectin. [Figure 10E] Figures 10A-10G. Characterization of hiPSCs during the reprogramming and maintenance process. (10A) Typical colony morphology 3 days after single-cell 96-well plate sorting. Scale bar represents 400 μm. (10B) Representative morphology of single-cell-derived hiPSC-like colonies 7-9 days after sorting from various starting cells. Scale bar represents 1000 μm. (10C) Immunocytochemical analysis of NANOG expression in hiPSC-like colonies in 96-well plates. (10D) Flow profile analysis of FTC007, induced to reprogram and maintained on Matrigel- or vitronectin-coated culture plates, on day 16. (10E) Brightfield image, (10F) immunofluorescence of OCT4 and NANOG, or (10G) flow cytometry analysis of SSEA4 and TRA1-81 in FTC016-c28, maintained continuously on Matrigel or in FMM for 5 passages on vitronectin. [Figure 10F]Figures 10A-10G. Characterization of hiPSCs during the reprogramming and maintenance process. (10A) Typical colony morphology 3 days after single-cell 96-well plate sorting. Scale bar represents 400 μm. (10B) Representative morphology of single-cell-derived hiPSC-like colonies 7-9 days after sorting from various starting cells. Scale bar represents 1000 μm. (10C) Immunocytochemical analysis of NANOG expression in hiPSC-like colonies in 96-well plates. (10D) Flow profile analysis of FTC007, induced to reprogram and maintained on Matrigel- or vitronectin-coated culture plates, on day 16. (10E) Brightfield image, (10F) immunofluorescence of OCT4 and NANOG, or (10G) flow cytometry analysis of SSEA4 and TRA1-81 in FTC016-c28, maintained continuously on Matrigel or in FMM for 5 passages on vitronectin. [Figure 10G] Figures 10A-10G. Characterization of hiPSCs during the reprogramming and maintenance process. (10A) Typical colony morphology 3 days after single-cell 96-well plate sorting. Scale bar represents 400 μm. (10B) Representative morphology of single-cell-derived hiPSC-like colonies 7-9 days after sorting from various starting cells. Scale bar represents 1000 μm. (10C) Immunocytochemical analysis of NANOG expression in hiPSC-like colonies in 96-well plates. (10D) Flow profile analysis of FTC007, induced to reprogram and maintained on Matrigel- or vitronectin-coated culture plates, on day 16. (10E) Brightfield image, (10F) immunofluorescence of OCT4 and NANOG, or (10G) flow cytometry analysis of SSEA4 and TRA1-81 in FTC016-c28, maintained continuously on Matrigel or in FMM for 5 passages on vitronectin. [Figure 11A]11A-11C. Examples of minimal gene reprogramming using the FMM culture platform. (11A) Morphology of cells treated with hygromycin 2-5 days after transfection with an episomal construct containing a hygromycin selection cassette. (11B) Reprogrammed pools were maintained for longer periods and profiled 16 days after transfection. (11C) Appearance of cultures maintained on Matrigel or vitronectin. [Figure 11B] 11A-11C. Examples of minimal gene reprogramming using the FMM culture platform. (11A) Morphology of cells treated with hygromycin 2-5 days after transfection with an episomal construct containing a hygromycin selection cassette. (11B) Reprogrammed pools were maintained for longer periods and profiled 16 days after transfection. (11C) Appearance of cultures maintained on Matrigel or vitronectin. [Figure 11C] 11A-11C. Examples of minimal gene reprogramming using the FMM culture platform. (11A) Morphology of cells treated with hygromycin 2-5 days after transfection with an episomal construct containing a hygromycin selection cassette. (11B) Reprogrammed pools were maintained for longer periods and profiled 16 days after transfection. (11C) Appearance of cultures maintained on Matrigel or vitronectin. [Figure 12A]Figures 12A-12E. Characterization of hiPSCs cultured under multiple conditions. (12A) Lentiviral-induced and SMC4-maintained FTi111 demonstrated the hallmarks of pluripotency and maintained genomic integrity. (12B) Depiction of the thawing strategy for FTi111 p43. A single virus was thawed into the four culture environments as indicated. Surviving cultures were passaged in each culture, except for the conventional culture with thiazovivin supplementation on feeder cells, which was transferred to conventional culture without thiazovivin in the presence of feeder cells and passaged as clumps. (12C) Morphology of recovered cells in various cultures after thawing. No surviving cells were identified in the conventional culture without thiazovivin in the presence of feeder cells. (12D) Morphology of a set of cultures at passage 3 after thawing. Larger colony morphology was associated with the conventional culture. Scale bar: 1000 μm. (12E) qRT-PCR analysis of endogenous pluripotency gene expression in each culture set. Data were normalized to GAPDH and compared to H1 hESCs. [Figure 12B] Figures 12A-12E. Characterization of hiPSCs cultured under multiple conditions. (12A) Lentiviral-induced and SMC4-maintained FTi111 demonstrated the hallmarks of pluripotency and maintained genomic integrity. (12B) Depiction of the thawing strategy for FTi111 p43. A single virus was thawed into the four culture environments as indicated. Surviving cultures were passaged in each culture, except for the conventional culture with thiazovivin supplementation on feeder cells, which was transferred to conventional culture without thiazovivin in the presence of feeder cells and passaged as clumps. (12C) Morphology of recovered cells in various cultures after thawing. No surviving cells were identified in the conventional culture without thiazovivin in the presence of feeder cells. (12D) Morphology of a set of cultures at passage 3 after thawing. Larger colony morphology was associated with the conventional culture. Scale bar: 1000 μm. (12E) qRT-PCR analysis of endogenous pluripotency gene expression in each culture set. Data were normalized to GAPDH and compared to H1 hESCs. [Figure 12C]Figures 12A-12E. Characterization of hiPSCs cultured under multiple conditions. (12A) Lentiviral-induced and SMC4-maintained FTi111 demonstrated the hallmarks of pluripotency and maintained genomic integrity. (12B) Depiction of the thawing strategy for FTi111 p43. A single virus was thawed into the four culture environments as indicated. Surviving cultures were passaged in each culture, except for the conventional culture with thiazovivin supplementation on feeder cells, which was transferred to conventional culture without thiazovivin in the presence of feeder cells and passaged as clumps. (12C) Morphology of recovered cells in various cultures after thawing. No surviving cells were identified in the conventional culture without thiazovivin in the presence of feeder cells. (12D) Morphology of a set of cultures at passage 3 after thawing. Larger colony morphology was associated with the conventional culture. Scale bar: 1000 μm. (12E) qRT-PCR analysis of endogenous pluripotency gene expression in each culture set. Data were normalized to GAPDH and compared to H1 hESCs. [Figure 12D] Figures 12A-12E. Characterization of hiPSCs cultured under multiple conditions. (12A) Lentiviral-induced and SMC4-maintained FTi111 demonstrated the hallmarks of pluripotency and maintained genomic integrity. (12B) Depiction of the thawing strategy for FTi111 p43. A single virus was thawed into the four culture environments as indicated. Surviving cultures were passaged in each culture, except for the conventional culture with thiazovivin supplementation on feeder cells, which was transferred to conventional culture without thiazovivin in the presence of feeder cells and passaged as clumps. (12C) Morphology of recovered cells in various cultures after thawing. No surviving cells were identified in the conventional culture without thiazovivin in the presence of feeder cells. (12D) Morphology of a set of cultures at passage 3 after thawing. Larger colony morphology was associated with the conventional culture. Scale bar: 1000 μm. (12E) qRT-PCR analysis of endogenous pluripotency gene expression in each culture set. Data were normalized to GAPDH and compared to H1 hESCs. [Figure 12E]Figures 12A-12E. Characterization of hiPSCs cultured under multiple conditions. (12A) Lentiviral-induced and SMC4-maintained FTi111 demonstrated the hallmarks of pluripotency and maintained genomic integrity. (12B) Depiction of the thawing strategy for FTi111 p43. A single virus was thawed into the four culture environments as indicated. Surviving cultures were passaged in each culture, except for the conventional culture with thiazovivin supplementation on feeder cells, which was transferred to conventional culture without thiazovivin in the presence of feeder cells and passaged as clumps. (12C) Morphology of recovered cells in various cultures after thawing. No surviving cells were identified in the conventional culture without thiazovivin in the presence of feeder cells. (12D) Morphology of a set of cultures at passage 3 after thawing. Larger colony morphology was associated with the conventional culture. Scale bar: 1000 μm. (12E) qRT-PCR analysis of endogenous pluripotency gene expression in each culture set. Data were normalized to GAPDH and compared to H1 hESCs. [Figure 13A] Figures 13A-13E. Gene ontology diagrams of gene expression profiles of hiPSCs cultured under various conditions. (13A) Table describing the derivation and maintenance of each line described in the global gene expression study. (13B) A total of 300 probe sets were differentially expressed by more than 2.5-fold or less than 2.5-fold between conventional and small molecule (FMM and SMC4) culture conditions. Hierarchical class multi-analysis of the 300 probe sets using complete linkage based on Euclidean distance measurement. (13C) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 133 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to small molecule culture). (13D) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 167 probe sets that were upregulated by more than 2.5-fold in small molecule culture (compared to conventional culture). (13E) Gene Ontology biological process enrichment analysis of 126 probe sets that were upregulated by 2.5-fold or more by FMM culture (compared to conventional culture). [Figure 13B]Figures 13A-13E. Gene ontology diagrams of gene expression profiles of hiPSCs cultured under various conditions. (13A) Table describing the derivation and maintenance of each line described in the global gene expression study. (13B) A total of 300 probe sets were differentially expressed by more than 2.5-fold or less than 2.5-fold between conventional and small molecule (FMM and SMC4) culture conditions. Hierarchical class multi-analysis of the 300 probe sets using complete linkage based on Euclidean distance measurement. (13C) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 133 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to small molecule culture). (13D) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 167 probe sets that were upregulated by more than 2.5-fold in small molecule culture (compared to conventional culture). (13E) Gene Ontology biological process enrichment analysis of 126 probe sets that were upregulated by 2.5-fold or more by FMM culture (compared to conventional culture). [Figure 13C] Figures 13A-13E. Gene ontology diagrams of gene expression profiles of hiPSCs cultured under various conditions. (13A) Table describing the derivation and maintenance of each line described in the global gene expression study. (13B) A total of 300 probe sets were differentially expressed by more than 2.5-fold or less than 2.5-fold between conventional and small molecule (FMM and SMC4) culture conditions. Hierarchical class multi-analysis of the 300 probe sets using complete linkage based on Euclidean distance measurement. (13C) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 133 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to small molecule culture). (13D) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 167 probe sets that were upregulated by more than 2.5-fold in small molecule culture (compared to conventional culture). (13E) Gene Ontology biological process enrichment analysis of 126 probe sets that were upregulated by 2.5-fold or more by FMM culture (compared to conventional culture). [Figure 13D]Figures 13A-13E. Gene ontology diagrams of gene expression profiles of hiPSCs cultured under various conditions. (13A) Table describing the derivation and maintenance of each line described in the global gene expression study. (13B) A total of 300 probe sets were differentially expressed by more than 2.5-fold or less than 2.5-fold between conventional and small molecule (FMM and SMC4) culture conditions. Hierarchical class multi-analysis of the 300 probe sets using complete linkage based on Euclidean distance measurement. (13C) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 133 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to small molecule culture). (13D) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 167 probe sets that were upregulated by more than 2.5-fold in small molecule culture (compared to conventional culture). (13E) Gene Ontology biological process enrichment analysis of 126 probe sets that were upregulated by 2.5-fold or more by FMM culture (compared to conventional culture). [Figure 13E] Figures 13A-13E. Gene ontology diagrams of gene expression profiles of hiPSCs cultured under various conditions. (13A) Table describing the derivation and maintenance of each line described in the global gene expression study. (13B) A total of 300 probe sets were differentially expressed by more than 2.5-fold or less than 2.5-fold between conventional and small molecule (FMM and SMC4) culture conditions. Hierarchical class multi-analysis of the 300 probe sets using complete linkage based on Euclidean distance measurement. (13C) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 133 probe sets that were upregulated by more than 2.5-fold in conventional culture (compared to small molecule culture). (13D) Gene Ontology Biological Process Enrichment Analysis (DAVID) of 167 probe sets that were upregulated by more than 2.5-fold in small molecule culture (compared to conventional culture). (13E) Gene Ontology biological process enrichment analysis of 126 probe sets that were upregulated by 2.5-fold or more by FMM culture (compared to conventional culture). [Figure 14A]Figures 14A-14F. Cloning maps showing examples of lentiviral (14A-14B) and episomal (14C-14F) constructs used for reprogramming. The lentiviral constructs contain the EF1α promoter and LOXP sites for CRE-mediated cleavage of the transgene. The episomal constructs also contain the EF1α promoter. [Figure 14B] Figures 14A-14F. Cloning maps showing examples of lentiviral (14A-14B) and episomal (14C-14F) constructs used for reprogramming. The lentiviral constructs contain the EF1α promoter and LOXP sites for CRE-mediated cleavage of the transgene. The episomal constructs also contain the EF1α promoter. [Figure 14C] Figures 14A-14F. Cloning maps showing examples of lentiviral (14A-14B) and episomal (14C-14F) constructs used for reprogramming. The lentiviral constructs contain the EF1α promoter and LOXP sites for CRE-mediated cleavage of the transgene. The episomal constructs also contain the EF1α promoter. [Figure 14D] Figures 14A-14F. Cloning maps showing examples of lentiviral (14A-14B) and episomal (14C-14F) constructs used for reprogramming. The lentiviral constructs contain the EF1α promoter and LOXP sites for CRE-mediated cleavage of the transgene. The episomal constructs also contain the EF1α promoter. [Figure 14E] Figures 14A-14F. Cloning maps showing examples of lentiviral (14A-14B) and episomal (14C-14F) constructs used for reprogramming. The lentiviral constructs contain the EF1α promoter and LOXP sites for CRE-mediated cleavage of the transgene. The episomal constructs also contain the EF1α promoter. [Figure 14F] Figures 14A-14F. Cloning maps showing examples of lentiviral (14A-14B) and episomal (14C-14F) constructs used for reprogramming. The lentiviral constructs contain the EF1α promoter and LOXP sites for CRE-mediated cleavage of the transgene. The episomal constructs also contain the EF1α promoter. [Figure 15A] 15A-15C. Representative flow analysis of various reprogramming factor combinations on days 8-15. Human fibroblasts were attracted with various combinations of lentivirus-mediated reprogramming factors, including OCT4, ECAT1, and UTF1. [Figure 15B] 15A-15C. Representative flow analysis of various reprogramming factor combinations on days 8-15. Human fibroblasts were attracted with various combinations of lentivirus-mediated reprogramming factors, including OCT4, ECAT1, and UTF1. [Figure 15C] 15A-15C. Representative flow analysis of various reprogramming factor combinations on days 8-15. Human fibroblasts were attracted with various combinations of lentivirus-mediated reprogramming factors, including OCT4, ECAT1, and UTF1. [Figure 16A] Figures 16A-16D show representative flow analysis and iPSC morphology characteristics for various reprogramming factor combinations on days 21-27. The data demonstrate that unique reprogramming combinations can be used to derive SSEA4+ / TRA181+ hiPSCs. [Figure 16B] Figures 16A-16D show representative flow analysis and iPSC morphology characteristics for various reprogramming factor combinations on days 21-27. The data demonstrate that unique reprogramming combinations can be used to derive SSEA4+ / TRA181+ hiPSCs. [Figure 16C]Figures 16A-16D show representative flow analysis and iPSC morphology characteristics for various reprogramming factor combinations on days 21-27. The data demonstrate that unique reprogramming combinations can be used to derive SSEA4+ / TRA181+ hiPSCs. [Figure 16D] Figures 16A-16D show representative flow analysis and iPSC morphology characteristics for various reprogramming factor combinations on days 21-27. The data demonstrate that unique reprogramming combinations can be used to derive SSEA4+ / TRA181+ hiPSCs. [Figure 17A] 17A-17B. Highly improved lentiviral reprogramming efficiency as demonstrated by flow analysis (SSEA4+ / TRA181+ and CD30+ populations) and iPSC morphology characterization. Human fibroblasts were reprogrammed with OCT4, ECAT1, UTF1, ESRRB, and NANOG. Cells were reprogrammed using FRM and maintained in FMM. [Figure 17B] 17A-17B. Highly improved lentiviral reprogramming efficiency as demonstrated by flow analysis (SSEA4+ / TRA181+ and CD30+ populations) and iPSC morphology characterization. Human fibroblasts were reprogrammed with OCT4, ECAT1, UTF1, ESRRB, and NANOG. Cells were reprogrammed using FRM and maintained in FMM. [Figure 18A] Figures 18A-18D show representative flow analysis and phase images of four established iPSC clones after 7-9 passages after 96-well sorting. Clones were generated with lentiviral reprogramming factors (OCT4, ECAT1, UTF1, ESRRB, and NANOG) using FMM and maintained in FMM. The population expressing high SSEA4+ / TRA181+ indicates pluripotency. [Figure 18B]Figures 18A-18D show representative flow analysis and phase images of four established iPSC clones after 7-9 passages after 96-well sorting. Clones were generated with lentiviral reprogramming factors (OCT4, ECAT1, UTF1, ESRRB, and NANOG) using FMM and maintained in FMM. The population expressing high SSEA4+ / TRA181+ indicates pluripotency. [Figure 18C] Figures 18A-18D show representative flow analysis and phase images of four established iPSC clones after 7-9 passages after 96-well sorting. Clones were generated with lentiviral reprogramming factors (OCT4, ECAT1, UTF1, ESRRB, and NANOG) using FMM and maintained in FMM. The population expressing high SSEA4+ / TRA181+ indicates pluripotency. [Figure 18D] Figures 18A-18D show representative flow analysis and phase images of four established iPSC clones after 7-9 passages after 96-well sorting. Clones were generated with lentiviral reprogramming factors (OCT4, ECAT1, UTF1, ESRRB, and NANOG) using FMM and maintained in FMM. The population expressing high SSEA4+ / TRA181+ indicates pluripotency. [Figure 19A] 19A-19B. Representative flow analysis of OCT4 and NANOG expression in human fibroblasts reprogrammed with lentiviral reprogramming factors OCT4, ECAT1, UTF1, NANOG, and ESRRB. Clones were reprogrammed using FMM and maintained in FMM. High OCT4 / NANOG populations indicate pluripotency. [Figure 19B] 19A-19B. Representative flow analysis of OCT4 and NANOG expression in human fibroblasts reprogrammed with lentiviral reprogramming factors OCT4, ECAT1, UTF1, NANOG, and ESRRB. Clones were reprogrammed using FMM and maintained in FMM. High OCT4 / NANOG populations indicate pluripotency. [Figure 20A](2) Karyotype analysis of hIPSC clones derived from human fibroblasts reprogrammed with lentiviral reprogramming factors OCT4, ECAT1, UTF1, NANOG, and ESRRB. The clones were reprogrammed using FRM and maintained in FMM. The clones exhibit a normal male karyotype. [Figure 20B] (2) Karyotype analysis of hIPSC clones derived from human fibroblasts reprogrammed with lentiviral reprogramming factors OCT4, ECAT1, UTF1, NANOG, and ESRRB. The clones were reprogrammed using FRM and maintained in FMM. The clones exhibit a normal male karyotype. [Figure 20C] (2) Karyotype analysis of hIPSC clones derived from human fibroblasts reprogrammed with lentiviral reprogramming factors OCT4, ECAT1, UTF1, NANOG, and ESRRB. The clones were reprogrammed using FRM and maintained in FMM. The clones exhibit a normal male karyotype. [Figure 21] FIG. 1 shows the 96-well plate sorting efficiency of the reprogramming factor combination OCT4 / ESRRB / NANOG / ECAT1 / UTF1 compared to the reprogramming factor combination OCT4 / NANOG / SOX2 / LARGE T. [Figure 22A] 22A-22B. (22A) Images of expanding colonies from 96 wells for cells reprogrammed with OCT4-P2A-OCT4 / NANOG-P2A-ESRRB-T2A-LIN28 / ECAT1-T2A-UTF1 at days 4, 6, and 11, and (22B) two wells at day 7 and one well at day 10. [Figure 22B]22A-22B. (22A) Images of expanding colonies from 96 wells for cells reprogrammed with OCT4-P2A-OCT4 / NANOG-P2A-ESRRB-T2A-LIN28 / ECAT1-T2A-UTF1 at days 4, 6, and 11, and (22B) two wells at day 7 and one well at day 10. [Figure 23A] 23A-23C. (23A) Flow analysis showing the effect of using genetic markers for reprogramming factor stoichiometry and cell selection by ectopic OCT4 expression, (23B) flow analysis of human fibroblasts reprogrammed with episomal OCT4-P2A-NANOG-T2A-SOX2 / SV40 large T antigen without OCT4 selection, and (23C) flow analysis of human fibroblasts reprogrammed with episomal OCT4-P2A-NANOG-T2A-SOX2 / SV40 large T antigen / OCT2-P2A-OCT4-puromycin. [Figure 23B] 23A-23C. (23A) Flow analysis showing the effect of using genetic markers for reprogramming factor stoichiometry and cell selection by ectopic OCT4 expression, (23B) flow analysis of human fibroblasts reprogrammed with episomal OCT4-P2A-NANOG-T2A-SOX2 / SV40 large T antigen without OCT4 selection, and (23C) flow analysis of human fibroblasts reprogrammed with episomal OCT4-P2A-NANOG-T2A-SOX2 / SV40 large T antigen / OCT2-P2A-OCT4-puromycin. [Figure 23C]23A-23C. (23A) Flow analysis showing the effect of using genetic markers for reprogramming factor stoichiometry and cell selection by ectopic OCT4 expression, (23B) flow analysis of human fibroblasts reprogrammed with episomal OCT4-P2A-NANOG-T2A-SOX2 / SV40 large T antigen without OCT4 selection, and (23C) flow analysis of human fibroblasts reprogrammed with episomal OCT4-P2A-NANOG-T2A-SOX2 / SV40 large T antigen / OCT2-P2A-OCT4-puromycin. [Figure 24] FIG. 1 shows immunofluorescence analysis of induced iPSC clones stained for pluripotency markers OCT4 (green) and TRA181 (red) with DAPI in blue. [Figure 25] Figure 1 shows images of SSEA4+ / TRA181+ / CD30+ 96-well plate sorted clones after CRE-mediated cleavage. Colonies were sorted from iPSC clones originally derived from human fibroblasts, reprogrammed with lentiviral factors OCT4, ECAT1, UTF1, NANOG, and ESRRB, and then cleaved for the transgene. Sorted colonies exhibit an iPSC phenotype. DETAILED DESCRIPTION OF THE INVENTION

[0182] A. Overview Current methods for generating and maintaining pluripotent cells have yet to achieve homogenous cultures of pluripotent cells that are free of spontaneous differentiation, have high-resolution / high-clonal single-cell passaging, and have a footprint that facilitates large-scale expansion. Ground-state pluripotent cells may offer qualities and characteristics that overcome these challenges. However, to date, there are no reliable and robust methods for high-throughput generation of ground-state pluripotent cells under feeder-free conditions. Therefore, current methods may not be suitable for generating industrial- or clinical-grade pluripotent cells. The invention contemplated herein addresses the need for robust generation of stable pluripotent cells of, or possessing the properties of, ground-state pluripotency and solves the problems in producing stable pluripotent cells suitable for industrial and clinical applications.

[0183] Generally, the present invention relates to compositions and methods for the improved production of pluripotent cells, particularly cells with reduced spontaneous differentiation, including ground-state pluripotent cells. More specifically, the present invention relates to multi-stage culture platforms that utilize small molecule modulators of cell signaling pathways in a stage-specific manner, allowing for the derivation and maintenance of pluripotent cells to a point where the culture methods and derivation methods do not contribute to downstream use variability and / or gating activity. Furthermore, the culture platforms contemplated herein allow for the derivation and maintenance of pluripotent cells under feeder-free conditions with improved genomic stability, improved undifferentiated state, reduced spontaneous differentiation, improved culture homogeneity, improved survival in culture, dissociation, and passaging of single pluripotent cells, and reprogramming of cells to ground-state pluripotency without the use of transgenes or footprints. Thus, the compositions and methods contemplated herein enable the production of pluripotent cells and / or ground-state pluripotent cells suitable for industrial and clinical applications.

[0184] To date, no small molecule-driven platform has been shown to improve reprogramming and support single-cell and FF culture of footprint-free induced pluripotent stem cells (iPSCs) derived from human cells (Nichols and Smith, 2012). The culture platforms contemplated herein, in part, provide for the application of specific combinations of stage-specific small molecule inhibitors, enabling rapid and robust reprogramming and stable long-term culture of pluripotent stem cells. In various embodiments, culture platforms are provided for inducing or maintaining improved undifferentiated pluripotent states, including ground-state pluripotency. The platforms contemplated herein also provide robust culture systems for generating and maintaining ground-state pluripotency in human iPSCs (hiPSCs). In one embodiment, the culture platform enables a reprogramming method that does not use transgenes or footprints. In a specific embodiment, the platforms contemplated herein represent an improved method for producing hiPSCs that overcomes major challenges in the multiple derivation and maintenance of transgene-free hiPSCs.

[0185] The practice of the present invention will employ, unless specifically indicated otherwise, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, cell biology, stem cell protocols, cell culture, and transgenic biology that are within the skill of the art, many of which are described below by way of example. Such techniques are fully explained in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3 rd Edition,2001);Sambrook,et al.,Molecular Cloning:A Laboratory Manual(2 ndEdition,1989);Maniatis et al.,Molecular Cloning:A Laboratory Manual(1982);Ausubel et al.,Current Protocols in Molecular Biology(John Wiley and Sons,updated July 2008);Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience;Glover,DNA Cloning:A Practical Approach,vol.I&II(IRL Press,Oxford,1985);Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991);Oligonucleotide Synthesis(N.Gait,Ed.,1984);Nucleic Acid Hybridization(B.Hames&S.Higgins,Eds.,1985);Transcription and Translation(B.Hames&S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Fire et al.,RNA Interference Technology:From Basic Science to Drug Development(Cambridge University Press,Cambridge,2005);Schepers,RNA Interference in Practice(Wiley-VCH,2005);Engelke,RNA Interference(RNAi):The Nuts&Bolts of siRNA Technology(DNA Press,2003);Gott,RNA Interference,Editing,and Modification:Methods and Protocols(Methods in Molecular Biology; Human Press, Totowa, NJ, 2004); Sohail, Gene Silencing by RNA Interference: Technology and Application(CRC, 2004); Clarke and Sanseau, microRNA: Biology, Function & Expression(Nuts & Bolts series; DNA Press, 2006); Immobilized Cells And Enzymes(IRL Press, 1986); Papers, Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV(D.M.Weir and C.Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Embryonic Stem Cells: Methods and Protocols(Methods in Molecular Biology)(Kurstad Turksen, Ed., 2002); Embryonic Stem Cell Protocols:Volume I:Isolation and Characterization(Methods in Molecular Biology)(Kurstad Turksen,Ed.,2006);Embryonic Stem Cell Protocols:Volume II:Differentiation Models(Methods in Molecular Biology)(Kurstad Turksen,Ed.,2006);Human Embryonic Stem Cell Protocols(Methods in Molecular Biology)(Kursad Turksen Ed.,2006);Mesenchymal Stem Cells:Methods and Protocols(Methods in Molecular Biology)(Darwin J.Prockop,Donald G.Phinney,and Bruce A.Bunnell Eds.,2008);Hematopoietic Stem Cell Protocols(Methods in Molecular Medicine)(Christopher A.Klug,and Craig T.Jordan Eds.,2001);Hematopoietic Stem Cell Protocols(Methods in Molecular Biology)(Kevin D.Bunting Ed.,2008) Neural Stem Cells:Methods and Protocols(Methods in Molecular Biology)(Leslie P.Weiner Ed.,2008);Hogan et al.,Methods of Manipulating the Mouse Embyro(2 nd Edition,1994);Nagy et al.,Methods of Manipulating the Mouse Embryo(3 rd Edition,2002) and The Zebrafish See the book, A guide for the laboratory use of zebrafish (Danio rerio), 4th Ed., (University of Oregon Press, Eugene, OR, 2000).

[0186] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0187] B. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For purposes of the present invention, the following terms are defined as follows:

[0188] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0189] The use of the alternative (eg, "or") should be understood to mean either one or both of the alternatives, or any combination thereof.

[0190] The term "and / or" should be understood to mean either or both of the alternatives.

[0191] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that differs by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length with respect to the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0192] As used herein, the term "substantially" or "essentially" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "essentially the same" or "substantially the same" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about the same as the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0193] As used herein, the terms "substantially free of" and "essentially free of" are used interchangeably and, when used to describe a composition such as a cell population or culture medium, refer to a composition that does not contain the specified substance, e.g., 95% free, 96% free, 97% free, 98% free, 99% free, or undetectable as measured by conventional means, of the specified substance. A similar meaning may be applied to the term "absent" when referring to the absence of a particular substance or component of a composition.

[0194] As used herein, the term "appreciable" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length range, or event that is readily detectable by one or more standard methods. The terms "not-appreciable" and "not appreciable" refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length range, or event that is not readily detectable or undetectable by standard methods. In one embodiment, an event is not appreciable if it occurs with a probability of 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001% or less.

[0195] Throughout this application, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" are understood to imply the inclusion of the stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. In specific embodiments, the terms "including," "having," "containing," and "comprising" are used interchangeably.

[0196] "Consisting of" means inclusive of and limited to what is attached to the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0197] "Consisting essentially of" means including any elements listed with that phrase, and limited to other elements that do not interfere with or contribute to the activity or function identified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or essential, but that other elements are not optional and may or may not be present depending on whether they affect the activity or function of the listed elements.

[0198] Throughout this specification, the terms "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, appearances of such phrases throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0199] The term "ex vivo" generally refers to activities occurring outside of a living organism, e.g., experiments or measurements performed in or on living tissue, preferably in an artificial environment outside of the organism that minimally alters natural conditions. In specific embodiments, "ex vivo" procedures involve living cells or tissues removed from an organism and cultured in a laboratory device, usually under sterile conditions, typically for a period of several hours up to about 24 hours, but in some circumstances up to 48 or 72 hours. In certain embodiments, such tissues or cells may be harvested and frozen, and then thawed for ex vivo processing. Tissue culture experiments or procedures lasting more than a few days using living cells or tissues are typically considered "in vitro," although in certain embodiments, the term may be used interchangeably with ex vivo.

[0200] "In vivo" generally refers to activities that occur within a living organism.

[0201] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potential" or "increasing developmental potential" refers to the method of increasing cell potential or dedifferentiating cells into a less differentiated state.For example, a cell with increased cell potential has developmental plasticity (i.e., can differentiate into more cell types) compared to the same cell in a non-reprogrammed state.In other words, a reprogrammed cell is a cell that is in a less differentiated state than the same cell in a non-reprogrammed state.

[0202] As used herein, the term "potency" refers to the sum of all developmental options available to a cell (i.e., developmental potential). Those skilled in the art will recognize that cell potential is a continuum ranging from totipotent stem cells, which are the most plastic cells with the highest developmental potential, to terminally differentiated cells, which are the least plastic cells with the lowest developmental potential. The continuum of cell potential includes, but is not limited to, totipotent cells, pluripotent cells, multipotent cells, oligopotent cells, unipotent cells, and terminally differentiated cells.

[0203] As used herein, the term "pluripotency" refers to the ability of a cell to form all the lineages of the body or organism (i.e., the embryonic body). For example, embryonic stem cells are a type of pluripotent stem cell that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm.

[0204] Pluripotency can be determined, in part, by assessing the pluripotent properties of cells, including, but not limited to, (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal; (iii) the expression of pluripotent stem cell markers, including, but not limited to, SSEA1 (mouse only), SSEA3 / 4; SSEA5, TRA1-60 / 81; TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50; (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm); (v) teratoma formation composed of the three somatic cell lineages; and (vi) the formation of embryoid bodies composed of cells from the three somatic cell lineages.

[0205] Two types of pluripotency have previously been described: "primed" or "metastable" pluripotency, resembling epiblast stem cells (EpiSCs) of late blastocysts, and "naive" or "ground" pluripotency, resembling the inner cell mass of early / preimplantation blastocysts. While both pluripotent states exhibit the characteristics described above, the naive or ground state additionally exhibits (i) pre-inactivation or reactivation of the X chromosome in female cells, (ii) improved clonability and survival in single-cell culture, (iii) globally reduced DNA methylation, (iv) reduced deposition of H3K27me3 repressive chromatin marks on developmental control gene promoters, and (v) reduced expression of differentiation markers relative to primed pluripotent cells. Standard methods of cellular reprogramming, in which exogenous pluripotency genes are introduced into somatic cells, expressed, and then silenced or removed from the resulting pluripotent cells, generally appear to possess the characteristics of primed pluripotency. Under standard pluripotent cell culture conditions, such cells remain in a primed state and exhibit ground state characteristics unless exogenous transgene expression is maintained.

[0206] As used herein, the term "pluripotent stem cell morphology" refers to the classical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a small, rounded shape, a high nuclear-to-cytoplasmic ratio, the presence of prominent nucleoli, and typical intercellular spacing.

[0207] As used herein, the terms "gene expression profile," "gene expression signature," "gene expression panel," "gene panel," or "gene signature" refer to the expression or level of expression of multiple genes that serve to distinguish a cell or population of cells from another cell or population of cells. For example, a population of pluripotent cells maintained in a medium to prevent spontaneous differentiation may exhibit a gene expression profile that includes reduced expression of differentiation genes compared to a control population of pluripotent cells of the same origin that are not maintained in the same medium.

[0208] As used herein, the term "differentiation marker gene" or "differentiation gene" refers to a gene whose expression is indicative of cell differentiation occurring in a cell, such as a pluripotent cell. The differentiation marker genes are the following: FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, and CD1D. These include, but are not limited to, FOXG1, LEFTY1, TUJ1, T gene (Brachyury) and ZIC1.

[0209] As used herein, the terms "differentiation marker gene profile" or "differentiation gene profile," "differentiation gene expression profile," "differentiation gene expression signature," "differentiation gene expression panel," "differentiation gene panel," or "differentiation gene signature" refer to the expression or level of expression of multiple differentiation marker genes.

[0210] In specific embodiments, a population of pluripotent cells exhibiting reduced spontaneous differentiation can be characterized by a reduced expression of differentiation marker genes or a differentiation marker gene profile. For example, reduced spontaneous differentiation in a pluripotent cell or population of pluripotent cells can be indicated when a given set of culture conditions results in at least a 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more decrease in the expression of one or more differentiation marker genes compared to the expression of the differentiation marker genes in a control pluripotent cell or population of pluripotent cells that does not have the same culture conditions.

[0211] "Gene expression," as used herein, refers to the relative levels and / or patterns of expression of genes in a biological sample, such as a pluripotent cell or a population of cells comprising pluripotent cells. In a specific embodiment, the pluripotent cell is an iPSC.

[0212] Any method available in the art for detecting the expression of genes that characterize the cells of the present invention is encompassed herein. As used herein, the term "detecting expression" refers to determining the quantity or presence of the RNA transcripts of a gene or its expression product. Methods for detecting gene expression, i.e., for gene expression profiling, include methods based on polynucleotide hybridization analysis, polynucleotide sequencing, immunohistochemistry, and proteomics. The methods generally detect the expression product (e.g., mRNA) of a gene of interest. In some embodiments, PCR-based methods, such as reverse transcription PCR (RT-PCR) (Weis et al., TIG 8:263-64, 1992), and array-based methods, such as microarrays (Schena et al., Science 270:467-70, 1995), are used.

[0213] "Adherence" refers to cells that adhere to a vessel, e.g., cells that adhere to a sterile plastic (or coated plastic) cell culture dish or flask in the presence of an appropriate culture medium. Certain classes of cells will not be maintained or grown in culture unless they adhere to a cell culture vessel. Certain classes of cells ("non-adherent cells") are maintained and / or grown in culture without attachment.

[0214] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. "Cell culture media," "culture media" (in each case singular "medium"), "supplement," and "media supplement" refer to nutritional compositions that foster cell cultures.

[0215] "Growing" refers to the maintenance, propagation (growth) and / or differentiation of cells outside a tissue or body, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. "Growing" may utilize culture medium as a source of nutrients, hormones and / or other factors that aid in the propagation and / or maintenance of cells.

[0216] As used herein, "dissociated" cells refer to cells that have been substantially separated or purified from other cells or surfaces (e.g., culture plate surfaces). For example, cells can be dissociated from animals or tissues by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be enzymatically or mechanically dissociated from each other, for example, by dissociating into a suspension of clusters, single cells, or a mixture of single cells and clusters. In yet another alternative embodiment, adherent cells are dissociated from a culture plate or other surface. Thus, dissociation can include disruption of cellular interactions with the extracellular matrix (ECM) and the substrate (e.g., the culture surface), or disruption of the ECM between cells.

[0217] As used herein, the terms "enrich" and "enriching" refer to increasing the amount of a particular component in a composition, such as a composition of cells, and "enriched," when used to describe a composition of cells, such as a cell population, refers to a population of cells in which the quantitative proportion of a particular component is increased compared to the proportion of such component in the population of cells prior to enrichment. For example, a composition, such as a population of cells, may be enriched for a target cell type (i.e., cells having a particular characteristic), and thus the proportion or percentage of the target cell type may be increased compared to the proportion of target cells present in the population of cells prior to enrichment. A population of cells may be enriched for a target cell type by cell selection and sorting methods known in the art. In some embodiments, a population of cells is enriched by a sorting or selection process such as those described in the Examples herein. In a specific embodiment, a method of enriching for a target cell population enriches the cell population by at least about 20% for the target cell population, i.e., the enriched cell population contains proportionally about 20% more of the target cell type than in the population prior to enrichment. In one embodiment, the method of enriching for a target cell population enriches the cell population proportionally for the target cell population by at least about 30+%, 40+%, 50+%, 60+%, 70+%, 80%, 85%, 90%, 95%, 97%, 98% or 99%, or at least about 98%, or in a specific embodiment, about 99%.

[0218] In certain embodiments, the population of cells is enriched for the amount of pluripotent cells or cells exhibiting pluripotent characteristics. In a specific embodiment of the invention, the population of cells to be reprogrammed is enriched for target cells that have pluripotent characteristics, such as expression of pluripotent markers, including, but not limited to, SSEA3, SSEA4, TRA 1-60, TRA-1-81, CD30, or CD50.

[0219] In specific embodiments, a population of cells, e.g., a population of cells to be reprogrammed, is depleted of non-pluripotent cells using surface markers specific for differentiated cell lineages or non-pluripotent cells, which may include, for example, CD13, CD26, CD34, CD45, CD31, CD46, or CD7. The resulting cell population may therefore be described as a population of cells enriched for pluripotent cells.

[0220] In specific embodiments, the enriched cells have a distinct gene or protein expression profile, e.g., cell surface expression of at least two pluripotency markers, such as SSEA3, SSEA4, TRA 1-60, TRA-1-81, CD30, and CD50. In some embodiments, the enriched cells comprise two or more pluripotency markers. In specific embodiments, the enriched cells express SSEA4 in combination with TRA-181 or TRA-160. In more specific embodiments, the enriched cells express SSEA4, TRA181, and CD30. In one embodiment, the population of cells comprises at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% enriched cells, e.g., pluripotent cells.

[0221] Thus, in some embodiments, a method of enriching a population of cells for pluripotent cells comprises sorting the cell population based on cell surface expression of pluripotency markers, such as SSEA3, SSEA4, TRA 1-60, TRA-1-81, CD30, and CD50, and recovering the fraction of cells expressing such markers to obtain a population of cells enriched for pluripotent cells. In other embodiments, a population of cells is enriched for pluripotent cells by sorting the cell population based on cell surface expression of markers of differentiating or differentiated cells, such as CD13, CD26, CD34, CD45, CD31, CD46, and CD7, and depleting the cell population of such cells to obtain a population of cells enriched for pluripotent cells. In a specific embodiment, a cell population is sorted based on expression of CD13, and CD13 is recovered from the cell population. + The cells are removed to obtain a population of cells enriched for pluripotent cells.

[0222] As used herein, "feeder cells" or "feeders" are terms used to describe one type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can grow (because the feeder cells provide growth factors and nutrients to support the second cell type). Feeder cells are optionally from a different species than the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts. Feeder cells, when co-cultured with other cells, can typically be inactivated by irradiation or treatment with an antimitotic agent such as mitomycin C to prevent overgrowth of the cells they support. Without being limited to the above, one specific type of feeder cell can be a human feeder, such as human dermal fibroblasts. Another type of feeder cell can be mouse embryonic fibroblasts (MEFs).

[0223] As used herein, a "feeder-free" (FF) environment refers to an environment, e.g., a cell culture or culture medium, that is essentially free of feeder cells and / or that has not been previously conditioned by the growth of feeder cells. "Preconditioned" medium refers to medium that has been harvested after feeder cells have been grown in the medium for a predetermined period of time, e.g., at least one day. Preconditioned medium contains many mediator substances, including growth factors and cytokines, secreted by feeder cells grown in the medium.

[0224] Genome stability refers to the cell's ability to faithfully replicate DNA and maintain the integrity of the DNA replication process.As used herein, the terms "genome-stable cell" and "cell with genome stability" refer to cells that exhibit mutation and chromosomal abnormality frequencies (such as translocation, aneuploidy, copy number variation and duplication) that are substantially similar to those of normal human somatic cells.

[0225] "Ingredient" refers to any compound or other material, chemical or biological in origin, that can be used in a cell culture medium to maintain and / or promote cell growth and / or differentiation. The terms "constituent," "nutrient," and "ingredient" can be used interchangeably. Conventional ingredients used in cell culture media can include, but are not limited to, amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, etc. Other ingredients that promote and / or maintain cell growth ex vivo can be selected by one of skill in the art as needed for the desired effect.

[0226] "Isolate" or "isolating" refers to the separation and recovery of a composition or material from its natural environment, e.g., the separation of individual cells or cell cultures from a tissue or body. In one aspect, a population or composition of cells is substantially free of cells and materials with which the cells may be naturally associated. In the context of a target population of cells, "isolated" or "purified" or "substantially pure" refers to a population of cells that is at least about 50%, at least about 75%, at least about 85%, at least about 90%, and in specific embodiments, at least about 95% pure with respect to the target cells that make up the total cell population. The purity of a population or composition of cells can be assessed by suitable methods well known in the art. For example, a substantially pure population of pluripotent cells refers to a population of cells that is at least about 50%, at least about 75%, at least about 85%, at least about 90%, and in specific embodiments, at least about 95%, and in certain embodiments, at least about 98% pure with respect to the pluripotent cells that make up the total cell population. The term "essentially pure" is used interchangeably herein with "substantially pure."

[0227] "Passage" or "passaging" refers to the act of dividing and seeding cells onto multiple cell culture surfaces or vessels when the cells have grown to a desired extent. In some embodiments, "passage" or "passaging" refers to dividing, diluting, and seeding the cells. When cells are passed from a primary culture surface or vessel to a subsequent set of surfaces or vessels, the subsequent culture may be referred to herein as a "secondary culture" or "first passage," etc. Each act of dividing and seeding into a new culture vessel is considered one passage.

[0228] "Seeding" refers to placing a cell(s) into a cell culture vessel so that the cells adhere to or spread onto the cell culture vessel.

[0229] "Pluripotency factor" refers to an agent that can increase the developmental potential of a cell, alone or in combination with other agents. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

[0230] "Proliferate" refers to the property of one cell to divide into two essentially identical cells or into a population of cells that increase in number (eg, reproduce).

[0231] "Propagation" refers to growing cells (eg, reproducing by cell proliferation) outside a tissue or body, for example, in a sterile container such as a plastic (or coated plastic) cell culture dish or flask.

[0232] "Initial culture" refers to a cell, tissue, and / or culture when isolated cells are placed in a first culture vessel having a culture medium. The cell, tissue, and / or culture may be retained and / or expanded, but as long as the cell, tissue, and / or culture is maintained in the first vessel, the cell, tissue, and / or culture is referred to as an initial culture.

[0233] The terms "small molecule reprogramming agent" or "small molecule reprogramming compound" are used interchangeably herein and refer to small molecules that can increase the developmental potential of cells, alone or in combination with other pluripotency factors. "Small molecule" refers to an agent having a molecular weight of less than about 5 kD, less than about 4 kD, less than about 3 kD, less than about 2 kD, less than about 1 kD, or less than about 0.5 kD. Small molecules include, but are not limited to, nucleic acids, peptidomimetics, peptoids, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be used as a source of small molecules in certain embodiments. In specific embodiments, small molecule reprogramming agents used herein have a molecular weight of less than 10,000 daltons, e.g., less than 8000, 6000, 4000, or 2000 daltons, e.g., between 50-1500, 500-1500, 200-2000, or 500-5000 daltons. C. Cell

[0234] In specific embodiments, one or more cells may be cultured, dissociated, and passaged using the compositions and methods contemplated herein. In one embodiment, a single cell is cultured, dissociated, and passaged using the compositions and methods contemplated herein. In another embodiment, a population of cells or a plurality of cells is cultured, dissociated, and passaged using the compositions and methods contemplated herein.

[0235] Starting cell populations suitable for use in specific embodiments can be derived from essentially any suitable source and can be heterogeneous or homogeneous with respect to cell type or pluripotent state. Suitable cells include fetal and adult cells. Furthermore, suitable cells can be mammalian in origin, e.g., from rodents, cats, dogs, pigs, goats, sheep, horses, cattle, or primates. In one embodiment, the cells are human cells.

[0236] The cells may be somatic cells, non-pluripotent cells, incompletely or partially pluripotent stem cells, multipotent cells, oligopotent cells, unipotent cells, terminally differentiated cells, or a mixed cell population containing any combination of the above. Pluripotent cells suitable for use in specific embodiments include, but are not limited to, spontaneous stem cells, embryonic stem cells, or iPSCs. A "mixed" cell population is a population of cells with varying degrees of developmental potential. For example, a mixed cell population may include cells that have undergone reprogramming, such that the mixed population includes pluripotent cells, partially pluripotent cells, and non-pluripotent cells, such as fully differentiated cells.

[0237] In one embodiment, the starting cell population is selected from adult or neonatal stem / progenitor cells, hi a specific embodiment, the starting stem / progenitor cell population is selected from the group consisting of mesodermal stem / progenitor cells, endodermal stem / progenitor cells, and ectodermal stem / progenitor cells.

[0238] Examples of mesodermal stem / progenitor cells include, but are not limited to, mesodermal stem / progenitor cells, endothelial stem / progenitor cells, bone marrow stem / progenitor cells, umbilical cord stem / progenitor cells, adipose tissue-inducing stem / progenitor cells, hematopoietic stem / progenitor cells (HSCs), mesenchymal stem / progenitor cells, muscle stem / progenitor cells, kidney stem / progenitor cells, osteoblastic stem / progenitor cells, cartilage stem / progenitor cells, and the like.

[0239] Examples of ectodermal stem / progenitor cells include, but are not limited to, neural stem / progenitor cells, retinal stem / progenitor cells, skin stem / progenitor cells, and the like.

[0240] Examples of endodermal stem / progenitor cells include, but are not limited to, hepatic stem / progenitor cells, pancreatic stem / progenitor cells, epithelial stem / progenitor cells, and the like.

[0241] In certain embodiments, the starting cell population may be a heterogeneous or homogeneous cell population selected from the group consisting of pancreatic islet cells, CNS cells, PNS cells, cardiac myocytes, skeletal muscle cells, smooth muscle cells, hematopoietic cells, bone cells, hepatocytes, adipocytes, kidney cells, lung cells, chondrocytes, skin cells, follicular cells, vascular cells, epithelial cells, immune cells, endothelial cells, and the like.

[0242] D. Culture Substrates for Reducing Spontaneous Differentiation and Inducing Ground-State Pluripotency Cell banking, disease modeling, and cell therapy applications place increasing demands on the production of high-quality pluripotent cells. For example, the derivation of iPSCs without a high-throughput footprint and their expansion in systems that enable large-scale production remains technically elusive. In specific embodiments, a culture platform is contemplated that enables the rapid, parallel generation, selection, and expansion of pluripotent cells using small molecule pathway inhibitors in stage-specific media compositions. The platform contemplated herein supports efficient and rapid reprogramming using minimal reprogramming factors in a completely feeder-free environment, enabling single-cell culture and expansion of pluripotent cells while maintaining homogeneous and genomically stable pluripotent populations. Furthermore, the culture platform contemplated herein provides for the cultivation of pluripotent cells, including hESCs and hiPSCs, to a state of reduced spontaneous differentiation and a general basal state of pluripotency, regardless of genetic background and independent of transgene expression.

[0243] The culture substrates contemplated herein are useful, in part, for the generation of industrial or clinical grade pluripotent cells with reduced spontaneous differentiation in culture. In one embodiment, non-pluripotent cells are induced to become pluripotent cells and cultured to maintain pluripotency. In another embodiment, non-pluripotent cells are induced to become pluripotent cells and cultured to achieve and / or maintain reduced spontaneous differentiation in culture. In another embodiment, non-pluripotent cells are induced to become pluripotent cells and cultured to achieve and / or maintain ground-state pluripotency.

[0244] In various embodiments, the culture substrates contemplated herein maintain ground state pluripotency, normal karyotype, and genomic stability of one or more pluripotent cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more passages (including any intervening number of passages).

[0245] In other embodiments, a culture substrate contemplated herein maintains reduced spontaneous differentiation in one or more pluripotent cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 or more passages (including any intervening number of passages).

[0246] In one embodiment, the culture substrate comprises a cell culture medium and a cell culture medium comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho Kinase (ROCK) inhibitor. In various embodiments, the cell culture medium contemplated herein does not contain or have inhibitors of the TGFβ / activin signaling pathway, including TGFβ receptor (TGFβR) inhibitors and ALK5 inhibitors. Without wishing to be bound by any particular theory, the inventors surprisingly discovered that while TGFβR / ALK5 inhibitors increase the efficiency of reprogramming, these inhibitors counteract the long-term maintenance, quality, and homogeneity of the pluripotent cell population; i.e., while inhibition of TGFβ pathway signaling improved the efficiency of cell reprogramming, release from this inhibition is required for subsequent maintenance of the pluripotent cell population in in vitro culture systems, particularly systems using feeder-cell-free single-cell enzymatic passaging, when a homogeneous pluripotent population with reduced spontaneous differentiation is preferred, more particularly in the absence of transgene expression. Furthermore, culturing metastable pluripotent cells in medium containing a GSK-3 inhibitor and a MEK inhibitor, and optionally a ROCK inhibitor, but without a TGFβR / ALK5 inhibitor, transitions the pluripotent cells, achieves reduced spontaneous differentiation, and / or achieves ground-state pluripotency, as disclosed herein. The culture medium base contemplated herein also enables efficient reprogramming and long-term culture of pluripotent cells in a feeder-free environment. Furthermore, while "ALK5 inhibitor" is not intended to encompass non-specific kinase inhibitors, "ALK5 inhibitor" should be understood to encompass inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, such as SB-431542 (see, e.g., Inman, et al., J. Mol. Pharmacol. 62(1):65-74 (2002)).

[0247] In a preferred embodiment, the culture substrate comprises a cell culture medium containing a GSK-3 inhibitor, a MEK inhibitor, a Rho Kinase (ROCK) inhibitor, and optionally LIF and / or bFGF, and does not contain small molecule inhibitors of the TGFβ / activin signaling pathway, including but not limited to TGFβR or ALK5 inhibitors.

[0248] In additional embodiments, the cell culture medium is substantially free of cytokines and / or growth factors, and optionally a feeder-free environment. In other embodiments, the cell culture medium contains supplements such as serum, extracts, growth factors, hormones, cytokines, etc.

[0249] In one preferred embodiment, the culture substrate comprises a feeder-free culture.

[0250] The culture substrates contemplated herein also provide many advantages, such as the production of homogenous populations of industrial- or clinical-grade pluripotent cells that have reduced spontaneous expression and / or have achieved ground-state pluripotency. As used herein, the term "homogeneous" refers to a population of cells in which each cell is identical or substantially identical to the other cells in the population. In one embodiment, a cell is identical to the other cells in the population if each cell expresses one or more of the same pluripotency markers as contemplated herein, e.g., SSEA4 and TRA1-81. In one embodiment, a population is homogenous if at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more of the cells are identical or substantially identical to the other cells in the population.

[0251] 1. TGFB receptor / ALK5 inhibitor TGFβ receptor (eg, ALK5) inhibitors may include antibodies against TGFβ receptor (eg, ALK5), dominant-negative mutants thereof, and antisense nucleic acids that suppress the expression thereof. Exemplary TGFβ receptor / ALK5 inhibitors include SB431542 (see, e.g., Inman, et al., Molecular Pharmacology 62(1):65-74(2002)), A-83-01, also known as 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (see, e.g., Tojo, et al., Cancer Science 96(11):791-800(2005) and commercially available, e.g., from Toicris Bioscience); 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a / BIO (see, e.g., Dalton, et al., which are incorporated herein by reference). al., WO2008 / 094597), BMP4 (Dalton, supra), GW788388 (-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide) (see, e.g., Gellibert, et al., Journal of Medicinal Chemistry 49(7):2210-2221 (2006)), SM16 (see, e.g., Suzuki, et al., Cancer Research 67(5):2351-2359 (2007)), IN-1130 (3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide) (see, e.g., Kim, et al., Xenobiotica 38(3):325-339 (2008)), GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, e.g., de Gouville, et al., Drug News Perspective 19(2):85-90(2006)), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride) (see, e.g., DaCosta, et al., Molecular Pharmacology 65(3):744-752(2004)), and pyrimidine derivatives (see, e.g., those listed in Stiefl, et al., WO2008 / 006583, which is incorporated herein by reference). Furthermore, while the term "ALK5 inhibitor" is not intended to encompass nonspecific kinase inhibitors, it should be understood to encompass inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, such as SB-431542 (see, for example, Inman, et al., J. Mol. Pharmacol. 62(1):65-74 (2002)). While not intending to limit the scope of the present invention, it is believed that ALK5 inhibitors affect the epithelial-mesenchymal transition (MET) process. The TGFβ / activin pathway is a driving mechanism for mesenchymal-epithelial transition (EMT). Therefore, inhibition of the TGFβ / activin pathway can promote the MET (i.e., reprogramming) process.

[0252] In view of the data herein showing the effect of inhibiting ALK5, inhibition of the TGFβ / activin pathway is believed to have a similar effect to inhibiting ALK5. Therefore, any inhibitor of the TGFβ / activin pathway (e.g., upstream or downstream) can be used in combination with or instead of an ALK5 inhibitor as described in each paragraph herein. Exemplary TGFβ / activin pathway inhibitors include, but are not limited to, TGFβ receptor inhibitors, inhibitors of SMAD2 / 3 phosphorylation, inhibitors of the interaction between SMAD2 / 3 and SMAD4, and activators / agonists of SMAD6 and SMAD7. Furthermore, the categorization described below is for organizational purposes only, and those skilled in the art will recognize that compounds can affect more than one point in the pathway and therefore function in more than one of the defined categories.

[0253] TGFβ receptor (TGFβR) inhibitors may include antibodies against TGFβ receptors, dominant-negative mutants thereof, and siRNA or antisense nucleic acids targeting them. Specific examples of TGFβ receptor inhibitors include SU5416; 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124); lerdelimumab (CAT-152); methelimumab (CAT-192); GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580276; LY364947; LY210976. 1; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; 3,5,7,2',4'-pentahydroxyflavone (morin); activin-M108A; P144; soluble TBR2-Fc; and antisense transfected tumor cells targeting TGFβ receptors (e.g., Wrzesinski, et al. al., Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska, et al., Acta Biochimica Polonica 52(2):329-337 (2005); and Chang, et al., Frontiers in Bioscience 12:4393-4401 (2007)).

[0254] Inhibitors of SMAD2 / 3 phosphorylation can include antibodies against SMAD2 or SMAD3, their dominant-negative mutants, and antisense nucleic acids that target them. Specific examples of inhibitors include PD169316; SB203580; SB-431542; LY364947; A77-01; and 3,5,7,2',4'-pentahydroxyflavone (morin) (see, for example, Wrzesinski, supra; Kaminska, supra; Shimanuki, et al., Oncogene 26:3311-3320 (2007); and Kataoka, et al., EP1992360, which are incorporated herein by reference).

[0255] Inhibitors of the SMAD2 / 3 and smad4 interaction may include antibodies against SMAD2, SMAD3, and / or smad4, dominant-negative mutants thereof, and antisense nucleic acids targeting them. Specific examples of inhibitors of the SMAD2 / 3 and SMAD4 interaction include, but are not limited to, Trx-SARA, Trx-xFoxH1b, and Trx-Lef1 (see, e.g., Cui, et al., Oncogene 24:3864-3874 (2005) and Zhao, et al., Molecular Biology of the Cell, 17:3819-3831 (2006)).

[0256] Activators / agonists of SMAD6 and SMAD7 include, but are not limited to, antibodies against SMAD6 or SMAD7, dominant-negative mutants thereof, and antisense nucleic acids targeting them. Specific examples of inhibitors include, but are not limited to, smad7-as-PTO-oligonucleotides (see, e.g., Miyazono, et al., US6534476, and Steinbrecher, et al., US2005119203, both of which are incorporated herein by reference).

[0257] 2. WNT pathway agonists As used herein, "Wnt signal promoter," "Wnt pathway activator," or "Wnt pathway agonist" refers to an agonist of the Wnt signaling pathway, including, but not limited to, one or more agonists of Wnt1, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15, or Wnt16. Wnt pathway agonists include, but are not limited to, one or more of the following polypeptides: a Dkk polypeptide, a crescent polypeptide, a cerberus polypeptide, an axin polypeptide, a Frzb polypeptide, a T-cell factor polypeptide, or a dominant-negative disheveled polypeptide, or fragments thereof.

[0258] Non-limiting examples of Wnt pathway agonists further include one or more of: a nucleic acid comprising a nucleotide sequence encoding a Wnt polypeptide, a polypeptide comprising the amino acid sequence of a Wnt polypeptide, a nucleic acid comprising a nucleotide sequence encoding an activated Wnt receptor, a polypeptide comprising the amino acid sequence of an activated Wnt receptor, a small organic molecule that promotes Wnt / β-catenin signaling, a small organic molecule that inhibits the expression or activity of a Wnt antagonist, an antisense oligonucleotide that inhibits the expression of a Wnt antagonist, a ribozyme that inhibits the expression of a Wnt antagonist, an RNAi construct, siRNA, or shRNA that inhibits the expression of a Wnt antagonist, an antibody that binds to and inhibits the activity of a Wnt antagonist, a nucleic acid comprising a nucleotide sequence encoding a β-catenin polypeptide, a polypeptide comprising the amino acid sequence of a β-catenin polypeptide, a nucleic acid comprising a nucleotide sequence encoding a Lef-1 polypeptide, or a polypeptide comprising the amino acid sequence of a Lef-1 polypeptide.

[0259] Wnt pathway agonists further include GSK3 inhibitors, such as nucleic acids comprising a nucleotide sequence encoding a dominant negative GSK-3, GSK3α or GSK3β polypeptide, polypeptides comprising the amino acid sequence of a dominant negative GSK-3, GSK3α or GSK3β polypeptide, small organic molecules that bind to GSK-3, GSK3α or GSK3β and inhibit their expression or activity, RNAi constructs, siRNA or shRNA, antisense oligonucleotides that bind to GSK-3, GSK3α or GSK3β and inhibit their expression, antibodies that bind to GSK-3, GSK3α or GSK3β and inhibit their expression and / or activity, ribozymes that bind to GSK-3, GSK3α or GSK3β and inhibit their expression, and any GSK-3-independent reagents that activate β-catenin target genes with effects similar to GSK-3 inhibition.

[0260] 3.GSK-3B inhibitors GSK-3β inhibitors are specific exemplary Wnt pathway agonists suitable for use in the compositions contemplated herein and may include, but are not limited to, polynucleotides, polypeptides, and small molecules. GSK-3β inhibitors contemplated herein may reduce GSK-3β expression and / or GSK-3β activity. Examples of GSK-3β inhibitors contemplated herein include, but are not limited to, anti-GSK-3β antibodies targeting GSK-3β, dominant-negative GSK-3β mutants, siRNA, shRNA, miRNA, and antisense nucleic acids.

[0261] Other exemplary GSK-3β inhibitors include kenpaullone, l-azakempaullone, CHIR99021, CHIR98014, AR-A014418, CT99021, CT20026, SB216763, AR-A014418, lithium, SB 415286, TDZD-8, BIO, BIO-acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopentadienyl ruthenium complex, TDZD-8 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxazole, OTDZT, alpha-4-dibromoacetophenone, AR-AO 144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione; TWS119 pyrrolopyrimidine compound, L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form; 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone; GF109203X; RO318220; TDZD-8; TIBPO; and OTDZT.

[0262] In specific exemplary embodiments, the GSK-3β inhibitor is CHIR99021, BIO, or Kenpaullon.

[0263] In a preferred embodiment, the GSK-3β inhibitor is CHIR99021.

[0264] 4. ERK / MEK inhibitors Suitable ERK / MEK inhibitors for use in the compositions contemplated herein include, but are not limited to, polynucleotides, polypeptides, and small molecules. The ERK / MEK inhibitors contemplated herein can reduce MEK or ERK expression and / or MEK or ERK activity. Examples of MEK / ERK inhibitors contemplated herein include, but are not limited to, anti-MEK or anti-ERK antibodies targeting MEK or ERK, dominant-negative MEK or ERK mutants, siRNA, shRNA, miRNA, and antisense nucleic acids.

[0265] Other exemplary ERK / MEK inhibitors include, but are not limited to, PD0325901, PD98059, UO126, SL327, ARRY-162, PD184161, PD184352, sunitinib, sorafenib, vandetanib, pazopanib, axitinib, GSKl 120212, ARRY-438162, RO5126766, XL518, AZD8330, RDEAl 19, AZD6244, FR180204, and PTK787.

[0266] Additional exemplary MEK / ERK inhibitors include compounds disclosed in International Patent Application Publication Nos. WO99 / 01426, WO02 / 06213, WO03 / 077914, WO05 / 051301, and WO2007 / 044084.

[0267] Further illustrative examples of MEK / ERK inhibitors include the following compounds: 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2,3-dihydroxy-propoxy)-amide; 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 1-[6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2,3-dihydroxy-propoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazol-5-yl]-2-hydroxy-ethanone, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)- Amide, 6-(4-bromo-2-fluoro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(2,4-dichloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide 2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (hereinafter referred to as MEK inhibitor 2); and 4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridazine-3-carboxamide, or a pharmaceutically acceptable salt thereof.

[0268] In a preferred embodiment, the MEK / ERK inhibitor is PD98059.

[0269] 5.ROCK inhibitors Rho-associated kinase (ROCK) is a serine / threonine kinase that acts as a downstream effector of Rho kinase (of which there are three isoforms: RhoA, RhoB, and RhoC). ROCK inhibitors suitable for use in the compositions contemplated herein include, but are not limited to, polynucleotides, polypeptides, and small molecules. ROCK inhibitors contemplated herein can reduce ROCK expression and / or ROCK activity. Examples of ROCK inhibitors contemplated herein include, but are not limited to, anti-ROCK antibodies targeting ROCK, dominant-negative ROCK mutants, siRNA, shRNA, miRNA, and antisense nucleic acids.

[0270] Exemplary ROCK inhibitors contemplated herein include, but are not limited to, thiazovivin, Y27632, fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, and the ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated herein by reference in its entirety.

[0271] In one embodiment, the ROCK inhibitor is thiazovivin, Y27632, or pyrin tegrin.

[0272] In a preferred embodiment, the ROCK inhibitor is thiazovivin.

[0273] The amount of small molecule in the compositions and cell culture media contemplated herein can vary and can be optimized according to the particular culture conditions, including the particular molecules and combinations used, the cell type being cultured in the media, and the particular application. In one embodiment, the small molecule is present in the composition at a concentration sufficient to induce pluripotency, improve the efficiency of reprogramming, increase or maintain the ability of cells, or induce or maintain ground-state pluripotency.

[0274] In specific embodiments, preferred concentrations and combinations of small molecules in the cell culture media of the present invention are shown in Table 1 as Fate Maintenance Medium (FMM). The components of the medium may be present in the medium in amounts within an optimal range around the optimal concentrations shown in Table 1. Fate Reprogramming Medium (FRM) is useful in the culture substrates contemplated herein, including cell reprogramming, but is not suitable for the establishment and long-term maintenance of ground-state pluripotent cells. [Table 1-1] [Table 1-2]

[0275] 6. Cytokines and growth factors In specific embodiments, the cell culture medium of the present invention is substantially free of cytokines and / or growth factors. In certain embodiments, the cell culture medium contains one or more supplements including, but not limited to, serum, extracts, growth factors, hormones, cytokines, etc.

[0276] In one exemplary embodiment, the culture medium may include one or more of an ECM protein, laminin 1, fibronectin, collagen IV isotype, protease, protease inhibitor, cell surface adhesion protein, cell signaling protein, cadherin, chloride intracellular channel 1, transmembrane receptor PTK7, insulin-like growth factor, or inhibin beta A, but does not include an attractant of the TGFβ / activin / nodal signaling pathway, and does not include activin A. In other embodiments, the medium may include an attractant of the TGFβ / activin / nodal signaling pathway.

[0277] In another exemplary embodiment, the culture medium comprises one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), transferrin, various interleukins (e.g., IL-1 through IL-18), various colony-stimulating factors (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ), and other cytokines that affect stem cells, such as stem cell factor (SCF) and erythropoietin (Epo). These cytokines are commercially available, for example, from R&D Systems, Minneapolis, Minn., and may be natural or recombinant. In specific embodiments, growth factors and cytokines may be added at concentrations contemplated herein. In certain embodiments, growth factors and cytokines may be added at concentrations determined empirically or as guided by established cytokine technology.

[0278] 7.Culture substrate Any suitable vessel or cell culture vessel can be used as a carrier for cell culture in basal medium and / or cell culture supplements. A substrate coating on the carrier is not required. However, coating the surface of the vessel with an adhesion-promoting substance (e.g., collagen, fibronectin, RGD-containing polypeptide, gelatin, etc.) can promote cell attachment and, in specific embodiments, improve the effectiveness of the cell culture medium and supplements disclosed herein. Suitable substrates for cell culture and passaging are known in the art and include, but are not limited to, vitronectin, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, mixtures of naturally occurring cell line-generated matrices such as Matrigel™, and synthetic or artificial surfaces such as polyamine monolayers and carboxy-terminated monolayers.

[0279] In one embodiment, the culture substrate contemplated herein comprises a substrate comprising Matrigel™ or vitronectin.

[0280] 8. Feeder-free environment Current methods for culturing pluripotent cells rely heavily on feeder cells or media preconditioned with feeder cells and containing fetal bovine serum, but such environments can be inappropriate for generating cells for clinical and therapeutic applications. For example, cells propagated in such externally contaminated environments are generally considered unsuitable for human cell transplantation because exposure to animal components can pose a significant risk of immune rejection and transmission of unidentified pathogens to treated patients, potentially reactivating animal retroviruses. The culture systems contemplated herein using animal-component-free cell media, such as feeder-free environments, facilitate the production of clinical-grade cell lines, specifically hESC and hiPSC cell lines.

[0281] In specific embodiments, the feeder-free environment is essentially free of human feeder cells, including, but not limited to, mouse embryonic fibroblasts, human fibroblasts, keratinocytes, and embryonic stem cells, and is not pre-conditioned with feeder cells. The feeder-free cell culture medium is suitable for use in culturing pluripotent cells, reprogramming cells, single cell culture, dissociation, and passaging pluripotent cells, cell sorting of pluripotent cells, generating ground-state pluripotent cells, and maintaining ground-state pluripotency. In specific embodiments, the feeder-free environment is used to induce pluripotency, improve reprogramming efficiency, and / or maintain cell potency. In certain embodiments, the feeder-free environment is substantially free of growth factors, including cytokines and bFGF.

[0282] 9. Dissociation One advantage provided by the culture substrate contemplated herein is the improved viability and survival of single ground-state pluripotent cells during culture, passaging, and dissociation. Dissociation of cells into single cells, e.g., into a single-cell suspension, can be achieved by enzymatic or mechanical means. Any enzymatic agent known in the art to enable dissociation of cells into single cells can be used in the methods of the present invention. In one embodiment, the dissociation agent is selected from Trypsin / EDTA, TrypLE-Select, collagenase IV, and dispase.

[0283] Chelating agents such as EDTA, Accutase, or AccuMax may also be used alone or in combination with enzymatic agents in dissociating cells by the methods contemplated herein. The dissociation agent may be dissolved in calcium- and magnesium-free PBS to facilitate dissociation into single cells.

[0284] To enhance cell survival during or after dissociation, pro-survival substances, e.g., ROCK inhibitors such as thiazovivin, may be added (e.g., growth factors, inhibitors of cellular pathways involved in cell death and apoptosis, or conditioned media).

[0285] Techniques for cell culture and medium recovery are described in Hu et al., Curr. Opin. Biotechnol. 8:148, 1997; K. Kitano, Biotechnology 17:73, 1991; Curr. Opin. Biotechnol. 2:375, 1991; Birch et al., Bioprocess Technol. 19:251, 1990; "Teratocarcinomas and embryonic stem cells: A practical approach" (E.J. Robertson, ed., IRL Press Ltd. 1987); "Guide to Techniques in Mouse Development" (P.M. Wasserman et al. eds., Academic Press 1993); "Embryonic Stem Cell Differentiation" (E.J. Robertson, ed., IRL Press Ltd. 1993); in vitro" (MV Wiles, Meth. Enzymol. 225:900, 1993); "Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy" (PD Rathjen et al., 1993).

[0286] Stem cell differentiation is discussed in Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 10:31, 1998. 10. Enrichment and Depletion Strategies

[0287] In specific embodiments, strategies are provided for enriching a population of cells for pluripotent cells, e.g., iPSCs. In one embodiment, enrichment provides a method for inducing clonal iPSC colonies in a relatively short time, thereby improving the efficiency of iPSC generation. Enrichment can include sorting a population of cells induced to reprogram, identify, and obtain cells expressing markers of pluripotency, thereby obtaining a population of cells enriched for pluripotent cells. Additional enrichment methods include depleting cells expressing markers of differentiation or non-pluripotent cells to obtain an enriched population of pluripotent cells. In some embodiments, cells are cultured after reprogramming induction for about 4 to 30 days, about 4 to 24 days, about 6 to 22 days, or about 8 to about 12 days.

[0288] In one embodiment, enriching a population of cells for pluripotent cells comprises dissociating cells within the population and resuspending the cells to create a single cell suspension. The dissociated cells may be resuspended in any suitable solution or medium for maintaining the cells or for cell sorting. In a specific embodiment, the single cell suspension contains a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor, and does not have a TFGβ inhibitor. In a specific embodiment, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the ROCK inhibitor is thiazovivin.

[0289] In specific embodiments, a population of cells is sorted to positively select for pluripotent cells and / or the population is depleted of non-reprogrammed or non-pluripotent cells, thereby obtaining a population of cells enriched for pluripotent cells. In one embodiment, a single cell suspension is prepared, and the single cells are then prepared for sorting, such as by staining for markers of pluripotency using, for example, appropriate antibodies. Cells can be sorted by any suitable method, such as by magnetic bead or flow cytometry (FACS) sorting.

[0290] Cells can be sorted based on various markers of pluripotency, including expression of SSEA3 / 4, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD105, OCT4, NANOG, SOX2, KLF4, SSEA1 (mouse), CD30, SSEA5, CD90, and CD50. In various embodiments, cells are sorted based on at least two, at least three, or at least four markers of pluripotency. In certain embodiments, cells are sorted based on expression of SSEA4, and in certain specific embodiments, based on expression of SSEA4 in combination with TRA1-81 or TRA1-60. In certain embodiments, cells are sorted based on expression of SSEA4, TRA1-81, or TRA1-60 and CD30. In certain embodiments, cells are first depleted for non-reprogrammed cells using surface markers of differentiating cells, including but not limited to CD13, CD26, CD34, CD45, CD31, CD46, or CD7, and then enriched for pluripotency markers such as SSEA4, TRA1-81, and CD30.

[0291] The enriched population for pluripotent cells may be placed in a cell culture system, such as conventional hESC medium or the cell culture medium of the present invention. The cell culture system may be supplemented with feeder cells or, optionally, a feeder-free environment. In some embodiments, sorted cells expressing markers of pluripotency are placed in a culture system supplemented with feeder cells and then transferred to a feeder-free environment. In one embodiment, the cell culture medium is a feeder-free environment and comprises a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor, but does not have a TGFβ inhibitor. In a specific embodiment, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the ROCK inhibitor is thiazovivin. In another specific embodiment of the present invention, the cell culture system is a feeder-free environment comprising Matrigel®-coated tissue plates. In one embodiment, the cell culture system comprises FMM medium as shown in Table 1.

[0292] The enriched cell population can be cultured in the cell culture system described herein to obtain ground state iPSC colonies, which typically appear about 3 to about 25 days after sorting, about 5 to 9 days after sorting, or about 5 to 7 days after sorting. The iPSC colonies can be harvested or sorted for clonal expansion. Using the enrichment strategies contemplated herein, cell populations are enriched for pluripotent cells by at least about 3-fold, 5-fold, or 10-fold or more.

[0293] In some embodiments, the population of cells undergoing reprogramming or the population of pluripotent cells is depleted of differentiated cells. In one embodiment, the population of pluripotent cells or cells induced to be reprogrammed can be depleted of cells bearing cell surface markers of differentiated cells. Examples of cell surface markers of differentiated cells include, but are not limited to, CD13, CD26, CD34, CD45, CD31, CD46, or CD7. In a specific embodiment, CD13 is used as a surface marker of differentiated cells.

[0294] In other embodiments, a population of cells is induced to differentiate into a desired lineage and depleted of pluripotent cells to obtain an enriched population of differentiated or differentiated cells. In some embodiments, the population of differentiated cells includes a population of cells, such as ESCs or iPSCs, that have been induced to differentiate into a specific lineage. The population of cells may be depleted of pluripotent cells using negative cell sorting techniques ("panning") as described above, such as sorting cells within the population using magnetic beads or FAC based on a pluripotency marker. In some embodiments, a population of cells containing differentiated cells is sorted by FAC using a pluripotency marker to obtain a fraction depleted of cells expressing the pluripotency marker. In other embodiments, a population of cells is sorted by FAC based on a lineage-specific marker for differentiation, such as CD13, CD26, CD34, CD45, CD31, CD46, or CD7, to obtain a fraction depleted of pluripotency markers. CD13 is used as a surface marker for differentiating cells in specific embodiments of the invention. D. Culture Substrates for Reprogramming Cells

[0295] To induce or increase pluripotency in cells (Takahashi, K., and Yamanaka, S., Cell 126, 663-676 (2006); Takahashi et al., Cell 131, 861-872 (2007); Yu et al., Science 318, 1917-1920 (2007); Zhou et al., Cell Stem Cell 4, 381-384 (2009); Kim et al., Cell Stem Cell 4, 472-476 (2009); Yamanaka et al., 2009; Saha, K., Jaenisch, R., Cell Stem Cell 5, 584-595 (2009)), and to improve reprogramming efficiency (Shi et al., Cell Stem Cell 2, 525-528 (2008a); Shi et al.,Cell Stem Cell 3,568-574(2008b);Huangfu et al.,Nat Biotechnol 26,795-797(2008a);Huangfu et al.,Nat Biotechnol 26,1269-1275(2008b);Silva et al.,Plos Bio 6,e253.doi:10.1371 / journal.pbio.0060253(2008);Lyssiotis et al.,PNAS 106,8912-8917(2009);Ichida et al.,Cell Stem Cell 5,491-503(2009);Maherali,N.,Hochedlinger,K.,Curr Biol 19,1718-1723(2009b);Esteban et al., Cell Stem Cell 6, 71-79 (2010); Feng et al., Cell Stem Cell 4, 301-312 (2009)), various strategies are being pursued.However, current methods still leave room for a high-throughput solution for the production of industrial or clinical-grade pluripotent cells, i.e., clonal transgene-free pluripotent cell populations with homogeneous pluripotency, no significant spontaneous differentiation, and the ability to culture and expand the cell population using enzymatic passaging of single cells in an exogenous-free feeder cell culture system.

[0296] The culture substrates contemplated herein are useful, in part, for generating high-grade induced pluripotent stem cells (iPSCs). In one embodiment, non-pluripotent cells are reprogrammed to become pluripotent and cultured to maintain pluripotency. In another embodiment, iPSCs are cultured to achieve ground-state pluripotency.

[0297] In various embodiments, the culture substrate enables a transgene- and / or footprint-free reprogramming method. The culture substrates contemplated herein provide highly efficient episomal reprogramming, significantly reducing the time and effort required for hiPSC generation. Without wishing to be bound by any particular theory, the efficiency of hiPSC generation is significantly improved using episomal vectors in FF and single-cell culture systems by both blocking differentiation cues early in the reprogramming process and promoting mesenchymal-epithelial transition (MET) through small molecule inhibition of specific pathways (MEK, ERK, TGFβ, and ROCK).

[0298] In one embodiment, the culture substrate comprises reprogramming one or more non-pluripotent cells to a pluripotent state, comprising increasing the expression of endogenous OCT4 in the cells. The expression of endogenous OCT4 in the cells can be increased by introducing one or more polynucleotides, polypeptides, or small molecule inducers of OCT4 expression. In one embodiment, introduction of a polynucleotide encoding OCT4 or an OCT4 polypeptide into the cells is sufficient to induce endogenous expression of OCT4 in the cells.

[0299] In one embodiment, the culture substrate comprises a method for reprogramming one or more non-pluripotent cells, comprising introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more reprogramming factors selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, and SV40LT. In another embodiment, the culture substrate comprises a method for reprogramming one or more non-pluripotent cells, comprising introducing into the one or more non-pluripotent cells one or more polypeptides selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, SV40LT, hTERT, SALL4, GLIS, ESRRB, DPPA2, ECAT1, SOX1, SOX3, KLF2, KLF5, L-MYC, N-MYC, LRH1, and UTF1.

[0300] In one embodiment, the culture substrate comprises a method for reprogramming one or more non-pluripotent cells, comprising introducing into the one or more non-pluripotent cells one or more polynucleotides encoding one or more reprogramming factors selected from the group consisting of OCT4, NANOG, ESRRB, ECAT1, and UTF1. In another embodiment, the culture substrate comprises a method for reprogramming one or more non-pluripotent cells, comprising introducing into the one or more non-pluripotent cells one or more polypeptides selected from the group consisting of OCT4, NANOG, ESRRB, ECAT1, and UTF1.

[0301] As used herein, in specific embodiments, the term "introducing" refers to a process comprising contacting a cell with a polynucleotide, polypeptide, or small molecule. The introducing step can also include microinjecting the polynucleotide or polypeptide into the cell, using liposomes to deliver the polynucleotide or polypeptide into the cell, or derivatizing the polynucleotide or polypeptide with a cell-permeable moiety to introduce it into the cell.

[0302] In specific embodiments, one or more polynucleotides encoding one, two, three, four, five, or more copies of one or more reprogramming factors selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, SV40LT, hTERT, SALL4, GLIS, ESRRB, DPPA2, ECAT1, SOX1, SOX3, KLF2, KLF5, L-MYC, N-MYC, LRH1, and UTF1 may be introduced into non-pluripotent cells to reprogram the cells. The copy number of each reprogramming factor introduced into the cells may be the same or different, in any combination suitable to achieve ground-state pluripotency as contemplated herein.

[0303] In one embodiment, one or more polynucleotides encoding one, two, three, four, five or more copies of one or more reprogramming factors selected from the group consisting of OCT4, SOX2, and NANOG are introduced into the non-pluripotent cells.

[0304] In one embodiment, one or more polynucleotides encoding one, two, three, four, five or more copies of each of OCT4, SOX2, and NANOG are introduced into the non-pluripotent cells.

[0305] In one embodiment, one or more polynucleotides encoding one, two, three, four, five or more copies of each of OCT4 and SOX2 are introduced into the non-pluripotent cells.

[0306] In one embodiment, one or more polynucleotides encoding one, two, three, four, five or more copies of OCT4 are introduced into the non-pluripotent cells.

[0307] In one embodiment, one or more polynucleotides encoding two copies of OCT4, two copies of SOX2, and two copies of NANOG are introduced into the non-pluripotent cells, and SV40LT is optionally introduced into the non-pluripotent cells.

[0308] In another embodiment, one or more polynucleotides encoding three copies of OCT4, two copies of SOX2, one copy of NANOG, and one copy of UTF1 are introduced into the non-pluripotent cells.

[0309] In various exemplary embodiments, a culture substrate comprising reprogramming pluripotent cells comprises introducing 1 to 5 copies of a polynucleotide encoding OCT4, 1 to 3 copies of a polynucleotide encoding SOX2, and optionally 1 to 2 copies of a polynucleotide encoding NANOG. Polynucleotides may be introduced into cells as any combination of one or more larger polynucleotides. In one non-limiting example, one or more polynucleotides encoding 1 to 4 copies of OCT4, 1 or 2 copies of SOX2, and 1 copy of NANOG are introduced into non-pluripotent cells. In another non-limiting example, reprogramming non-pluripotent cells to a pluripotent state comprises introducing into the non-pluripotent cells a first polynucleotide encoding 2 copies of OCT4, a second polynucleotide encoding 1 copy of OCT4 and 1 copy of SOX2, and a third polynucleotide encoding 1 copy of OCT4, 1 copy of SOX2, and 1 copy of NANOG. In a further non-limiting example, reprogramming one or more non-pluripotent cells comprises introducing into the one or more non-pluripotent cells a first polynucleotide encoding two copies of OCT4 and a second polynucleotide encoding one copy of OCT4, one copy of SOX2, and one copy of NANOG. In a further non-limiting example, a first polynucleotide encoding two copies of OCT4 and a second polynucleotide encoding one copy of OCT4 and one copy of SOX2 are introduced into one or more non-pluripotent cells to generate pluripotent cells.

[0310] In one embodiment, a single vector containing a polynucleotide comprising any number and combination of reprogramming factors contemplated herein is introduced into a non-pluripotent cell and is sufficient to reprogram the cell to a pluripotent state.

[0311] In one embodiment, one or more vectors comprising one, two, three, four, five or more polynucleotides comprising any number and combination of reprogramming factors contemplated herein are introduced into a non-pluripotent cell and are sufficient to reprogram the cell to a pluripotent state.

[0312] In a preferred embodiment, one or more vectors containing one or more polynucleotides contemplated herein for reprogramming non-somatic cells are used to introduce one or more polynucleotides into a cell, sufficient to reprogram the cell.

[0313] In a most preferred embodiment, one or more episomal vectors containing one or more polynucleotides contemplated herein for reprogramming non-somatic cells are used to introduce one or more polynucleotides into cells, sufficient to reprogram the cells. Pluripotent cells exhibiting reduced spontaneous differentiation and / or a ground state can be produced using episomal vectors as contemplated herein, which are then cultured until the vector is depleted to obtain pluripotent cells exhibiting reduced spontaneous differentiation and / or a ground state that do not contain exogenous nucleic acids encoding reprogramming factors.

[0314] Furthermore, when the polynucleotide or vector comprising it comprises a polynucleotide encoding at least two reprogramming factors, or at least two copies of a reprogramming factor, it is contemplated that the polynucleotide comprises a polynucleotide encoding an IRES sequence, or a self-cleaving polypeptide sequence, between each of the reprogramming factors as contemplated herein.

[0315] In some embodiments, the efficiency of reprogramming of non-pluripotent cells is increased by selecting for ectopic expression of one or more reprogramming factor polynucleotides after the reprogramming factor polynucleotides are introduced into the non-pluripotent cells. Such selection can occur, for example, by linking one or more of the reprogramming factor polynucleotides to a selectable marker, introducing the reprogramming factor polynucleotides and the selectable marker into the non-pluripotent cells, and selecting for those cells that express the selectable marker, which identifies cells that have increased reprogramming efficiency relative to cells that do not have expression of the marker and its associated reprogramming factor polynucleotide. Those skilled in the art will recognize that in specific embodiments, any selectable marker that identifies expression of the introduced reprogramming polynucleotide by the non-pluripotent cells can be used.

[0316] One non-limiting example of such a selectable marker includes, but is not limited to, an antibiotic resistance gene, such as puromycin resistance. The selectable marker may be linked to one or more of the following reprogramming factor polynucleotides: OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, SV40LT, hTERT, SALL4, GLIS, ESRRB, DPPA2, ECAT1, SOX1, SOX3, KLF2, KLF5, L-MYC, N-MYC, LRH1, and UTF1. In some embodiments, a particular combination of reprogramming factor polynucleotides is introduced as a polycistronic vector, and the selectable marker is linked to a particular combination of reprogramming factor polynucleotides. A particular combination of reprogramming factor polynucleotides may encode two or more copies of the reprogramming factor polynucleotides disclosed herein.

[0317] In one non-limiting embodiment, the polycistronic vector encodes two or more copies of an OCT4 polynucleotide linked to a selectable marker, such as a gene encoding puromycin resistance.

[0318] In some embodiments, a polycistronic vector encoding one or more reprogramming factor polynucleotides and a selectable marker is introduced into non-pluripotent cells in addition to one or more separate reprogramming factor polynucleotides, and selection for cells expressing the selectable marker generates a population of cells that have a higher reprogramming efficiency than cells that do not have expression of the selectable marker.

[0319] In one non-limiting example of this selection process, OCT4, NANOG, and SOX2 polynucleotides are introduced into non-pluripotent cells along with a polycistronic vector encoding two or more copies of OCT4 linked to a puromycin resistance gene. Subsequent selection for non-pluripotent cells expressing the selectable marker identifies non-pluripotent cells with a higher reprogramming efficiency than non-pluripotent cells that do not express the selectable marker. The selected cells may have a reprogramming efficiency of at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, or at least 40%.

[0320] In the reprogramming step of specific preferred embodiments, small molecules are often included. Without wishing to be bound by any particular theory, it is contemplated that the inclusion of small molecule inhibitors of various differentiation pathways increases the efficiency and kinetics of reprogramming. Thus, in specific embodiments, a culture substrate comprising reprogramming non-pluripotent cells comprises introducing one or more reprogramming factors into cells as contemplated herein and contacting the cells with a GSK3 inhibitor, a MEK inhibitor, a TGFβR inhibitor, and a ROCK inhibitor.

[0321] Improved efficiency of reprogramming can be measured by (1) a decrease in the time required to reprogram and generate pluripotent cells (e.g., by shortening the time to generate pluripotent cells by at least one day compared to a similar or identical process without a small molecule), or alternatively or in combination, (2) an increase in the number of pluripotent cells generated by a particular process (e.g., increasing the number of cells reprogrammed by at least 10%, 30%, 50%, 100%, 200%, 500%, for example, in a given period compared to a similar or identical process without a small molecule). In some embodiments, a 2- to 20-fold improvement in reprogramming efficiency is observed. In some embodiments, reprogramming efficiency is improved by more than 20-fold. In some embodiments, a greater than 100-fold improvement in efficiency over methods that do not use small molecule reprogramming agents (e.g., a greater than 100-fold increase in the number of pluripotent cells generated) is observed.

[0322] In one embodiment, the culture substrate contemplated herein comprises reprogramming non-pluripotent cells by introducing one or more reprogramming factors into cells as contemplated herein and contacting the cells with a GSK3 inhibitor, a MEK inhibitor, and a TGFβR inhibitor, and optionally a ROCK inhibitor.

[0323] In one preferred embodiment, the culture substrate contemplated herein comprises reprogramming non-pluripotent cells by introducing one or more reprogramming factors into cells as contemplated herein and contacting the cells with a GSK3 inhibitor, a MEK inhibitor, a TGFβR inhibitor, and a ROCK inhibitor.

[0324] In a more preferred embodiment, the culture substrate contemplated herein comprises reprogramming non-pluripotent cells by introducing one or more reprogramming factors into cells as contemplated herein and contacting the cells with a GSK3 inhibitor, a MEK inhibitor, a TGFβR inhibitor, and a ROCK inhibitor, wherein the ROCK inhibitor is thiazovivin.

[0325] However, to enable long-term culture of pluripotent cells in a feeder-cell-free enzymatic passaging system with reduced or no significant spontaneous differentiation, or to induce and / or maintain ground-state pluripotency, iPSCs require subsequent culture in a cell culture medium containing a GSK-3 inhibitor, a MEK inhibitor, and optionally a Rho kinase (ROCK) inhibitor, which cell culture medium does not contain or has inhibitors of the TGFβ / activin signaling pathway, including a TGFβ receptor (TGFβR) inhibitor and an ALK5 inhibitor, as contemplated herein. Without wishing to be bound by any particular theory, it is contemplated that long-term culture of pluripotent cells with a TGFβR / ALK5 inhibitor results in spontaneous differentiation of the cultured transgene-free iPSCs and ultimately a loss of ground-state pluripotency.

[0326] In various embodiments, a two-step culture platform is used to stably reprogram somatic cells to achieve reduced spontaneous differentiation in a medium containing ground-state pluripotency. In certain embodiments, non-pluripotent cells are reprogrammed by any suitable method disclosed in the art, and the reprogrammed somatic cells are cultured to achieve reduced spontaneous differentiation in culture by culturing the cells in a medium containing a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, the medium lacking a TGFβR / ALK5 inhibitor. In some embodiments, the reprogrammed somatic cells are cultured to provide ground-state pluripotent cells.

[0327] In a specific embodiment, non-pluripotent cells are reprogrammed by the methods disclosed herein and the reprogrammed somatic cells are cultured to a stable basal state of pluripotency by culturing the cells in a medium comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, and the medium does not have a TGFβR / ALK5 inhibitor.

[0328] In some embodiments, non-pluripotent cells are reprogrammed by introducing one or more reprogramming factors and culturing the cells in medium comprising a GSK-3 inhibitor, a MEK inhibitor, a Rho kinase (ROCK) inhibitor, and a TGFβR / ALK5 inhibitor, and the reprogrammed somatic cells are then cultured to provide cells with reduced spontaneous differentiation by culturing the cells in medium comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, wherein the medium does not have a TGFβR / ALK5 inhibitor.

[0329] In some embodiments, non-pluripotent cells are reprogrammed by introducing one or more reprogramming factors and culturing the cells in medium comprising a GSK-3 inhibitor, a MEK inhibitor, a Rho kinase (ROCK) inhibitor, and a TGFβR / ALK5 inhibitor, and the reprogrammed somatic cells are then cultured to a stable basal state of pluripotency by culturing the cells in medium comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, the medium lacking a TGFβR / ALK5 inhibitor.

[0330] In a preferred embodiment, non-pluripotent cells are reprogrammed by introducing one or more reprogramming factors and culturing the cells in medium containing a GSK-3 inhibitor, a MEK inhibitor, a Rho kinase (ROCK) inhibitor, and a TGFβR / ALK5 inhibitor, and then the reprogrammed somatic cells are cultured to a stable basal state of pluripotency by culturing the cells in medium containing a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, the medium lacking the TGFβR / ALK5 inhibitor and where there is no residual significant expression of the reprogramming transgene.

[0331] In one embodiment, non-pluripotent cells are reprogrammed by introducing one or more reprogramming factors selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, SV40LT, hTERT, SALL4, GLIS, ESRRB, DPPA2, ECAT1, SOX1, SOX3, KLF2, KLF5, L-MYC, N-MYC, LRH1 and UTF1 as disclosed elsewhere herein, and culturing the cells in a medium comprising a GSK-3 inhibitor, a MEK inhibitor, a Rho kinase (ROCK) inhibitor, and a TGFβR / ALK5 inhibitor, and thereafter the reprogrammed somatic cells are cultured in a medium comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, wherein the medium does not have a TGFβR / ALK5 inhibitor.

[0332] In a preferred embodiment, non-pluripotent cells are reprogrammed by introducing one or more reprogramming factors selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, SV40LT, hTERT, SALL4, GLIS, ESRRB, DPPA2, ECAT1, SOX1, SOX3, KLF2, KLF5, L-MYC, N-MYC, LRH1 and UTF1 as disclosed elsewhere herein, and culturing the cells in a medium comprising a GSK-3 inhibitor, a MEK inhibitor, a Rho kinase (ROCK) inhibitor, and a TGFβR / ALK5 inhibitor, thereafter the reprogrammed somatic cells are cultured in a medium comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor, wherein the ROCK inhibitor is thiazovivin, and the medium is free of the TGFβR / ALK5 inhibitor.

[0333] In various embodiments, methods are provided for producing pluripotent cells with reduced spontaneous differentiation and / or ground state induced pluripotent stem cells (iPSCs) using the culture substrates contemplated herein.

[0334] In specific embodiments, starting material including one or more non-pluripotent or partially pluripotent stem cells is used to produce pluripotent cells with reduced spontaneous differentiation and / or ground state induced pluripotent stem cells (iPSCs) by culturing the one or more pluripotent or partially pluripotent stem cells in a culture medium that does not contain a TGFβR inhibitor. The starting material may be obtained or formed. For example, non-pluripotent or partially pluripotent stem cells may be provided from a commercial supplier or other source or may be obtained fresh; non-pluripotent cells may also be isolated from tissues or organs; and partially pluripotent cells may also be generated by reprogramming somatic or adult stem cells. In some embodiments, pluripotent embryonic stem cells or pluripotent cells obtained by somatic cell nuclear transfer may be induced to achieve ground state pluripotency using the culture media and substrates described herein.

[0335] In specific embodiments, the one or more populations of IPSCs may comprise reprogrammed somatic cells or reprogrammed adult stem cells. In specific embodiments, IPSCs may be generated by any known method, by performing a method, or by obtaining IPSCs generated by a method.

[0336] Exemplary methods of generating IPSCs include, but are not limited to, increasing the expression of endogenous OCT4 in non-pluripotent cells; introducing into one or more non-pluripotent cells one or more polynucleotides, optionally in one or more copies, encoding one or more reprogramming factors selected from the group consisting of OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, SV40LT, hTERT, SALL4, GLIS, ESRRB, DPPA2, ECAT1, SOX1, SOX3, KLF2, KLF5, L-MYC, N-MYC, LRH1, and UTF1; or introducing into one or more non-pluripotent cells one or more polynucleotides, optionally in one or more copies, encoding one or more reprogramming factors selected from the group consisting of OCT4, SOX2, and NANOG. The method of generating iPSCs may further comprise contacting one or more non-pluripotent or partially pluripotent cells with a GSK3 inhibitor, a MEK inhibitor, and a TGFβR inhibitor, and optionally a ROCK inhibitor, to generate one or more iPSCs.

[0337] In certain embodiments, the cell culture medium comprises a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor.

[0338] In a preferred embodiment, the cell culture medium comprises a GSK3 inhibitor, a MEK inhibitor, and a ROCK inhibitor, wherein the ROCK inhibitor is thiazovivin.

[0339] In specific embodiments, culturing one or more pluripotent cells, e.g., IPSCs, in cell culture medium maintains or induces a ground state of pluripotency, viability, normal karyotype, genomic stability, and a reduced rate of spontaneous differentiation, which can be maintained for at least 5 passages, at least 10 passages, at least 50 passages, at least 100 passages, or more (including any intervening number of passages).

[0340] E. Characterization of Pluripotent Cells Pluripotent cells produced using the culture substrates contemplated herein may further include selection or validation of pluripotent cell products, including, for example, ground state pluripotent cells or pluripotent cells with reduced spontaneous differentiation. Pluripotent cells may be selected and / or validated after reprogramming and subsequent culture with the compositions and methods contemplated herein, or, if the pluripotent cells were not reprogrammed, after the pluripotent cells are transferred to the culture methods contemplated herein. The pluripotency of cells may be characterized and / or selected based on relevant and detectable morphological, molecular, and / or biochemical changes associated with pluripotency.

[0341] Specific characteristics of cellular pluripotency that may be monitored separately or in combination in assessing cellular potency include, but are not limited to, gene expression, methylation, and in vivo and in vitro characteristics, such as: i) round pluripotent stem cell morphology; ii) expression of pluripotent stem cell markers, including SSEA3 / 4 (human pluripotent stem cells), TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD105, OCT4, NANOG, SOX2, CD30, SSEA5, CD90, and / or CD50, and combinations thereof; iii) teratoma formation of pluripotent stem cells; iv) embryoid body formation and in vitro differentiation into three germ layers; and v) inactive X chromosome reactivation. In certain embodiments, any subset of the above characteristics is used to monitor cellular potency. In one embodiment, the pluripotent cells are characterized by having round colony morphology, expression of SSEA4, TRA1-81, and OCT4, and the ability to form embryoid bodies and teratomas.

[0342] In another embodiment, pluripotent cells with reduced spontaneous differentiation in in vitro culture may be identified by a gene expression signature comprising at least about a 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% decrease in expression of one or more of the following differentiation marker genes compared to pluripotent cells cultured in the presence of a TGFβR inhibitor: FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA 3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL, SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, MYC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyuri), and ZIC1.

[0343] In one embodiment, pluripotent cells with reduced spontaneous differentiation are characterized by decreased expression of one or more differentiation marker genes, including, but not limited to, T genes, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, and TUJ1. In a specific embodiment, pluripotent cells with reduced spontaneous differentiation may be identified by a gene expression signature comprising at least about a 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% decrease in expression of one or more differentiation marker genes (e.g., T genes, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, TUJ1) compared to pluripotent cells cultured in the presence of a TGFβR inhibitor. In another embodiment, pluripotent cells with reduced spontaneous differentiation may be identified by a gene expression signature comprising at least about a 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% reduction in expression of one or more differentiation marker genes (e.g., T gene, CXCR4, NODAL, GATA4, SOX17, FOXA2, OTX2, TUJ1).

[0344] In specific embodiments, ground-state pluripotent cells have greatly suppressed Xist expression and expression of early markers of differentiated cells, such as Foxa2, Sox17, and Brachyury, while conventional cultured pluripotent cells show only moderate suppression of Xist expression and prominent expression of early differentiation markers.

[0345] In specific embodiments, the ground state pluripotent cells maintain the properties of ground state pluripotency for multiple cell passages, for example, at least 1, 3, 5, 7, 10, 15, 20 or more passages.

[0346] F. Polynucleotides In various exemplary embodiments, the present invention contemplates, in part, polynucleotides, polynucleotides encoding polypeptides, and fusion polypeptides contemplated herein, as well as compositions comprising them. In various other exemplary embodiments, the present invention contemplates, in part, reprogramming non-pluripotent cells with polynucleotides encoding one or more copies of at least one reprogramming factor. Reprogramming factors for use with the culture substrates described herein include, but are not limited to, OCT4, SOX2, NANOG, KLF4, LIN28, C-MYC, SV40LT, hTERT, SALL4, GLIS, ESRRB, DPPA2, ECAT1, SOX1, SOX3, KLF2, KLF5, L-MYC, N-MYC, LRH1, and UTF1. In preferred embodiments, the polynucleotide comprises the sequence of a reprogramming factor as described herein.

[0347] As used herein, a "gene" may refer to a polynucleotide sequence that includes enhancers, promoters, introns, exons, etc. In specific embodiments, the term "gene" refers to a polynucleotide sequence that encodes a polypeptide, regardless of whether the polynucleotide sequence is identical to a genomic sequence that encodes the polypeptide.

[0348] An "isolated polynucleotide," as used herein, refers to a polynucleotide that has been purified from a DNA fragment that has been removed from sequences that flank it in its naturally occurring state, e.g., sequences that normally flank the fragment. In a specific embodiment, an "isolated polynucleotide" refers to a complementary DNA (cDNA), recombinant DNA, or other polynucleotide that is not found in nature and has been made by the hand of man.

[0349] In specific embodiments, one or more polynucleotides may be arranged in any suitable order within a larger polynucleotide, such as a vector. In a preferred embodiment, the vector is an episomal vector.

[0350] Regardless of the length of the coding sequence itself, the polynucleotides contemplated herein may be combined with other DNA sequences, such as expression control sequences, promoters and / or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or known in the art, and therefore their overall length can vary greatly. Thus, it is contemplated that polynucleotide fragments of almost any length can be used, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0351] Polynucleotides can be prepared, manipulated, and / or expressed using any of a variety of well-established techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors are the pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In specific embodiments, the coding sequences of the polypeptides disclosed herein can be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0352] In specific embodiments, the vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without integration into the host's chromosomal DNA and without gradual loss from dividing host cells, which also means that the vector replicates extrachromosomally or episomally. The vector is engineered to contain a sequence encoding an origin of DNA replication or "ori" from a lymphotropic herpesvirus or gammaherpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, specifically the lymphotropic herpesvirus or gammaherpesvirus origin of replication corresponding to the oriP of EBV. In specific embodiments, the lymphotropic herpesvirus may be Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), herpesvirus saimiri (HS), or Marek's disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses. Typically, host cells contain viral replication transactivator proteins that activate replication.

[0353] The "expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are the untranslated regions of the vector, i.e., origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine-Dalgarno or Kozak sequences), introns, polyadenylation sequences, and 5' and 3' untranslated regions that interact with host cell proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters, can be used.

[0354] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to the functional linkage between an expression control sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0355] Exemplary universal expression control sequences for use in specific embodiments of the present invention include the cytomegalovirus (CMV) immediate early promoter, the viral Simian Virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters from vaccinia virus, the elongation factor 1-alpha promoter, and the mitochondrial factor 1-alpha promoter. These include, but are not limited to, the alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), the human ROSA26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / avian β-actin (CAG) promoter, and the β-actin promoter.

[0356] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters, such as promoters of genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionine promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0357] Conditional expression can also be achieved using site-specific DNA recombinases. According to certain embodiments of the invention, a polynucleotide comprises at least one (typically two) site for recombination mediated by a site-specific recombinase. As used herein, the term "recombinase" or "site-specific recombinase" includes cleavable or integrable proteins, enzymes, cofactors, or related proteins involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, six, seven, eight, nine, ten, or more), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing the recombinant protein sequence or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in specific embodiments of the present invention include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0358] In specific embodiments, polynucleotides contemplated herein include one or more polynucleotides encoding one or more polypeptides. In specific embodiments, to achieve efficient translation of each of multiple polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides. As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into an initiation codon, such as ATG, of a cistron (protein-coding region), thereby resulting in cap-independent translation of the gene. See, e.g., Jackson et al., 1990. Trends Biochem Sci 15(12):477-83 and Jackson and Kaminski, 1995. RNA 1(10):985-1000. Examples of IRESs commonly used by those skilled in the art include those described in U.S. Patent No. 6,692,736. Further examples of "IRES" known in the art include, but are not limited to, IRES that can be obtained from picornaviruses (Jackson et al., 1990).

[0359] H. Polypeptides The present invention contemplates, in part, compositions comprising polypeptides, fusion polypeptides, and vectors expressing polypeptides. In a preferred embodiment, the polypeptide comprises an amino acid sequence described herein. "Polypeptide," "polypeptide fragment," "peptide," and "protein" are used interchangeably and according to their conventional meaning, i.e., as a sequence of amino acids, unless otherwise specified. In one embodiment, "polypeptide" includes fusion polypeptides and other variants. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides are not limited to a particular length; for example, they can include full-length protein sequences, fragments of full-length proteins, or fusion proteins, and can include post-translational modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications, both naturally occurring and non-naturally occurring, known in the art.

[0360] "Isolated peptide" or "isolated polypeptide" and the like, as used herein, refer to the in vitro isolation and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell, i.e., it is not significantly associated with in vivo materials.

[0361] In one embodiment where expression of two or more polypeptides is desired, the encoding polynucleotide sequences may be separated by an IRES sequence, as discussed elsewhere herein. In another embodiment, two or more polypeptides may be expressed as a fusion protein comprising a polypeptide cleavage signal between each of the polypeptide domains described herein. Additionally, polypeptide segments may be inserted within any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (deFelipe). and Ryan, 2004. Traffic, 5(8); 616-26.

[0362] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, byovirus NIa protease, biovirus RNA-2 encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow mottle virus) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. Due to its high cleavage stringency, in one embodiment a TEV (tobacco etch virus) protease cleavage site, e.g., EXXYXQ(G / S) (SEQ ID NO: 29), such as ENLYFQG (SEQ ID NO: 30) and ENLYFQS (SEQ ID NO: 31) (X represents any amino acid) is preferred (TEV cleavage occurs between Q and G or between Q and S).

[0363] In certain embodiments, the self-cleaving polypeptide site comprises a 2A or 2A-like site, sequence, or domain (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). In specific embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0364] In one embodiment, the viral 2A peptide is a foot and mouth disease virus (FMDV) 2A peptide, an equine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna (Thosea asigna virus (TaV) 2A peptide, porcine teschovirus-1 (PTV-1) 2A peptide, tylovirus 2A peptide, and encephalomyocarditis virus 2A peptide. [Table 2]

[0365] In a preferred embodiment, the vector encoding one or more reprogramming factor polypeptides contains one or more of the same or different protease cleavage sites between each of the reprogramming factors.

[0366] All publications, patent applications and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application or issued patent was specifically and individually indicated to be incorporated by reference.

[0367] Although the foregoing invention has been described in some detail using figures and examples for clarity of understanding, it will be readily apparent to those skilled in the art, in light of the teachings of this invention, that certain changes and modifications may be made to the present invention without departing from the spirit or scope of the appended claims. The following examples are provided for illustrative purposes only, and not for limiting purposes. Those skilled in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results. [Example]

[0368] The examples disclosed herein identified a platform for rapid parallel generation, selection, and expansion of hiPSCs using small molecule pathway inhibitors in stage-specific media compositions. The platform supported efficient and accelerated episomal reprogramming using minimal reprogramming factors in a completely feeder-free environment. The resulting hiPSCs were transgene-free and easily cultured and expanded as single cells, while maintaining a homogenous and genomically stable pluripotent population. hiPSCs generated or maintained in the media compositions contemplated in the examples exhibited characteristics associated with a ground state of pluripotency and represent a robust, high-throughput system for the production of uniform, industrial- or clinical-grade hiPSCs.

[0369] Example 1 Identifying a culture medium platform for long-term maintenance and expansion of iPSCs overview Although the majority of lentiviral-induced hiPSC lines in SMC4-supplemented cultures maintained a homogenous population of undifferentiated cells, silencing transgene reprogramming factors in a subset of lines exhibited varying degrees of spontaneous differentiation in long-term culture (Figure 1A and B). Therefore, various cell culture components were evaluated to identify conditions for maintaining pluripotency during continuous FF culture and enzymatic passaging of single cells, independent of residual transgene expression. A multi-step culture system targeting unique pathways at different stages of the reprogramming and maintenance process was identified as an efficient and robust approach for hiPSC generation.

[0370] result Inhibition of the TGFβ pathway during long-term maintenance was identified as a critical factor in the spontaneous differentiation of hiPSC lines with silenced transgene expression (Figure 1C). One of the iPSC cell lines found to produce spontaneous expression was transitioned to culture in a new media formulation, Fate Maintenance Medium (FMM) (Table 1). Spontaneous differentiation was eliminated, and a homogenous population of SSEA4 / TRA1-81-positive cells was established within 2–3 passages (Figure 1A).

[0371] We also compared OCT4 / KLF4 / SOX2 (OKS) lentiviral reprogramming in conventional culture (hESC medium on MEF feeder cells), SMC4-supplemented medium in FF culture, or freshly formulated FMM in FF culture (Figure 1D). Seventeen days after lentiviral reprogramming, SSEA4 / TRA1-81-positive cells were selected by FAC and replated in SMC4 or FMM for comparison (Figure 1D). SMC4 improved the reprogramming kinetics, resulting in significantly more SSEA4 / TRA1-81-positive cells than FMM reprogramming at 17 days after induction (2.72% vs. 0.76% in FMM and 0.10% in conventional culture; Figure 1D).

[0372] After the initial sorting, cells were maintained under each condition for 10 days, followed by a second round of SSEA4 / TRA1-81-positive flow cytometry selection (Figure 1D). Cultures were maintained for an additional 9 days (a total of 36 days after infection) and scored for undifferentiated colonies based on OCT4 and NANOG coexpression (Figures 1D and 1E). The combination of initial reprogramming in SMC4 followed by transfer to FMM ultimately resulted in more OCT4 / NANOG-positive colonies and a significantly reduced number of OCT4 / NANOG-negative colonies relative to continued maintenance in SMC4 (Figures 1D and 1E). OCT4 / NANOG-positive colonies were detected in cultures maintained only in FMM, although the number and size of colonies appeared to be lower than with the stage-specific media approach.

[0373] These results demonstrate that a novel multi-step culture system targeting unique pathways at different stages of the reprogramming and maintenance process resulted in the efficient production of high-quality hiPSCs.

[0374] Example 2 Platform overview for the production of transgene-free hiPSCs in single cell passaging and FF format The efficiency of non-integrative reprogramming methods using episomal vector systems is extremely low (<0.001%), especially in FF environments (Narsinh et al., 2011; O'Doherty et al., 2013). We tested episomal induction in a multi-stage culture system containing two media: Fate Reprogramming Medium (FRM), which contains SMC4 and media additives shown to improve reprogramming, and FMM (Figure 2A and Table 1).

[0375] result Various fibroblasts were transfected with an episomal expression system consisting of the OCT4 / SOX2 / NANOG / KLF4 / LIN28 / MYC / SV40LT (OSNKLMT) gene combination. 24 hours after induction of episomal expression, reprogramming cultures were shifted to ERM to improve reprogramming kinetics. Initial colony formation was observed within the first week, and by day 10, a large population of SSEA4 / TRA1-81-positive cells was detected (>1%) (Figures 8A and 8B). On day 14, FRM-supported reprogramming cultures were split into FRM or FMM medium. On day 21, SSEA4 / TRA1-81 / CD30-positive cells were identified in the cultures using FACS (Figure 8C). FRM-maintenance cultures contained both differentiated and undifferentiated cells, while FMM cultures contained mostly undifferentiated cells (Figure 8D).

[0376] The throughput and robustness of this method were tested with fibroblasts and CD34+ cells expanded from minimal amounts of umbilical cord blood from donors of different ages, genders, and ethnicities (Figures 9A and 9B). Somatic cell reprogramming was induced with the episomal gene combination set OSNKLMT as outlined in Figure 2A, and individual hiPSCs were sorted by 96-well plate flow cytometry between days 16 and 21 (Figure 2B). A large population of SSEA4 / TRA1-81 / CD30-positive cells was observed for the majority of lines tested. Compared to parallel reprogramming experiments using conventional media and feeder cells, the FRM and FMM media systems resulted in a significant increase in the number of hiPSC clones (8.55% in FRM / FMM versus 0.02% in conventional culture for the FTC007 fibroblast line (Figures 2A and 2B)). On average, 22 clonal hiPSCs per 96-well plate were observed for each somatic cell line, including the fibroblast FTC008, which had previously been observed to be refractory to lentiviral reprogramming using SMC4 medium (Figures 2B, 10A, and 10B). Colonies were subsequently confirmed as bona fide hiPSC clones by analysis of intracellular and surface marker expression and direct qRTPCR for NANOG (Figures 2D, 2E, and 10C). Reprogramming efficiency using the 96-well sorting and selection process was also increased (Figure 2E). Similar reprogramming efficiency was observed with the defined surface coating vitronectin (Figure 10D).

[0377] These data demonstrated that the platform is robust and reproducible when applied to episomal reprogramming, and allows multiple reprogramming experiments to be performed in parallel in a high-throughput manner with minimal effort and without compromising the quality of the final iPSC product.

[0378] Example 3 Long-term passaging and expansion of transgene-free hiPSC lines in FMM overview Using hiPSC clones from Example 2 expanded as single cells in FF culture, long-term passaging and expansion of hiPSCs using FRM and FMM multi-stage media platforms was tested (Figures 3A and 3B).

[0379] result hiPSC lines reprogrammed according to Example 2 lost episomal DNA by passages 4–7 and were therefore pluripotent independent of transgene-based reprogramming factors (Figure 3C). The hiPSC lines maintained a homogenous population of undifferentiated cells positive for SSEA4, TRA1-81, OCT4, and NANOG. Furthermore, these lines maintained their pluripotent characteristics (Figure 3F) without any washing strategies commonly utilized in pluripotent culture (Figures 3D and 3E). Similar expansion of homogenous hiPSC cultures was observed when Matrigel was replaced with the defined surface coating vitronectin during routine single-cell passaging (Figures S10E–G).

[0380] Genomic abnormalities are often detected in hESC and hiPSC lines cultured as single cells in an FF environment (Laurent et al., 2011; Taapken et al., 2011). Karyotype analysis of all analyzed hiPSC lines demonstrated genomic stability in FMM culture (Figure 4A). Furthermore, single-cell and FF-cultured hiPSC clones maintained in FMM for long periods (25–30 passages) continued to maintain their undifferentiated profile and genomic stability without the need for culture washing or selection (Figure 4B).

[0381] Episome-derived hiPSC clones maintained in FMM also readily gave rise to all three s...

Claims

1. 1. A method of culturing a population of human induced pluripotent stem cells (hiPSCs), the method comprising: (a) culturing the population of hiPSCs in a first culture medium, wherein (i) the culturing comprises specifically inhibiting each of GSK3, MEK, and ROCK, (ii) the culturing does not comprise specifically inhibiting TGFβR, and (iii) the first culture medium does not comprise feeder cells; (b) passaging the population of cultured hiPSCs; (c) culturing the population of passaged hiPSCs in a second culture medium, wherein (i) the culturing comprises specifically inhibiting each of GSK3, MEK, and ROCK; (ii) the culturing does not comprise specifically inhibiting TGFβR; and (iii) the second culture medium does not comprise feeder cells. A method comprising:

2. The method of claim 1, wherein the hiPSCs cultured in the first culture medium and the second culture medium exhibit ground-state pluripotency.

3. The method of claim 2, wherein the hiPSCs with ground state pluripotency exhibit suppressed expression of one or more of GATA4, SOX17.

4. The method of claim 1 , wherein the hiPSCs in the second culture medium do not contain an exogenous polynucleotide encoding a reprogramming factor.

5. The method of claim 1 , wherein said passaging comprises dissociating said population of hiPSCs to produce a population of dissociated hiPSCs.

6. 2. The method of claim 1, wherein (i) at least 70% of cells in the population of passaged hiPSCs are positive for both SSEA4 and TRA1-81, and (ii) the passaged hiPSCs maintain ground-state pluripotency.

7. The method of claim 1, wherein at least 80% or at least 90% of cells in the population of passaged hiPSCs are positive for both SSEA4 and TRA1-81.

8. The method of claim 5 , wherein the dissociating comprises enzymatic dissociation.

9. The method of any one of claims 1 to 8, wherein said passaging comprises sorting said cells to enrich for hiPSCs with one or more markers of pluripotency.

10. The method of claim 9, wherein the one or more markers of pluripotency include SSEA4 and TRA1-81.

11. The method of any one of claims 1 to 8, wherein the passaged hiPSCs maintain one or more of genomic stability or a normal karyotype.

12. 9. The method of any one of claims 1 to 8, wherein the passaging comprises at least 10 passages, at least 50 passages, or at least 100 passages.

13. 9. The method of any one of claims 1 to 8, wherein the population of hiPSCs comprises one or more exogenous polynucleotides encoding one or more of OCT4, SOX2, KLF4, NANOG, ECAT1, UTF1, ESRRB, and SV40LT.

14. 9. The method of any one of claims 1 to 8, wherein specifically inhibiting each of GSK3, MEK, and ROCK comprises culturing with (a) thiazovivin or Y27632, (b) A-83-01 or SB431542, and (c) CHIR99021 or BIO.

15. 9. The method of any one of claims 1 to 8, further comprising reprogramming non-pluripotent human cells to produce said population of hiPSCs.

16. 16. The method of claim 15, wherein the reprogramming comprises introducing one or more copies of at least one reprogramming factor encoded by one or more exogenous polynucleotides into the non-pluripotent human cell, wherein the at least one reprogramming factor is selected from the group consisting of OCT4, SOX2, KLF4, NANOG, ECAT1, UTF1, ESRRB, and SV40LT.

17. 17. The method of claim 16, wherein said reprogramming further comprises culturing said non-pluripotent human cells in a reprogramming medium, wherein said culturing in said reprogramming medium comprises specifically inhibiting each of GSK3, MEK, ROCK, and TGFβR.

18. the one or more exogenous polynucleotides (a) at least two copies of an OCT4 polynucleotide, one copy of an ECAT1 polynucleotide, one copy of a UTF1 polynucleotide, one copy of a NANOG polynucleotide, and one copy of an ESRRB polynucleotide; or (b) at least one copy of an OCT4 polynucleotide, one copy of an ECAT1 polynucleotide, and one copy of a UTF1 polynucleotide; 17. The method of claim 16, comprising a vector comprising:

19. the one or more exogenous polynucleotides (a) introduced into cells by retrovirus, Sendai virus, adenovirus, episome, minicircle, vector system carrying an expression cassette, or mRNA; (b) comprises a polycistronic polynucleotide separated by at least one 2A peptide; or (c) comprising polycistronic polynucleotides each encoding an OCT4 polypeptide; 17. The method of claim 16.

20. 1. A culture medium comprising a GSK3 inhibitor, a MEK inhibitor, a ROCK inhibitor, and a population of human induced pluripotent stem cells (hiPSCs), (a) the culture medium does not contain a TGFβR inhibitor; (b) the culture medium is a feeder-free medium; and (c) the hiPSCs comprise hiPSCs with ground state pluripotency; Culture medium.

21. (a) the GSK3 inhibitor is CHIR99021 or BIO; (b) the MEK inhibitor is PD98059 or PD0325901; (c) the ROCK inhibitor is thiazovivin or Y27632; or (d) the culture medium further comprises bFGF and / or LIF; 21. The culture medium of claim 20.

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