Cell culture platform for single cell sorting and enhanced reprogramming of ipsc

Abstract

To provide cell culture conditions for culturing stem cells, including feeder-free conditions for generating and culturing human induced pluripotent stem cells (iPSCs).SOLUTION: According to the present invention there is provided a culture platform that allows long-term culture of pluripotent cells in a feeder-free environment; reprogramming of cells in a feeder-free environment; single-cell dissociation of pluripotent cells; cell sorting of pluripotent cells; maintenance of an undifferentiated status; improved efficiency of reprogramming; and generation of a naive pluripotent cell.SELECTED DRAWING: None

Description

[Technical field]

[0001] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text form in lieu of a hard copy and is incorporated herein by reference. The name of the text file containing the sequence listing is FATE_094_00WO_ST25.txt. This text file is 4 KB, was created on Dec. 15, 2011, and is being submitted electronically via EFS-Web.

[0002] 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 / 426,369, filed December 22, 2010, and U.S. Provisional Patent Application No. 61 / 496,991, filed June 14, 2011, each of which is incorporated by reference in its entirety.

[0003] 2. Background of the Invention Technical Field The present invention relates generally to cell culture conditions, media, and culture platforms for culturing stem cells, including feeder-free conditions for generating and culturing human induced pluripotent stem cells (iPSCs). [Background technology]

[0004] 2. Description of Related Art The application of pluripotent stem cell biology opens new doors in regenerative medicine. Obtaining human embryonic stem cells (hESCs) by culturing preimplantation blastocysts in a cocktail of growth factors has led to many promising cell therapy approaches, where the expanded self-renewing cell population can be differentiated into therapy-relevant cell lineages in vitro or in vivo. In further applications of ESC biology, and by using preimplantation genetic analysis, it has become possible to obtain ESC lines from several genetic disease backgrounds and thus model these diseases in tissue culture dishes. However, ESC technology has some limitations: the range of genetic backgrounds from which ESCs can be obtained is technically and politically limited, the genetic background of ESCs is not always known, and the use of ESC-derived cell therapies is essentially an allogeneic transplant, which carries the same risk of rejection as traditional tissue / organ transplants.

[0005] In a major advance, pluripotent cell populations were generated from adult terminally differentiated cells, and the cells so obtained are referred to as induced pluripotent stem cells (iPSCs). iPSC technology allows cells from any donor to be reprogrammed into a pluripotent, self-renewing state, thus allowing the expansion of homogenous cell populations from any genetic background. iPSCs overcome the ethical considerations associated with ESCs and can be used to obtain models of any genetic human disease for high-throughput drug screening or hepatocytes and cardiomyocytes for xenobiotic drug toxicity screening. Furthermore, iPSCs may ultimately lead to cell therapy generated from a patient's own cells in autologous transplantation, which can prevent graft rejection. Expression and differentiation analyses show that there is a similar degree of variation between clonal iPSC cultures as observed when comparing multiple ESC lines, indicating that iPSCs are very close to ESCs at the molecular level.

[0006] iPSCs have generally been shown to be necessary for complete reprogramming iPSCs have been generated by ectopic expression of a combination of several key genes, namely Oct4, Sox2, Klf4, c-Myc, Lin28 and Nanog. iPSCs were first generated using integrative viral systems to express key transcription factors. Currently, retroviral and lentiviral systems, including polycistronic and inducible systems, have been successfully used in the generation of iPSCs. However, permanent genomic changes resulting from insertional mutagenesis and the potential for exogenous gene reactivation after iPSC differentiation may present potential problems for subsequent drug screening and therapeutic application of cells generated by these methods. Indeed, significant differences have been reported between iPSC clones generated using the same viral system, with the majority of clones forming tumors in rodents when transplanted as differentiated neurospheres. Studies suggest that iPSCs generated using the same viral method may behave differently once differentiated. Different ectopic gene integration sites may result in different insertional mutagenesis and epigenetic control of transgene expression. For iPSC generation methods involving integration systems, it may be necessary to obtain and screen many clones to identify clones that are stable in both pluripotent and differentiated states. Therefore, a method for rapidly obtaining clonal iPSCs from a given donor cell source would be beneficial. The use of non-integration systems, such as adenovirus or episomal transient expression, to generate iPSCs has also been demonstrated, albeit with low efficiency. Although these systems may overcome the safety and stability issues in the generation of iPSCs, there is a potential for genome integration when using any DNA-based reprogramming method, which must be evaluated before using them in the development of iPSC-derived cell therapy.

[0007] Excisable viral systems and genome-wide expression profiling have shown that iPSCs with integrated expression cassettes are less similar to ESCs than the same clones in which the viral factors were excised. Furthermore, protein-only reprogramming has now been demonstrated, expressing the most commonly used transcription factors as fusion proteins with cell-permeable peptides in E. coli. iPSCs were generated by applying multiple doses of purified protein to mouse fibroblasts. The efficiency of reprogramming using this protein-only system was very low. This may be due to the efficiency of protein transduction, the specific activity of the protein and / or the stability of the protein.

[0008] The process of reprogramming differentiated cells by ectopically expressing pluripotency genes or introducing them by protein transduction or mRNA requires several months and the knowledge of a skilled stem cell biologist. Identification of reprogrammed cells is initially a visual screening of ESC-like colony morphology. Such colonies must be manually picked and usually mechanically passaged and expanded. Introducing pluripotency factors also creates transformed cell colonies as well as incompletely reprogrammed cells. Although researchers may be able to identify true iPSC colonies from a background of transformed cells, this is not an efficient process. Further characterization and recognition as a true pluripotent population is then required, usually including immunocytochemical staining for markers of pluripotency, gene expression and epigenetic analysis as well as the ability of the pluripotent population to differentiate into the three germ layers (ectoderm, mesoderm and endoderm). Once pluripotent cells are identified and selected, such cells generally grow as colonies and to maintain the cells over time, manual passage by picking the cells and replating them after mechanical dissociation is required.

[0009] Embryonic stem cells derived from various pre-implantation and post-implantation stages exhibit distinct pluripotent states. For example, cells derived from the inner cell mass of the blastocyst are considered to be more "naive" and have important properties that are quite different from post-implantation derived cells, which are considered to be more "primed" with a higher tendency to randomly differentiate. Naive cells are more "ground state" (grown). Primed cells appear to be in a "primed state" and do not require exogenous signaling to maintain their undifferentiated state. Primed cells, on the other hand, require exogenous signaling of key cytokines, including TGFβ, activin, and bFGF, to maintain their undifferentiated state and are highly dependent on the ERK / MAPK cellular pathway. Summary of the Invention [Problem to be solved by the invention]

[0010] Improving the process of iPSC generation can dramatically lower the technical barriers and speed up the process, allowing for subsequent scale-up and differentiation of cells for industrial applications of the technology, such as drug screening and cell therapy. Methods are needed to more efficiently generate iPSCs without exogenous materials and to more efficiently identify and select reprogrammed cells. A method to generate iPSCs that promotes the naive state of human pluripotent stem cells would be a significant advantage in future applications in regenerative medicine, such as disease correction, directed differentiation, and scale-up of manufacturing. Furthermore, methods are needed to more efficiently generate iPSCs in defined culture conditions that allow single cell passaging and scalability. [Means for solving the problem]

[0011] Brief Summary of the Invention One embodiment of the present invention provides a method of culturing pluripotent cells in a feeder-free environment, comprising culturing pluripotent cells that are not mouse embryonic stem cells in a feeder-free environment in a medium comprising at least one agent that maintains the pluripotency of the cells selected from the group consisting of: i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor, and maintaining the pluripotency of the cells during culturing.

[0012] In certain embodiments, the medium comprises a sufficient amount of an agent to maintain the pluripotency of the cells while allowing at least one cell division. In further embodiments, the medium comprises at least two agents that maintain the pluripotency of the cells. In certain embodiments, the medium comprises at least three or four agents that maintain the pluripotency of the cells.

[0013] In another particular embodiment, the agent that maintains pluripotency of cells comprises a Rock inhibitor. In a particular embodiment, the Rock inhibitor is thiazovivin or Y27632.

[0014] In one embodiment, the agent that maintains pluripotency of a cell comprises a TFGβ inhibitor, and in a particular embodiment, the TFGβ inhibitor is A-83-01 or SB431542.

[0015] In certain embodiments, the agent that maintains pluripotency of cells comprises a GSK3 inhibitor, and in certain embodiments, the GSK3 inhibitor is CHIR99021 or BIO.

[0016] In one embodiment of the invention, the agent that maintains pluripotency of cells comprises a MEK inhibitor. In certain embodiments, the MEK inhibitor is PD98059 or PD032901.

[0017] In certain embodiments, the medium comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor. In a more specific embodiment of the present invention, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0018] In some embodiments of the invention, the pluripotency of the cells is maintained for at least five cell divisions. In other embodiments, the pluripotency of the cells is maintained for at least ten cell divisions. This is maintained.

[0019] In some embodiments, the cells are cultured in the absence of growth factors and cytokines, and optionally in the presence of soluble fibronectin. In further embodiments, the cells are cultured in the absence of Matrigel™, and in yet other embodiments, the medium is substantially free of bFGF.

[0020] In certain embodiments of the invention, the pluripotent cells are human embryonic stem cells or human induced pluripotent stem cells.

[0021] Another embodiment of the invention provides a method of culturing pluripotent cells, comprising culturing pluripotent cells that are not mouse embryonic stem cells in the absence of growth factors and cytokines.

[0022] In an embodiment, the method includes culturing the pluripotent cells in a medium containing at least one agent that maintains the pluripotency of the cells, selected from the group consisting of: i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor, to allow at least one cell division while maintaining the pluripotency of the cells.

[0023] In one embodiment, the pluripotent cells are human embryonic stem cells or human induced pluripotent stem cells.

[0024] In further embodiments, the medium comprises at least two, at least three, or four agents selected from the group consisting of i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor; in certain embodiments, the medium comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor; in other certain embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0025] Yet another embodiment of the present invention provides a method of obtaining dissociated human pluripotent cells, comprising dissociating the human pluripotent cells to obtain dissociated cells, and contacting the dissociated cells with a medium comprising at least one agent that enhances the viability of the dissociated cells, selected from the group consisting of: i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor, thereby enhancing the viability of the dissociated cells. In certain embodiments, the viability of the dissociated cells is enhanced by at least 10%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0026] In other embodiments, the methods of the invention further comprise culturing the dissociated cells in culture medium for at least 1, at least 2, at least 5, or at least 10 passages while maintaining the pluripotency of the dissociated cells.

[0027] In certain embodiments, the karyotype of the dissociated cells after culture is substantially similar to the karyotype of the cell population before dissociation.

[0028] In some embodiments, the method comprises dissociating in the presence of an agent. In yet other embodiments, the method comprises contacting the pluripotent cells with the agent prior to dissociating. In certain embodiments, contacting the dissociated cells with medium comprises suspending the dissociated cells in medium.

[0029] In another embodiment, the present invention relates to a method for determining the potency of cells in a feeder-free environment. The present invention provides a method for increasing the activity of a cell in a feeder-free environment, the method comprising contacting the cell with a medium containing at least one small molecule agent selected from the group consisting of: i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor, to obtain a cell having increased activity compared to the cell before contacting the medium. In a particular embodiment, the medium contains a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, and in an embodiment, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0030] In one embodiment, the contacting step comprises culturing the cells under conditions sufficient to increase the potency of the cells.

[0031] In some embodiments, the cell is selected from the group consisting of an embryonic stem cell, a pluripotent cell, a multipotent cell, a non-pluripotent cell, and a somatic cell.

[0032] In certain embodiments, the cell is not a mouse embryonic stem cell, in other certain embodiments, the cell is a human cell, and in some embodiments, the cell is an induced pluripotent stem cell.

[0033] In some embodiments, the method further comprises contacting the cell with at least one pluripotency factor. In some embodiments, the pluripotency factor comprises a polynucleotide, a polypeptide, or a small molecule. In some embodiments, the pluripotency factor is an exogenous transcription factor. In certain embodiments, the exogenous transcription factor comprises an Oct4, Sox, Klf, Myc, Lin28, or Nanog polypeptide, or a polynucleotide encoding Oct4, Sox, Klf, Myc, Lin28, or Nanog. In other embodiments, the polypeptide comprises an amino acid sequence that allows transport across the cell membrane. In other specific embodiments, the exogenous transcription factor comprises an Oct4, Sox2, and Klf4 polynucleotide.

[0034] In still other embodiments, the cells with increased potency are characterized by one or more of the following: expression of at least one pluripotent stem cell marker selected from the group consisting of Oct4, Nanog, KLF4, SSEA4, and TRA 1-81; pluripotent stem cell morphology; ability to contribute to germline transmission; formation of teratomas; ability to differentiate or transdifferentiate into a lineage different from the starting lineage; and tri-lineage differentiation in vitro. In some embodiments, the cells with increased potency express at least 2-fold higher levels of Oct4 compared to the cells prior to contact with the medium. In still other embodiments, the cells with increased potency have at least 2-fold lower Xist activity compared to conventionally cultured iPSCs. In further embodiments, the cells with increased potency have a compact, dome-shaped colony morphology.

[0035] In some embodiments, cells with increased potency replicate and maintain pluripotency in the absence of exogenous stimulation of the TFGβ, activin, and MEK signaling pathways, and optionally, in the absence of exogenous stimulation of the bFGF pathway.

[0036] In other embodiments, the method further comprises culturing the cells with increased potency in a feeder-free environment in the presence of at least one of i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, or iv) a Rock inhibitor to allow at least one cell division while maintaining cell potency. In certain embodiments, the cells are cultured in the presence of a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, in certain embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is PD0325901. The agent is thiazovibin.

[0037] The invention also provides a cell with increased potency produced by any of the above embodiments.

[0038] The invention further provides a method of improving the efficiency of reprogramming of a cell population in a feeder-free environment, comprising contacting the cell population in a feeder-free environment with at least one small molecule agent selected from the group consisting of: i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor, under conditions sufficient to induce reprogramming, thereby improving the efficiency of reprogramming by at least 10%, at least 50%, at least 100%, at least 300%, or at least 500% compared to the efficiency of reprogramming when the cell population is not contacted with the small molecule agent.

[0039] In certain embodiments, the cells are contacted with a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, and in certain embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0040] In some embodiments, the cell population prior to reprogramming comprises non-pluripotent cells, hi other embodiments, the efficiency of reprogramming is measured by the time required for reprogramming or by the number of reprogrammed cells.

[0041] In yet other embodiments, the conditions sufficient to induce reprogramming comprise contacting the cell population with at least one exogenous transcription factor selected from the group consisting of Oct4, Sox, Klf, Myc, Lin28, or Nanog polypeptides, or polynucleotides encoding Oct4, Sox, Klf, Myc, Lin28, or Nanog. In certain embodiments, the conditions comprise contacting the cell population with Oct4, Sox2, and Klf4 polypeptides, or polynucleotides encoding Oct4, Sox2, and Klf4.

[0042] Yet another embodiment of the present invention provides a method of sorting a cell population in a feeder-free environment to obtain a cell population enriched for pluripotent cells, comprising the steps of obtaining a suspension of dissociated cells comprising a mixed population of cells in a feeder-free environment, and sorting the cells in the suspension to obtain cells expressing one or more markers of pluripotency, thereby obtaining an enriched cell population enriched for pluripotent cells. In some embodiments, the suspension comprises at least one of i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, or iv) a Rock inhibitor. In further embodiments, the sorting is by magnetic beads or flow cytometry. In certain embodiments, the sorting is by magnetic beads. In other specific embodiments, the sorting is by flow cytometry.

[0043] In some embodiments, the method further comprises culturing the enriched cell population in a medium comprising at least one of: i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, or iv) a Rock inhibitor, optionally in combination with soluble fibronectin.

[0044] In certain embodiments, the mixed population of cells in suspension is contacted with at least one of i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, or iv) a Rock inhibitor prior to sorting. In another embodiment, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0045] In certain embodiments, the mixed population of cells comprises cells expressing one or more markers of pluripotency. In certain embodiments, the one or more markers of pluripotency comprise SSEA4, TRA160, TRA181, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD30, CD50, CD133 / prominin, CD140a, CD56, CD73, CD105, CD31, CD34, OCT4, Nanog, or Sox2. In specific embodiments, the markers of pluripotency are selected from the group consisting of SSEA4, TRA181, TRA160, and CD30.

[0046] In certain embodiments, the method comprises contacting a mixed population of cells with one or more pluripotency factors to induce reprogramming. In some embodiments, the contacting comprises introducing one or more pluripotency factors into cells in the mixed population of cells. In some embodiments, the pluripotency factor comprises Oct4, Sox, Klf, Myc, Lin28, or Nanog polypeptide, or a polynucleotide encoding Oct4, Sox, Klf, Myc, Lin28, or Nanog. In other specific embodiments, the pluripotency factor comprises Oct4, Sox2, and Klf4 polypeptide, or a polynucleotide encoding Oct4, Sox2, and Klf4 polypeptide.

[0047] In certain embodiments, the pluripotent cells are induced pluripotent cells.

[0048] In some embodiments, the enriched cell population is at least 20%, at least 50%, at least 100%, at least 200%, or at least 500% enriched for cells expressing one or more markers of pluripotency.

[0049] In another embodiment, the present invention provides a method for obtaining induced pluripotent stem cells, comprising the steps of treating a cell population to induce reprogramming, preparing a suspension of dissociated cells comprising the cell population, sorting the cells in the suspension to obtain the selected cells expressing one or more pluripotency markers, and culturing the selected cells expressing one or more pluripotency markers, wherein iPSCs are obtained.In certain embodiments, the sorted cells are cultured in the absence of cytokines and growth factors, optionally in a feeder-free environment, and optionally in the presence of soluble fibronectin.

[0050] In some embodiments, the cell population is contacted with at least one of: i) a TGFβ inhibitor; ii) a GSK3 inhibitor; iii) a MEK inhibitor; or iv) a Rock inhibitor. In certain embodiments, the cell population is contacted with a TGFβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, and in some embodiments, the TGFβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0051] In other embodiments, treating the cell population to induce reprogramming comprises contacting the cell population with one or more pluripotency factors. In certain embodiments, the pluripotency factors comprise Oct4, Sox, Klf, Myc, Lin28, or Nanog polypeptides, or polynucleotides encoding Oct4, Sox, Klf, Myc, Lin28, or Nanog. In certain embodiments, the pluripotency factors comprise Oct4, Sox, Klf, Myc, Lin28, or Nanog polypeptides, or polynucleotides encoding Oct4, Sox, Klf, Myc, Lin28, or Nanog. 4, Sox2, and Klf4 polypeptides, or a polynucleotide encoding Oct4, Sox2, and Klf4 polypeptides.

[0052] In other embodiments, treating the cell population to induce reprogramming further comprises contacting the cell population with at least one of i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, or iv) a Rock inhibitor. In certain embodiments, the cells are contacted with a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, in certain embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0053] In other embodiments, the suspension comprises at least one of i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, or iv) a Rock inhibitor. In certain embodiments, the suspension comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, in certain embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0054] In some embodiments, sorting is by flow cytometry or by magnetic beads. In certain embodiments, cells are sorted to obtain cells expressing at least one, two, three, four, or more markers of pluripotency. In other certain embodiments, the one or more markers of pluripotency include SSEA4, TRA160, TRA181, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD30, CD50, CD133 / prominin, CD140a, CD56, CD73, CD105, CD31, CD34, OCT4, Nanog, or Sox2. In other certain embodiments, the one or more markers of pluripotency are selected from the group consisting of SSEA4, TRA160, TRA181, and CD30. In some embodiments, the one or more markers of pluripotency are SSEA4, CD30, and TRA160 or TRA181. In another embodiment, cells are sorted using specific markers to deplete non-reprogrammed cells from the reprogramming population.

[0055] In some embodiments, the culturing step comprises culturing the cells in a medium comprising at least one small molecule agent selected from the group consisting of: i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor. In certain embodiments, the medium comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, and in certain embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0056] In certain embodiments, the cells are cultured in a feeder-free environment. In certain embodiments, the cells are treated, suspended, selected and cultured in a feeder-free environment.

[0057] In some embodiments, the induced pluripotent stem cells are obtained in about 2 to about 22 days. In certain embodiments, the induced pluripotent stem cells are obtained in about 4 to about 18 days.

[0058] In other embodiments, the induced pluripotent stem cells are obtained within about 4 to about 22 days after treating the cell population to induce reprogramming. In some embodiments, the induced pluripotent stem cells are obtained within about 6 to about 18 days after treating the cell population to induce reprogramming, and in other embodiments, the induced pluripotent stem cells are obtained within about 4 to about 22 days after treating the cell population to induce reprogramming. It can be obtained within about 10 to 16 days.

[0059] In another embodiment, the present invention also provides induced pluripotent stem cells obtained by any of the above methods.

[0060] The invention further provides a method of depleting pluripotent cells from a cell population, the method comprising obtaining a suspension of dissociated cells comprising a mixed population of cells having pluripotent cells, and sorting the cells in the suspension to remove cells expressing one or more markers of pluripotency, thereby depleting the pluripotent cells from the cell population.

[0061] In some embodiments the mixed population of cells comprises multipotent cells or (adult) somatic cells.

[0062] In other embodiments, the mixed population of cells in the suspension is cultured in a medium containing at least one small molecule agent selected from the group consisting of i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor before obtaining the suspension.In certain embodiments, the medium contains a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, and in some embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0063] In further embodiments of the present invention, the suspension comprises at least one small molecule agent selected from the group consisting of i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor. In certain embodiments, the suspension comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, in certain embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0064] In some embodiments, sorting is by flow cytometry, hi other embodiments, sorting is by antibody-coated magnetic bead enrichment.

[0065] Some embodiments of the present invention provide a method of obtaining pluripotent cells with genomic stability, comprising contacting cells in a feeder-free environment with at least one small molecule agent selected from the group consisting of: i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor, in the absence of c-myc, under conditions sufficient to obtain pluripotent cells with genomic stability.

[0066] In some embodiments, the cells are embryonic stem cells, pluripotent cells, multipotent cells, non-pluripotent cells, and somatic cells. In some specific embodiments, the cells comprise non-pluripotent cells.

[0067] In some embodiments, the condition comprises contacting the cell with at least one pluripotency factor. In some embodiments, the pluripotency factor is an exogenous transcription factor selected from the group consisting of Oct4, Sox, Klf, Myc, Lin28, or Nanog polypeptide, or a polynucleotide encoding Oct4, Sox, Klf, Myc, Lin28, or Nanog. In certain embodiments, the pluripotency factor comprises Oct4, Sox2, and Klf4 polynucleotide.

[0068] In some embodiments of the invention, the small molecule agent comprises a Rock inhibitor. In certain embodiments, the Rock inhibitor is thiazovivin or Y27632, and in further particular embodiments, In one embodiment, the Rock inhibitor is thiazovivin.

[0069] In other embodiments of the invention, the small molecule agent comprises a TFGβ inhibitor, hi some embodiments, the TFGβ inhibitor is A-83-01 or SB431542.

[0070] In some embodiments, the small molecule agent comprises a GSK3 inhibitor, and in particular embodiments, the GSK3 inhibitor is CHIR99021 or BIO.

[0071] In other embodiments of the invention, the small molecule agent comprises a MEK inhibitor, hi some specific embodiments, the MEK inhibitor is PD98059 or PD032901.

[0072] In some embodiments of the present invention, the small molecule agent comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor. In certain embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0073] The present invention further includes a method of culturing pluripotent cells to maintain genomic stability of the cells, comprising culturing the pluripotent cells in a feeder-free environment in a medium comprising at least one agent that maintains genomic stability of the pluripotent cells selected from the group consisting of: i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor, and maintaining the genomic stability of the pluripotent cells during culturing.

[0074] In some embodiments, the condition comprises contacting the cell with at least one pluripotency factor. In some embodiments, the pluripotency factor is an exogenous transcription factor selected from the group consisting of Oct4, Sox, Klf, Myc, Lin28, or Nanog polypeptide, or a polynucleotide encoding Oct4, Sox, Klf, Myc, Lin28, or Nanog. In certain embodiments, the pluripotency factor comprises Oct4, Sox2, and Klf4 polynucleotide.

[0075] In some embodiments, the medium comprises at least two, at least three, or four agents.

[0076] In some embodiments of the invention, the agent comprises a Rock inhibitor. In certain embodiments, the Rock inhibitor is thiazovivin or Y27632, and in more particular embodiments, the Rock inhibitor is thiazovivin.

[0077] In other embodiments of the invention, the agent comprises a TFGβ inhibitor, hi some embodiments, the TFGβ inhibitor is A-83-01 or SB431542.

[0078] In some embodiments, the agent comprises a GSK3 inhibitor, and in particular embodiments, the GSK3 inhibitor is CHIR99021 or BIO.

[0079] In other embodiments of the invention, the agent comprises a MEK inhibitor, hi some specific embodiments, the MEK inhibitor is PD98059 or PD032901.

[0080] In some embodiments of the present invention, the agent comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor. In certain embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0081] In some embodiments of the invention, the pluripotent cells are cultured in culture for at least 1, at least 2, at least 5, at least 10, or at least 15 passages while maintaining the genomic stability of the pluripotent cells. [Brief description of the drawings]

[0082] [Figure 1A] FIG. 1 shows the effect of various small molecule cocktails on single cell culture of human pluripotent stem cells on feeder-free substratum. A. Different morphologies were evident when human pluripotent stem cells dissociated into single cells were treated with various combinations of small molecules in a feeder-free environment. Media containing a combination of ROCKi / MEKi / TGFβi / GSKi resulted in robust growth and viability of human pluripotent stem cells. B. Some small molecules, i.e., MEKi / TGFβi / GSKi, supported the maintenance of pluripotent stem cells, while others, i.e., Rocki, facilitated the dissociation of pluripotent stem cells into single cells, and a unique combination of these small molecules, i.e., ROCKi / MEKi / TGFβi / GSKi, supported the dissociation into single cells while maintaining an undifferentiated state as indicated by highly proliferative colonies that were positive for the pluripotency marker Tra181. Tra181, red; DAPI, blue. C. hiPSCs were seeded on Matrigel at 1x105 cells and scored after 4 days for number of viable cells and percent viability measured by trypan blue, and the MEKi / TFGβi / GSKi / ROCKi combination supported viability and proliferation. D. In combination with MEKi / TFGβi / GSKi, colonies cultured with thiazovivin appear to have a more compact morphology compared to cultures supported by MEKi / TFGβi / GSKi and Y27632. E. Flow cytometry analysis of SSEA4 and Tra181 surface expression of hiPSCs cultured in MEKi / TFGβi / GSKi combined with Y27632 or thiazovivin shows that thiazovivin better supports the maintenance of cells in an undifferentiated state compared to the combination with Y27632. [Figure 1B] FIG. 1 shows the effect of various small molecule cocktails on single cell culture of human pluripotent stem cells on feeder-free substratum. A. Different morphologies were evident when human pluripotent stem cells dissociated into single cells were treated with various combinations of small molecules in a feeder-free environment. Media containing a combination of ROCKi / MEKi / TGFβi / GSKi resulted in robust growth and viability of human pluripotent stem cells. B. Some small molecules, i.e., MEKi / TGFβi / GSKi, supported the maintenance of pluripotent stem cells, while others, i.e., Rocki, facilitated the dissociation of pluripotent stem cells into single cells, and a unique combination of these small molecules, i.e., ROCKi / MEKi / TGFβi / GSKi, supported the dissociation into single cells while maintaining an undifferentiated state as indicated by highly proliferative colonies that were positive for the pluripotency marker Tra181. Tra181, red; DAPI, blue. C. hiPSCs were seeded on Matrigel at 1x105 cells and scored after 4 days for number of viable cells and percent viability measured by trypan blue, and the MEKi / TFGβi / GSKi / ROCKi combination supported viability and proliferation. D. In combination with MEKi / TFGβi / GSKi, colonies cultured with thiazovivin appear to have a more compact morphology compared to cultures supported by MEKi / TFGβi / GSKi and Y27632. E. Flow cytometry analysis of SSEA4 and Tra181 surface expression of hiPSCs cultured in MEKi / TFGβi / GSKi combined with Y27632 or thiazovivin shows that thiazovivin better supports the maintenance of cells in an undifferentiated state compared to the combination with Y27632. [Figure 1C]FIG. 1 shows the effect of various small molecule cocktails on single cell culture of human pluripotent stem cells on feeder-free substratum. A. Different morphologies were evident when human pluripotent stem cells dissociated into single cells were treated with various combinations of small molecules in a feeder-free environment. Media containing a combination of ROCKi / MEKi / TGFβi / GSKi resulted in robust growth and viability of human pluripotent stem cells. B. Some small molecules, i.e., MEKi / TGFβi / GSKi, supported the maintenance of pluripotent stem cells, while others, i.e., Rocki, facilitated the dissociation of pluripotent stem cells into single cells, and a unique combination of these small molecules, i.e., ROCKi / MEKi / TGFβi / GSKi, supported the dissociation into single cells while maintaining an undifferentiated state as indicated by highly proliferative colonies that were positive for the pluripotency marker Tra181. Tra181, red; DAPI, blue. C. hiPSCs were seeded on Matrigel at 1x105 cells and scored after 4 days for number of viable cells and percent viability measured by trypan blue, and the MEKi / TFGβi / GSKi / ROCKi combination supported viability and proliferation. D. In combination with MEKi / TFGβi / GSKi, colonies cultured with thiazovivin appear to have a more compact morphology compared to cultures supported by MEKi / TFGβi / GSKi and Y27632. E. Flow cytometry analysis of SSEA4 and Tra181 surface expression of hiPSCs cultured in MEKi / TFGβi / GSKi combined with Y27632 or thiazovivin shows that thiazovivin better supports the maintenance of cells in an undifferentiated state compared to the combination with Y27632. [Figure 1D]FIG. 1 shows the effect of various small molecule cocktails on single cell culture of human pluripotent stem cells on feeder-free substratum. A. Different morphologies were evident when human pluripotent stem cells dissociated into single cells were treated with various combinations of small molecules in a feeder-free environment. Media containing a combination of ROCKi / MEKi / TGFβi / GSKi resulted in robust growth and viability of human pluripotent stem cells. B. Some small molecules, i.e., MEKi / TGFβi / GSKi, supported the maintenance of pluripotent stem cells, while others, i.e., Rocki, facilitated the dissociation of pluripotent stem cells into single cells, and a unique combination of these small molecules, i.e., ROCKi / MEKi / TGFβi / GSKi, supported the dissociation into single cells while maintaining an undifferentiated state as indicated by highly proliferative colonies that were positive for the pluripotency marker Tra181. Tra181, red; DAPI, blue. C. hiPSCs were seeded on Matrigel at 1x105 cells and scored after 4 days for number of viable cells and percent viability measured by trypan blue, and the MEKi / TFGβi / GSKi / ROCKi combination supported viability and proliferation. D. In combination with MEKi / TFGβi / GSKi, colonies cultured with thiazovivin appear to have a more compact morphology compared to cultures supported by MEKi / TFGβi / GSKi and Y27632. E. Flow cytometry analysis of SSEA4 and Tra181 surface expression of hiPSCs cultured in MEKi / TFGβi / GSKi combined with Y27632 or thiazovivin shows that thiazovivin better supports the maintenance of cells in an undifferentiated state compared to the combination with Y27632. [Figure 1E]FIG. 1 shows the effect of various small molecule cocktails on single cell culture of human pluripotent stem cells on feeder-free substratum. A. Different morphologies were evident when human pluripotent stem cells dissociated into single cells were treated with various combinations of small molecules in a feeder-free environment. Media containing a combination of ROCKi / MEKi / TGFβi / GSKi resulted in robust growth and viability of human pluripotent stem cells. B. Some small molecules, i.e., MEKi / TGFβi / GSKi, supported the maintenance of pluripotent stem cells, while others, i.e., Rocki, facilitated the dissociation of pluripotent stem cells into single cells, and a unique combination of these small molecules, i.e., ROCKi / MEKi / TGFβi / GSKi, supported the dissociation into single cells while maintaining an undifferentiated state as indicated by highly proliferative colonies that were positive for the pluripotency marker Tra181. Tra181, red; DAPI, blue. C. hiPSCs were seeded on Matrigel at 1x105 cells and scored after 4 days for number of viable cells and percent viability measured by trypan blue, and the MEKi / TFGβi / GSKi / ROCKi combination supported viability and proliferation. D. In combination with MEKi / TFGβi / GSKi, colonies cultured with thiazovivin appear to have a more compact morphology compared to cultures supported by MEKi / TFGβi / GSKi and Y27632. E. Flow cytometry analysis of SSEA4 and Tra181 surface expression of hiPSCs cultured in MEKi / TFGβi / GSKi combined with Y27632 or thiazovivin shows that thiazovivin better supports the maintenance of cells in an undifferentiated state compared to the combination with Y27632. [Figure 2A]Figure 2 shows the adaptation of human pluripotent stem cells to feeder-free and single-cell culture. A. iPSCs generated on feeder cells were readily adapted to feeder-free and single-cell culture conditions utilizing SMC4 medium. However, cell survival was perturbed and cell attachment and growth were restricted upon dissociation into single cells and culturing in conventional culture conditions. B. Once adapted to the feeder-free environment, iPSCs were routinely cultured as single cells on feeder-free surfaces when continuously maintained in SMC4 medium. [Figure 2B] Figure 2 shows the adaptation of human pluripotent stem cells to feeder-free and single-cell culture. A. iPSCs generated on feeder cells were readily adapted to feeder-free and single-cell culture conditions utilizing SMC4 medium. However, cell survival was perturbed and cell attachment and growth were restricted upon dissociation into single cells and culturing in conventional culture conditions. B. Once adapted to the feeder-free environment, iPSCs were routinely cultured as single cells on feeder-free surfaces when continuously maintained in SMC4 medium. [Figure 3A]FIG. 3 shows long-term maintenance of single cells and culture of human pluripotent stem cells feeder-free. A. Single-cell dissociated human pluripotent stem cells maintained in SMC4 medium for 10 passages in feeder-free culture expressed pluripotency markers Nanog and Tra181 as analyzed by immunofluorescence. B. FIG. 3B shows the percentage of 7-aminoactinomycin D (7AAD) stained cells after 4 hours of dissociating human pluripotent stem cells into single cells and placing them in either conventional or SMC4 medium. Single-cell dissociated human pluripotent stem cells showed a significant increase in viability when cultured in SMC4 medium versus conventional medium as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that nearly all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of the undifferentiated state. D. Global expression analysis revealed a high correlation between iPSCs passaged as single cells and maintained in feeder-free SMC4 medium and human ESCs cultured on feeder cells, including H1 cells, and maintained as cell aggregates during passaging. E. iPSCs cultured in a feeder-free system with SMC4 medium and passaged as single cells expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs maintained in a feeder-free environment, unlike the exogenous factor positive control (lentivirus-infected fibroblasts expressing reprogramming factors after 4 days). Endogenous expression of reprogramming factors in feeder-free iPSCs was similar to ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells retained a normal karyotype. Fourteen days after replacing the H.SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment displayed gene expression indicative of ectoderm, endoderm, and mesoderm, in addition to extraembryonic markers, that are normally only expressed by naive and totipotent stem cells.I. Immunofluorescence analysis: iPSCs cultured in a feeder-free system using SMC4 medium and passaged as single cells readily developed into all three somatic cell types when transferred to conventional differentiation media: mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III); endoderm, Sox17. J. Analysis of teratoma formation demonstrates that human pluripotent stem cells cultured in SMC4 medium give rise to cell types representing the three germ layers, thus retaining their pluripotent potential. [Figure 3B]FIG. 3 shows long-term maintenance of single cells and culture of human pluripotent stem cells feeder-free. A. Single-cell dissociated human pluripotent stem cells maintained in SMC4 medium for 10 passages in feeder-free culture expressed pluripotency markers Nanog and Tra181 as analyzed by immunofluorescence. B. FIG. 3B shows the percentage of 7-aminoactinomycin D (7AAD) stained cells after 4 hours of dissociating human pluripotent stem cells into single cells and placing them in either conventional or SMC4 medium. Single-cell dissociated human pluripotent stem cells showed a significant increase in viability when cultured in SMC4 medium versus conventional medium as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that nearly all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of the undifferentiated state. D. Global expression analysis revealed a high correlation between iPSCs passaged as single cells and maintained in feeder-free SMC4 medium and human ESCs cultured on feeder cells, including H1 cells, and maintained as cell aggregates during passaging. E. iPSCs cultured in a feeder-free system with SMC4 medium and passaged as single cells expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs maintained in a feeder-free environment, unlike the exogenous factor positive control (lentivirus-infected fibroblasts expressing reprogramming factors after 4 days). Endogenous expression of reprogramming factors in feeder-free iPSCs was similar to ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells retained a normal karyotype. Fourteen days after replacing the H.SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment displayed gene expression indicative of ectoderm, endoderm, and mesoderm, in addition to extraembryonic markers, that are normally only expressed by naive and totipotent stem cells.I. Immunofluorescence analysis: iPSCs cultured in a feeder-free system using SMC4 medium and passaged as single cells readily developed into all three somatic cell types when transferred to conventional differentiation media: mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III); endoderm, Sox17. J. Analysis of teratoma formation demonstrates that human pluripotent stem cells cultured in SMC4 medium give rise to cell types representing the three germ layers, thus retaining their pluripotent potential. [Figure 3C]FIG. 3 shows long-term maintenance of single cells and culture of human pluripotent stem cells feeder-free. A. Single-cell dissociated human pluripotent stem cells maintained in SMC4 medium for 10 passages in feeder-free culture expressed pluripotency markers Nanog and Tra181 as analyzed by immunofluorescence. B. FIG. 3B shows the percentage of 7-aminoactinomycin D (7AAD) stained cells after 4 hours of dissociating human pluripotent stem cells into single cells and placing them in either conventional or SMC4 medium. Single-cell dissociated human pluripotent stem cells showed a significant increase in viability when cultured in SMC4 medium versus conventional medium as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that nearly all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of the undifferentiated state. D. Global expression analysis revealed a high correlation between iPSCs passaged as single cells and maintained in feeder-free SMC4 medium and human ESCs cultured on feeder cells, including H1 cells, and maintained as cell aggregates during passaging. E. iPSCs cultured in a feeder-free system with SMC4 medium and passaged as single cells expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs maintained in a feeder-free environment, unlike the exogenous factor positive control (lentivirus-infected fibroblasts expressing reprogramming factors after 4 days). Endogenous expression of reprogramming factors in feeder-free iPSCs was similar to ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells retained a normal karyotype. Fourteen days after replacing the H.SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment displayed gene expression indicative of ectoderm, endoderm, and mesoderm, in addition to extraembryonic markers, that are normally only expressed by naive and totipotent stem cells.I. Immunofluorescence analysis: iPSCs cultured in a feeder-free system using SMC4 medium and passaged as single cells readily developed into all three somatic cell types when transferred to conventional differentiation media: mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III); endoderm, Sox17. J. Analysis of teratoma formation demonstrates that human pluripotent stem cells cultured in SMC4 medium give rise to cell types representing the three germ layers, thus retaining their pluripotent potential. [Figure 3D]FIG. 3 shows long-term maintenance of single cells and culture of human pluripotent stem cells feeder-free. A. Single-cell dissociated human pluripotent stem cells maintained in SMC4 medium for 10 passages in feeder-free culture expressed pluripotency markers Nanog and Tra181 as analyzed by immunofluorescence. B. FIG. 3B shows the percentage of 7-aminoactinomycin D (7AAD) stained cells after 4 hours of dissociating human pluripotent stem cells into single cells and placing them in either conventional or SMC4 medium. Single-cell dissociated human pluripotent stem cells showed a significant increase in viability when cultured in SMC4 medium versus conventional medium as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that nearly all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of the undifferentiated state. D. Global expression analysis revealed a high correlation between iPSCs passaged as single cells and maintained in feeder-free SMC4 medium and human ESCs cultured on feeder cells, including H1 cells, and maintained as cell aggregates during passaging. E. iPSCs cultured in a feeder-free system with SMC4 medium and passaged as single cells expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs maintained in a feeder-free environment, unlike the exogenous factor positive control (lentivirus-infected fibroblasts expressing reprogramming factors after 4 days). Endogenous expression of reprogramming factors in feeder-free iPSCs was similar to ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells retained a normal karyotype. Fourteen days after replacing the H.SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment displayed gene expression indicative of ectoderm, endoderm, and mesoderm, in addition to extraembryonic markers, that are normally only expressed by naive and totipotent stem cells.I. Immunofluorescence analysis: iPSCs cultured in a feeder-free system using SMC4 medium and passaged as single cells readily developed into all three somatic cell types when transferred to conventional differentiation media: mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III); endoderm, Sox17. J. Analysis of teratoma formation demonstrates that human pluripotent stem cells cultured in SMC4 medium give rise to cell types representing the three germ layers, thus retaining their pluripotent potential. [Figure 3E]FIG. 3 shows long-term maintenance of single cells and culture of human pluripotent stem cells feeder-free. A. Single-cell dissociated human pluripotent stem cells maintained in SMC4 medium for 10 passages in feeder-free culture expressed pluripotency markers Nanog and Tra181 as analyzed by immunofluorescence. B. FIG. 3B shows the percentage of 7-aminoactinomycin D (7AAD) stained cells after 4 hours of dissociating human pluripotent stem cells into single cells and placing them in either conventional or SMC4 medium. Single-cell dissociated human pluripotent stem cells showed a significant increase in viability when cultured in SMC4 medium versus conventional medium as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that nearly all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of the undifferentiated state. D. Global expression analysis revealed a high correlation between iPSCs passaged as single cells and maintained in feeder-free SMC4 medium and human ESCs cultured on feeder cells, including H1 cells, and maintained as cell aggregates during passaging. E. iPSCs cultured in a feeder-free system with SMC4 medium and passaged as single cells expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs maintained in a feeder-free environment, unlike the exogenous factor positive control (lentivirus-infected fibroblasts expressing reprogramming factors after 4 days). Endogenous expression of reprogramming factors in feeder-free iPSCs was similar to ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells retained a normal karyotype. Fourteen days after replacing the H.SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment displayed gene expression indicative of ectoderm, endoderm, and mesoderm, in addition to extraembryonic markers, that are normally only expressed by naive and totipotent stem cells.I. Immunofluorescence analysis: iPSCs cultured in a feeder-free system using SMC4 medium and passaged as single cells readily developed into all three somatic cell types when transferred to conventional differentiation media: mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III); endoderm, Sox17. J. Analysis of teratoma formation demonstrates that human pluripotent stem cells cultured in SMC4 medium give rise to cell types representing the three germ layers, thus retaining their pluripotent potential. [Figure 3F]FIG. 3 shows long-term maintenance of single cells and culture of human pluripotent stem cells feeder-free. A. Single-cell dissociated human pluripotent stem cells maintained in SMC4 medium for 10 passages in feeder-free culture expressed pluripotency markers Nanog and Tra181 as analyzed by immunofluorescence. B. FIG. 3B shows the percentage of 7-aminoactinomycin D (7AAD) stained cells after 4 hours of dissociating human pluripotent stem cells into single cells and placing them in either conventional or SMC4 medium. Single-cell dissociated human pluripotent stem cells showed a significant increase in viability when cultured in SMC4 medium versus conventional medium as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that nearly all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of the undifferentiated state. D. Global expression analysis revealed a high correlation between iPSCs passaged as single cells and maintained in feeder-free SMC4 medium and human ESCs cultured on feeder cells, including H1 cells, and maintained as cell aggregates during passaging. E. iPSCs cultured in a feeder-free system with SMC4 medium and passaged as single cells expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs maintained in a feeder-free environment, unlike the exogenous factor positive control (lentivirus-infected fibroblasts expressing reprogramming factors after 4 days). Endogenous expression of reprogramming factors in feeder-free iPSCs was similar to ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells retained a normal karyotype. Fourteen days after replacing the H.SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment displayed gene expression indicative of ectoderm, endoderm, and mesoderm, in addition to extraembryonic markers, that are normally only expressed by naive and totipotent stem cells.I. Immunofluorescence analysis: iPSCs cultured in a feeder-free system using SMC4 medium and passaged as single cells readily developed into all three somatic cell types when transferred to conventional differentiation media: mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III); endoderm, Sox17. J. Analysis of teratoma formation demonstrates that human pluripotent stem cells cultured in SMC4 medium give rise to cell types representing the three germ layers, thus retaining their pluripotent potential. [Figure 3G]FIG. 3 shows long-term maintenance of single cells and culture of human pluripotent stem cells feeder-free. A. Single-cell dissociated human pluripotent stem cells maintained in SMC4 medium for 10 passages in feeder-free culture expressed pluripotency markers Nanog and Tra181 as analyzed by immunofluorescence. B. FIG. 3B shows the percentage of 7-aminoactinomycin D (7AAD) stained cells after 4 hours of dissociating human pluripotent stem cells into single cells and placing them in either conventional or SMC4 medium. Single-cell dissociated human pluripotent stem cells showed a significant increase in viability when cultured in SMC4 medium versus conventional medium as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that nearly all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of the undifferentiated state. D. Global expression analysis revealed a high correlation between iPSCs passaged as single cells and maintained in feeder-free SMC4 medium and human ESCs cultured on feeder cells, including H1 cells, and maintained as cell aggregates during passaging. E. iPSCs cultured in a feeder-free system with SMC4 medium and passaged as single cells expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs maintained in a feeder-free environment, unlike the exogenous factor positive control (lentivirus-infected fibroblasts expressing reprogramming factors after 4 days). Endogenous expression of reprogramming factors in feeder-free iPSCs was similar to ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells retained a normal karyotype. Fourteen days after replacing the H.SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment displayed gene expression indicative of ectoderm, endoderm, and mesoderm, in addition to extraembryonic markers, that are normally only expressed by naive and totipotent stem cells.I. Immunofluorescence analysis: iPSCs cultured in a feeder-free system using SMC4 medium and passaged as single cells readily developed into all three somatic cell types when transferred to conventional differentiation media: mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III); endoderm, Sox17. J. Analysis of teratoma formation demonstrates that human pluripotent stem cells cultured in SMC4 medium give rise to cell types representing the three germ layers, thus retaining their pluripotent potential. [Figure 3H]FIG. 3 shows long-term maintenance of single cells and culture of human pluripotent stem cells feeder-free. A. Single-cell dissociated human pluripotent stem cells maintained in SMC4 medium for 10 passages in feeder-free culture expressed pluripotency markers Nanog and Tra181 as analyzed by immunofluorescence. B. FIG. 3B shows the percentage of 7-aminoactinomycin D (7AAD) stained cells after 4 hours of dissociating human pluripotent stem cells into single cells and placing them in either conventional or SMC4 medium. Single-cell dissociated human pluripotent stem cells showed a significant increase in viability when cultured in SMC4 medium versus conventional medium as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that nearly all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of the undifferentiated state. D. Global expression analysis revealed a high correlation between iPSCs passaged as single cells and maintained in feeder-free SMC4 medium and human ESCs cultured on feeder cells, including H1 cells, and maintained as cell aggregates during passaging. E. iPSCs cultured in a feeder-free system with SMC4 medium and passaged as single cells expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs maintained in a feeder-free environment, unlike the exogenous factor positive control (lentivirus-infected fibroblasts expressing reprogramming factors after 4 days). Endogenous expression of reprogramming factors in feeder-free iPSCs was similar to ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells retained a normal karyotype. Fourteen days after replacing the H.SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment displayed gene expression indicative of ectoderm, endoderm, and mesoderm, in addition to extraembryonic markers, that are normally only expressed by naive and totipotent stem cells.I. Immunofluorescence analysis: iPSCs cultured in a feeder-free system using SMC4 medium and passaged as single cells readily developed into all three somatic cell types when transferred to conventional differentiation media: mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III); endoderm, Sox17. J. Analysis of teratoma formation demonstrates that human pluripotent stem cells cultured in SMC4 medium give rise to cell types representing the three germ layers, thus retaining their pluripotent potential. [Figure 3I]FIG. 3 shows long-term maintenance of single cells and culture of human pluripotent stem cells feeder-free. A. Single-cell dissociated human pluripotent stem cells maintained in SMC4 medium for 10 passages in feeder-free culture expressed pluripotency markers Nanog and Tra181 as analyzed by immunofluorescence. B. FIG. 3B shows the percentage of 7-aminoactinomycin D (7AAD) stained cells after 4 hours of dissociating human pluripotent stem cells into single cells and placing them in either conventional or SMC4 medium. Single-cell dissociated human pluripotent stem cells showed a significant increase in viability when cultured in SMC4 medium versus conventional medium as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that nearly all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of the undifferentiated state. D. Global expression analysis revealed a high correlation between iPSCs passaged as single cells and maintained in feeder-free SMC4 medium and human ESCs cultured on feeder cells, including H1 cells, and maintained as cell aggregates during passaging. E. iPSCs cultured in a feeder-free system with SMC4 medium and passaged as single cells expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs maintained in a feeder-free environment, unlike the exogenous factor positive control (lentivirus-infected fibroblasts expressing reprogramming factors after 4 days). Endogenous expression of reprogramming factors in feeder-free iPSCs was similar to ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells retained a normal karyotype. Fourteen days after replacing the H.SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment displayed gene expression indicative of ectoderm, endoderm, and mesoderm, in addition to extraembryonic markers, that are normally only expressed by naive and totipotent stem cells.I. Immunofluorescence analysis: iPSCs cultured in a feeder-free system using SMC4 medium and passaged as single cells readily developed into all three somatic cell types when transferred to conventional differentiation media: mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III); endoderm, Sox17. J. Analysis of teratoma formation demonstrates that human pluripotent stem cells cultured in SMC4 medium give rise to cell types representing the three germ layers, thus retaining their pluripotent potential. [Figure 3J]FIG. 3 shows long-term maintenance of single cells and culture of human pluripotent stem cells feeder-free. A. Single-cell dissociated human pluripotent stem cells maintained in SMC4 medium for 10 passages in feeder-free culture expressed pluripotency markers Nanog and Tra181 as analyzed by immunofluorescence. B. FIG. 3B shows the percentage of 7-aminoactinomycin D (7AAD) stained cells after 4 hours of dissociating human pluripotent stem cells into single cells and placing them in either conventional or SMC4 medium. Single-cell dissociated human pluripotent stem cells showed a significant increase in viability when cultured in SMC4 medium versus conventional medium as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that nearly all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of the undifferentiated state. D. Global expression analysis revealed a high correlation between iPSCs passaged as single cells and maintained in feeder-free SMC4 medium and human ESCs cultured on feeder cells, including H1 cells, and maintained as cell aggregates during passaging. E. iPSCs cultured in a feeder-free system with SMC4 medium and passaged as single cells expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs maintained in a feeder-free environment, unlike the exogenous factor positive control (lentivirus-infected fibroblasts expressing reprogramming factors after 4 days). Endogenous expression of reprogramming factors in feeder-free iPSCs was similar to ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells retained a normal karyotype. Fourteen days after replacing the H.SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment displayed gene expression indicative of ectoderm, endoderm, and mesoderm, in addition to extraembryonic markers, that are normally only expressed by naive and totipotent stem cells.I. Immunofluorescence analysis: iPSCs cultured in a feeder-free system using SMC4 medium and passaged as single cells readily developed into all three somatic cell types when transferred to conventional differentiation media: mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III); endoderm, Sox17. J. Analysis of teratoma formation demonstrates that human pluripotent stem cells cultured in SMC4 medium give rise to cell types representing the three germ layers, thus retaining their pluripotent potential. [Figure 4A]Figure 4. Enhanced single-cell sorting and subsequent feeder-free culture of human pluripotent stem cells. Demonstration of sorting of human pluripotent stem cells based on expression of pluripotency markers and subsequent maintenance of sorted cells in feeder-free culture supplemented with optimized SMC4 medium or SMC4+fibronectin medium. A. Prior to sorting, human pluripotent stem cells were dissociated into single cells and labeled with antibody markers against SSEA4 and Tra-181. Cells were sorted based on surface marker expression of the pluripotency markers SSEA4 and TRA181 using FACs technology. After sorting, cells were first plated in SMC4+fibronectin medium and this medium was replaced with SMC4 medium 24-72 hours later. One day after sorting, sorted cells were split and by day 5, large colonies consisting of hundreds of cells were present. B. Sorted cells retained expression of the pluripotency marker Tra160. Tra160, red; DAPI, blue. C. After 5 passages and approximately 1 month in culture after sorting, quantitative flow cytometry analysis revealed that the majority of sorted iPSCs retained their pluripotent state based on SSEA4 and Tra181 staining. D. Various densities of SSEA4+ and Tra181+ human pluripotent stem cell derived iPSCs were plated, including a density of 500 events per well of a 6-well plate and approximately 52 events per cm2 of clones on a feeder-free surface, supplemented with SMC4+fibronectin medium. Seven days after sorting, many alkaline phosphatase positive colonies appeared in each of the seeded wells. E. The resulting alkaline phosphatase positive colonies were scored. Approximately 8-10% of the seeded events gave rise to colonies, with 16,000 events per well giving rise to near-confluent wells. NC, not counted due to confluency preventing accurate counting. F. SSEA4+ and Tra181+ human pluripotent stem cells were sorted directly into 96-well plates at various densities, including 1 event per well and approximately 3 events per cm2. Eight days after sorting, wells were stained for alkaline phosphatase. G. On day 8, each well was scored for alkaline phosphatase colonies. [Figure 4B]Figure 4. Enhanced single-cell sorting and subsequent feeder-free culture of human pluripotent stem cells. Demonstration of sorting of human pluripotent stem cells based on expression of pluripotency markers and subsequent maintenance of sorted cells in feeder-free culture supplemented with optimized SMC4 medium or SMC4+fibronectin medium. A. Prior to sorting, human pluripotent stem cells were dissociated into single cells and labeled with antibody markers against SSEA4 and Tra-181. Cells were sorted based on surface marker expression of the pluripotency markers SSEA4 and TRA181 using FACs technology. After sorting, cells were first plated in SMC4+fibronectin medium and this medium was replaced with SMC4 medium 24-72 hours later. One day after sorting, sorted cells were split and by day 5, large colonies consisting of hundreds of cells were present. B. Sorted cells retained expression of the pluripotency marker Tra160. Tra160, red; DAPI, blue. C. After 5 passages and approximately 1 month in culture after sorting, quantitative flow cytometry analysis revealed that the majority of sorted iPSCs retained their pluripotent state based on SSEA4 and Tra181 staining. D. Various densities of SSEA4+ and Tra181+ human pluripotent stem cell derived iPSCs were plated, including a density of 500 events per well of a 6-well plate and approximately 52 events per cm2 of clones on a feeder-free surface, supplemented with SMC4+fibronectin medium. Seven days after sorting, many alkaline phosphatase positive colonies appeared in each of the seeded wells. E. The resulting alkaline phosphatase positive colonies were scored. Approximately 8-10% of the seeded events gave rise to colonies, with 16,000 events per well giving rise to near-confluent wells. NC, not counted due to confluency preventing accurate counting. F. SSEA4+ and Tra181+ human pluripotent stem cells were sorted directly into 96-well plates at various densities, including 1 event per well and approximately 3 events per cm2. Eight days after sorting, wells were stained for alkaline phosphatase. G. On day 8, each well was scored for alkaline phosphatase colonies. [Figure 4C]Figure 4. Enhanced single-cell sorting and subsequent feeder-free culture of human pluripotent stem cells. Demonstration of sorting of human pluripotent stem cells based on expression of pluripotency markers and subsequent maintenance of sorted cells in feeder-free culture supplemented with optimized SMC4 medium or SMC4+fibronectin medium. A. Prior to sorting, human pluripotent stem cells were dissociated into single cells and labeled with antibody markers against SSEA4 and Tra-181. Cells were sorted based on surface marker expression of the pluripotency markers SSEA4 and TRA181 using FACs technology. After sorting, cells were first plated in SMC4+fibronectin medium and this medium was replaced with SMC4 medium 24-72 hours later. One day after sorting, sorted cells were split and by day 5, large colonies consisting of hundreds of cells were present. B. Sorted cells retained expression of the pluripotency marker Tra160. Tra160, red; DAPI, blue. C. After 5 passages and approximately 1 month in culture after sorting, quantitative flow cytometry analysis revealed that the majority of sorted iPSCs retained their pluripotent state based on SSEA4 and Tra181 staining. D. Various densities of SSEA4+ and Tra181+ human pluripotent stem cell derived iPSCs were plated, including a density of 500 events per well of a 6-well plate and approximately 52 events per cm2 of clones on a feeder-free surface, supplemented with SMC4+fibronectin medium. Seven days after sorting, many alkaline phosphatase positive colonies appeared in each of the seeded wells. E. The resulting alkaline phosphatase positive colonies were scored. Approximately 8-10% of the seeded events gave rise to colonies, with 16,000 events per well giving rise to near-confluent wells. NC, not counted due to confluency preventing accurate counting. F. SSEA4+ and Tra181+ human pluripotent stem cells were sorted directly into 96-well plates at various densities, including 1 event per well and approximately 3 events per cm2. Eight days after sorting, wells were stained for alkaline phosphatase. G. On day 8, each well was scored for alkaline phosphatase colonies. [Figure 4D]Figure 4. Enhanced single-cell sorting and subsequent feeder-free culture of human pluripotent stem cells. Demonstration of sorting of human pluripotent stem cells based on expression of pluripotency markers and subsequent maintenance of sorted cells in feeder-free culture supplemented with optimized SMC4 medium or SMC4+fibronectin medium. A. Prior to sorting, human pluripotent stem cells were dissociated into single cells and labeled with antibody markers against SSEA4 and Tra-181. Cells were sorted based on surface marker expression of the pluripotency markers SSEA4 and TRA181 using FACs technology. After sorting, cells were first plated in SMC4+fibronectin medium and this medium was replaced with SMC4 medium 24-72 hours later. One day after sorting, sorted cells were split and by day 5, large colonies consisting of hundreds of cells were present. B. Sorted cells retained expression of the pluripotency marker Tra160. Tra160, red; DAPI, blue. C. After 5 passages and approximately 1 month in culture after sorting, quantitative flow cytometry analysis revealed that the majority of sorted iPSCs retained their pluripotent state based on SSEA4 and Tra181 staining. D. Various densities of SSEA4+ and Tra181+ human pluripotent stem cell derived iPSCs were plated, including a density of 500 events per well of a 6-well plate and approximately 52 events per cm2 of clones on a feeder-free surface, supplemented with SMC4+fibronectin medium. Seven days after sorting, many alkaline phosphatase positive colonies appeared in each of the seeded wells. E. The resulting alkaline phosphatase positive colonies were scored. Approximately 8-10% of the seeded events gave rise to colonies, with 16,000 events per well giving rise to near-confluent wells. NC, not counted due to confluency preventing accurate counting. F. SSEA4+ and Tra181+ human pluripotent stem cells were sorted directly into 96-well plates at various densities, including 1 event per well and approximately 3 events per cm2. Eight days after sorting, wells were stained for alkaline phosphatase. G. On day 8, each well was scored for alkaline phosphatase colonies. [Figure 4E]Figure 4. Enhanced single-cell sorting and subsequent feeder-free culture of human pluripotent stem cells. Demonstration of sorting of human pluripotent stem cells based on expression of pluripotency markers and subsequent maintenance of sorted cells in feeder-free culture supplemented with optimized SMC4 medium or SMC4+fibronectin medium. A. Prior to sorting, human pluripotent stem cells were dissociated into single cells and labeled with antibody markers against SSEA4 and Tra-181. Cells were sorted based on surface marker expression of the pluripotency markers SSEA4 and TRA181 using FACs technology. After sorting, cells were first plated in SMC4+fibronectin medium and this medium was replaced with SMC4 medium 24-72 hours later. One day after sorting, sorted cells were split and by day 5, large colonies consisting of hundreds of cells were present. B. Sorted cells retained expression of the pluripotency marker Tra160. Tra160, red; DAPI, blue. C. After 5 passages and approximately 1 month in culture after sorting, quantitative flow cytometry analysis revealed that the majority of sorted iPSCs retained their pluripotent state based on SSEA4 and Tra181 staining. D. Various densities of SSEA4+ and Tra181+ human pluripotent stem cell derived iPSCs were plated, including a density of 500 events per well of a 6-well plate and approximately 52 events per cm2 of clones on a feeder-free surface, supplemented with SMC4+fibronectin medium. Seven days after sorting, many alkaline phosphatase positive colonies appeared in each of the seeded wells. E. The resulting alkaline phosphatase positive colonies were scored. Approximately 8-10% of the seeded events gave rise to colonies, with 16,000 events per well giving rise to near-confluent wells. NC, not counted due to confluency preventing accurate counting. F. SSEA4+ and Tra181+ human pluripotent stem cells were sorted directly into 96-well plates at various densities, including 1 event per well and approximately 3 events per cm2. Eight days after sorting, wells were stained for alkaline phosphatase. G. On day 8, each well was scored for alkaline phosphatase colonies. [Figure 4F]Figure 4. Enhanced single-cell sorting and subsequent feeder-free culture of human pluripotent stem cells. Demonstration of sorting of human pluripotent stem cells based on expression of pluripotency markers and subsequent maintenance of sorted cells in feeder-free culture supplemented with optimized SMC4 medium or SMC4+fibronectin medium. A. Prior to sorting, human pluripotent stem cells were dissociated into single cells and labeled with antibody markers against SSEA4 and Tra-181. Cells were sorted based on surface marker expression of the pluripotency markers SSEA4 and TRA181 using FACs technology. After sorting, cells were first plated in SMC4+fibronectin medium and this medium was replaced with SMC4 medium 24-72 hours later. One day after sorting, sorted cells were split and by day 5, large colonies consisting of hundreds of cells were present. B. Sorted cells retained expression of the pluripotency marker Tra160. Tra160, red; DAPI, blue. C. After 5 passages and approximately 1 month in culture after sorting, quantitative flow cytometry analysis revealed that the majority of sorted iPSCs retained their pluripotent state based on SSEA4 and Tra181 staining. D. Various densities of SSEA4+ and Tra181+ human pluripotent stem cell derived iPSCs were plated, including a density of 500 events per well of a 6-well plate and approximately 52 events per cm2 of clones on a feeder-free surface, supplemented with SMC4+fibronectin medium. Seven days after sorting, many alkaline phosphatase positive colonies appeared in each of the seeded wells. E. The resulting alkaline phosphatase positive colonies were scored. Approximately 8-10% of the seeded events gave rise to colonies, with 16,000 events per well giving rise to near-confluent wells. NC, not counted due to confluency preventing accurate counting. F. SSEA4+ and Tra181+ human pluripotent stem cells were sorted directly into 96-well plates at various densities, including 1 event per well and approximately 3 events per cm2. Eight days after sorting, wells were stained for alkaline phosphatase. G. On day 8, each well was scored for alkaline phosphatase colonies. [Figure 4G]Figure 4. Enhanced single-cell sorting and subsequent feeder-free culture of human pluripotent stem cells. Demonstration of sorting of human pluripotent stem cells based on expression of pluripotency markers and subsequent maintenance of sorted cells in feeder-free culture supplemented with optimized SMC4 medium or SMC4+fibronectin medium. A. Prior to sorting, human pluripotent stem cells were dissociated into single cells and labeled with antibody markers against SSEA4 and Tra-181. Cells were sorted based on surface marker expression of the pluripotency markers SSEA4 and TRA181 using FACs technology. After sorting, cells were first plated in SMC4+fibronectin medium and this medium was replaced with SMC4 medium 24-72 hours later. One day after sorting, sorted cells were split and by day 5, large colonies consisting of hundreds of cells were present. B. Sorted cells retained expression of the pluripotency marker Tra160. Tra160, red; DAPI, blue. C. After 5 passages and approximately 1 month in culture after sorting, quantitative flow cytometry analysis revealed that the majority of sorted iPSCs retained their pluripotent state based on SSEA4 and Tra181 staining. D. Various densities of SSEA4+ and Tra181+ human pluripotent stem cell derived iPSCs were plated, including a density of 500 events per well of a 6-well plate and approximately 52 events per cm2 of clones on a feeder-free surface, supplemented with SMC4+fibronectin medium. Seven days after sorting, many alkaline phosphatase positive colonies appeared in each of the seeded wells. E. The resulting alkaline phosphatase positive colonies were scored. Approximately 8-10% of the seeded events gave rise to colonies, with 16,000 events per well giving rise to near-confluent wells. NC, not counted due to confluency preventing accurate counting. F. SSEA4+ and Tra181+ human pluripotent stem cells were sorted directly into 96-well plates at various densities, including 1 event per well and approximately 3 events per cm2. Eight days after sorting, wells were stained for alkaline phosphatase. G. On day 8, each well was scored for alkaline phosphatase colonies. [Figure 5A]Figure 5 shows the kinetics of reprogramming in the absence and presence of SMC4 medium. A. Seven days after initiating reprogramming by ectopic expression of Oct4, Sox2, Klf4 and cMyc, cells were maintained in conventional medium or switched to SMC4 medium. Twenty days after reprogramming, few if any iPSC-like colonies formed in conventional medium cultures, whereas many iPSC-like colonies appeared in cells switched to SMC4 medium. B. Many iPSC-like colonies in SMC4 medium expressed alkaline phosphatase, whereas few colonies expressed alkaline phosphatase in conventional cultures. [Figure 5B] Figure 5 shows the kinetics of reprogramming in the absence and presence of SMC4 medium. A. Seven days after initiating reprogramming by ectopic expression of Oct4, Sox2, Klf4 and cMyc, cells were maintained in conventional medium or switched to SMC4 medium. Twenty days after reprogramming, few if any iPSC-like colonies formed in conventional medium cultures, whereas many iPSC-like colonies appeared in cells switched to SMC4 medium. B. Many iPSC-like colonies in SMC4 medium expressed alkaline phosphatase, whereas few colonies expressed alkaline phosphatase in conventional cultures. [Figure 6A]FIG. 6. Culturing in SMC4 medium promotes naïve properties. A. Schematic of adaptation of human pluripotent stem cells to SMC4 medium and comparison with conventionally maintained human pluripotent stem cells. B. Affymetrix global gene expression and Pearson coefficient based hiPSCs derived from IMR90 parental cells (conventionally derived hiPSCs (Conv) and hiPSCs adapted to SMC4 medium (SMC4 medium)), both cultured in the respective medium for 11 passages, demonstrate that both hiPSCs are similar to each other and distinct from their parental IMR90 cells. C. Expression of Xist was moderately suppressed in conventionally cultured hiPSCs compared to their parental IMR90 cell lines, but was significantly suppressed in hiPSCs cultured in SMC4 medium, similar to the levels of female hESCs, HUES9. Genes located on the DX chromosome were more highly expressed in hiPSCs cultured in SMC4 medium compared to their expression in conventionally cultured hiPSCs. E. When placed back onto feeder cells, hiPSCs maintained in SMC4 medium exhibit a morphology more similar to mouse ESCs, as seen by more three-dimensional shapes and less flat morphology, compared to hiPSCs cultured in conventional medium. [Figure 6B]FIG. 6. Culturing in SMC4 medium promotes naïve properties. A. Schematic of adaptation of human pluripotent stem cells to SMC4 medium and comparison with conventionally maintained human pluripotent stem cells. B. Affymetrix global gene expression and Pearson coefficient based hiPSCs derived from IMR90 parental cells (conventionally derived hiPSCs (Conv) and hiPSCs adapted to SMC4 medium (SMC4 medium)), both cultured in the respective medium for 11 passages, demonstrate that both hiPSCs are similar to each other and distinct from their parental IMR90 cells. C. Expression of Xist was moderately suppressed in conventionally cultured hiPSCs compared to their parental IMR90 cell lines, but was significantly suppressed in hiPSCs cultured in SMC4 medium, similar to the levels of female hESCs, HUES9. Genes located on the DX chromosome were more highly expressed in hiPSCs cultured in SMC4 medium compared to their expression in conventionally cultured hiPSCs. E. When placed back onto feeder cells, hiPSCs maintained in SMC4 medium exhibit a morphology more similar to mouse ESCs, as seen by more three-dimensional shapes and less flat morphology, compared to hiPSCs cultured in conventional medium. [Figure 6C]FIG. 6. Culturing in SMC4 medium promotes naïve properties. A. Schematic of adaptation of human pluripotent stem cells to SMC4 medium and comparison with conventionally maintained human pluripotent stem cells. B. Affymetrix global gene expression and Pearson coefficient based hiPSCs derived from IMR90 parental cells (conventionally derived hiPSCs (Conv) and hiPSCs adapted to SMC4 medium (SMC4 medium)), both cultured in the respective medium for 11 passages, demonstrate that both hiPSCs are similar to each other and distinct from their parental IMR90 cells. C. Expression of Xist was moderately suppressed in conventionally cultured hiPSCs compared to their parental IMR90 cell lines, but was significantly suppressed in hiPSCs cultured in SMC4 medium, similar to the levels of female hESCs, HUES9. Genes located on the DX chromosome were more highly expressed in hiPSCs cultured in SMC4 medium compared to their expression in conventionally cultured hiPSCs. E. When placed back onto feeder cells, hiPSCs maintained in SMC4 medium exhibit a morphology more similar to mouse ESCs, as seen by more three-dimensional shapes and less flat morphology, compared to hiPSCs cultured in conventional medium. [Figure 6D]FIG. 6. Culturing in SMC4 medium promotes naïve properties. A. Schematic of adaptation of human pluripotent stem cells to SMC4 medium and comparison with conventionally maintained human pluripotent stem cells. B. Affymetrix global gene expression and Pearson coefficient based hiPSCs derived from IMR90 parental cells (conventionally derived hiPSCs (Conv) and hiPSCs adapted to SMC4 medium (SMC4 medium)), both cultured in the respective medium for 11 passages, demonstrate that both hiPSCs are similar to each other and distinct from their parental IMR90 cells. C. Expression of Xist was moderately suppressed in conventionally cultured hiPSCs compared to their parental IMR90 cell lines, but was significantly suppressed in hiPSCs cultured in SMC4 medium, similar to the levels of female hESCs, HUES9. Genes located on the DX chromosome were more highly expressed in hiPSCs cultured in SMC4 medium compared to their expression in conventionally cultured hiPSCs. E. When placed back onto feeder cells, hiPSCs maintained in SMC4 medium exhibit a morphology more similar to mouse ESCs, as seen by more three-dimensional shapes and less flat morphology, compared to hiPSCs cultured in conventional medium. [Figure 6E]FIG. 6. Culturing in SMC4 medium promotes naïve properties. A. Schematic of adaptation of human pluripotent stem cells to SMC4 medium and comparison with conventionally maintained human pluripotent stem cells. B. Affymetrix global gene expression and Pearson coefficient based hiPSCs derived from IMR90 parental cells (conventionally derived hiPSCs (Conv) and hiPSCs adapted to SMC4 medium (SMC4 medium)), both cultured in the respective medium for 11 passages, demonstrate that both hiPSCs are similar to each other and distinct from their parental IMR90 cells. C. Expression of Xist was moderately suppressed in conventionally cultured hiPSCs compared to their parental IMR90 cell lines, but was significantly suppressed in hiPSCs cultured in SMC4 medium, similar to the levels of female hESCs, HUES9. Genes located on the DX chromosome were more highly expressed in hiPSCs cultured in SMC4 medium compared to their expression in conventionally cultured hiPSCs. E. When placed back onto feeder cells, hiPSCs maintained in SMC4 medium exhibit a morphology more similar to mouse ESCs, as seen by more three-dimensional shapes and less flat morphology, compared to hiPSCs cultured in conventional medium. [Figure 7A]Figure 7 shows that generation and maintenance in SMC4 medium improves the undifferentiated state of human pluripotent stem cells. A. Schematic of parallel generation of clones FTi91 and 93 generated in SMC4 medium and maintained in FF culture in SMC4 medium, and clone FTi99 generated in conventional medium and maintained on feeder cells. B. Hierarchical clustering based on Affymetrix global gene expression and Pearson coefficients shows close relatedness between the resulting pluripotent lines and distant relatedness from the starting cell line (FTC1). C. Hierarchical clustering based on all genes differentially expressed 4-fold among Hues9, H1, FTi99, FTi91 and FTi93, demonstrating separation between pluripotent lines cultured in conventional systems and in SMC4 medium. D. Affymetrix gene expression of selected genes associated with naïve and lineage differentiation compared between SMC4 medium groups (FTi91 and 93) and conventional medium groups (H1, Hues9 and FTi99), showing increased expression of many genes associated with pluripotency and a significant decrease in lineage-specific gene expression in the SMC4 medium set. E. Schematic of different methods to obtain naïve state hiPSCs. The bottom panel lists the advantages of the naïve state over the primed state. [Figure 7B]Figure 7 shows that generation and maintenance in SMC4 medium improves the undifferentiated state of human pluripotent stem cells. A. Schematic of parallel generation of clones FTi91 and 93 generated in SMC4 medium and maintained in FF culture in SMC4 medium, and clone FTi99 generated in conventional medium and maintained on feeder cells. B. Hierarchical clustering based on Affymetrix global gene expression and Pearson coefficients shows close relatedness between the resulting pluripotent lines and distant relatedness from the starting cell line (FTC1). C. Hierarchical clustering based on all genes differentially expressed 4-fold among Hues9, H1, FTi99, FTi91 and FTi93, demonstrating separation between pluripotent lines cultured in conventional systems and in SMC4 medium. D. Affymetrix gene expression of selected genes associated with naïve and lineage differentiation compared between SMC4 medium groups (FTi91 and 93) and conventional medium groups (H1, Hues9 and FTi99), showing increased expression of many genes associated with pluripotency and a significant decrease in lineage-specific gene expression in the SMC4 medium set. E. Schematic of different methods to obtain naïve state hiPSCs. The bottom panel lists the advantages of the naïve state over the primed state. [Figure 7C]Figure 7 shows that generation and maintenance in SMC4 medium improves the undifferentiated state of human pluripotent stem cells. A. Schematic of parallel generation of clones FTi91 and 93 generated in SMC4 medium and maintained in FF culture in SMC4 medium, and clone FTi99 generated in conventional medium and maintained on feeder cells. B. Hierarchical clustering based on Affymetrix global gene expression and Pearson coefficients shows close relatedness between the resulting pluripotent lines and distant relatedness from the starting cell line (FTC1). C. Hierarchical clustering based on all genes differentially expressed 4-fold among Hues9, H1, FTi99, FTi91 and FTi93, demonstrating separation between pluripotent lines cultured in conventional systems and in SMC4 medium. D. Affymetrix gene expression of selected genes associated with naïve and lineage differentiation compared between SMC4 medium groups (FTi91 and 93) and conventional medium groups (H1, Hues9 and FTi99), showing increased expression of many genes associated with pluripotency and a significant decrease in lineage-specific gene expression in the SMC4 medium set. E. Schematic of different methods to obtain naïve state hiPSCs. The bottom panel lists the advantages of the naïve state over the primed state. [Figure 7D]Figure 7 shows that generation and maintenance in SMC4 medium improves the undifferentiated state of human pluripotent stem cells. A. Schematic of parallel generation of clones FTi91 and 93 generated in SMC4 medium and maintained in FF culture in SMC4 medium, and clone FTi99 generated in conventional medium and maintained on feeder cells. B. Hierarchical clustering based on Affymetrix global gene expression and Pearson coefficients shows close relatedness between the resulting pluripotent lines and distant relatedness from the starting cell line (FTC1). C. Hierarchical clustering based on all genes differentially expressed 4-fold among Hues9, H1, FTi99, FTi91 and FTi93, demonstrating separation between pluripotent lines cultured in conventional systems and in SMC4 medium. D. Affymetrix gene expression of selected genes associated with naïve and lineage differentiation compared between SMC4 medium groups (FTi91 and 93) and conventional medium groups (H1, Hues9 and FTi99), showing increased expression of many genes associated with pluripotency and a significant decrease in lineage-specific gene expression in the SMC4 medium set. E. Schematic of different methods to obtain naïve state hiPSCs. The bottom panel lists the advantages of the naïve state over the primed state. [Figure 7E]Figure 7 shows that generation and maintenance in SMC4 medium improves the undifferentiated state of human pluripotent stem cells. A. Schematic of parallel generation of clones FTi91 and 93 generated in SMC4 medium and maintained in FF culture in SMC4 medium, and clone FTi99 generated in conventional medium and maintained on feeder cells. B. Hierarchical clustering based on Affymetrix global gene expression and Pearson coefficients shows close relatedness between the resulting pluripotent lines and distant relatedness from the starting cell line (FTC1). C. Hierarchical clustering based on all genes differentially expressed 4-fold among Hues9, H1, FTi99, FTi91 and FTi93, demonstrating separation between pluripotent lines cultured in conventional systems and in SMC4 medium. D. Affymetrix gene expression of selected genes associated with naïve and lineage differentiation compared between SMC4 medium groups (FTi91 and 93) and conventional medium groups (H1, Hues9 and FTi99), showing increased expression of many genes associated with pluripotency and a significant decrease in lineage-specific gene expression in the SMC4 medium set. E. Schematic of different methods to obtain naïve state hiPSCs. The bottom panel lists the advantages of the naïve state over the primed state. [Figure 8A]FIG. 8 shows non-iPSC colonies generated during the reprogramming process. A. Typical cell populations early in the reprogramming process: Immunostaining showed that the majority of fast-growing cells formed colonies that were SSEA4 negative and inevitably non-pluripotent cells. These cells were transformed, highly proliferative, grew quickly, and dominated the culture. Colony 1 shows a SSEA4 positive colony with the potential to become an iPSC colony, while colony 2 shows a transformed population with a high proliferation rate but negative for pluripotency marker staining. B. Many non-iPSC colonies showed expression of a single pluripotency marker, while true iPSC colonies showed expression of multiple pluripotency markers. Various colonies were formed at later stages of reprogramming, and some colonies, i.e., colony 1, were double positive for both SSEA4 and Tra181, while others, e.g., colonies 4 and 5, were positive for only one of the markers or for neither. [Figure 8B] FIG. 8 shows non-iPSC colonies generated during the reprogramming process. A. Typical cell populations early in the reprogramming process: Immunostaining showed that the majority of fast-growing cells formed colonies that were SSEA4 negative and inevitably non-pluripotent cells. These cells were transformed, highly proliferative, grew quickly, and dominated the culture. Colony 1 shows a SSEA4 positive colony with the potential to become an iPSC colony, while colony 2 shows a transformed population with a high proliferation rate but negative for pluripotency marker staining. B. Many non-iPSC colonies showed expression of a single pluripotency marker, while true iPSC colonies showed expression of multiple pluripotency markers. Various colonies were formed at later stages of reprogramming, and some colonies, i.e., colony 1, were double positive for both SSEA4 and Tra181, while others, e.g., colonies 4 and 5, were positive for only one of the markers or for neither. [Figure 9]Figure 9. Selection of pluripotent cells such as iPSCs using cell sorting. Schematic of the single cell culture system and cell sorting methodology used to enrich and select pluripotent cells from a mixture of pluripotent and non-pluripotent cells. [Figure 10] Figure 10 is a graph showing the identification of pluripotency markers. To investigate additional markers specific for SSEA4+ / Tra181+ cells, various surface markers were evaluated. Detection of Tra160 expression was used as a positive control since it has been demonstrated to correlate well with Tra181 expression. CD200 and CD90 do not appear to be specific for pluripotent cells, as they stain similarly in both SSEA- / Tra181- and SSEA+ / Tra181+ populations, whereas CD9 shows little specificity for the SSEA+ / Tra181+ population. However, CD50 and CD30 appear to preferentially identify the SSEA4+ / Tra181+ population compared to the SSEA- / Tra181- population. [Figure 11A] FIG. 11: CD30 is specific to Nanog expressing clones. A. Representative flow profiles of SSEA4 / Tra181 expression of different hiPSC lines, demonstrating that all clones obtained represent multiple populations expressing both SSEA4 and Tra181. B. Based on the gating found in (A), CD30 and CD9 flow profiles of each cell line were evaluated. CD30 appears to be expressed only by clones FTC8 clone 1 and FTi31, whereas CD9 is expressed by all clones. C. The relative Nanog expression of the different lines was also evaluated. D. Schematic table demonstrating that only CD30 expression correlates with Nanog expression. [Figure 11B]FIG. 11: CD30 is specific to Nanog expressing clones. A. Representative flow profiles of SSEA4 / Tra181 expression of different hiPSC lines, demonstrating that all clones obtained represent multiple populations expressing both SSEA4 and Tra181. B. Based on the gating found in (A), CD30 and CD9 flow profiles of each cell line were evaluated. CD30 appears to be expressed only by clones FTC8 clone 1 and FTi31, whereas CD9 is expressed by all clones. C. The relative Nanog expression of the different lines was also evaluated. D. Schematic table demonstrating that only CD30 expression correlates with Nanog expression. [Figure 11C] FIG. 11: CD30 is specific to Nanog expressing clones. A. Representative flow profiles of SSEA4 / Tra181 expression of different hiPSC lines, demonstrating that all clones obtained represent multiple populations expressing both SSEA4 and Tra181. B. Based on the gating found in (A), CD30 and CD9 flow profiles of each cell line were evaluated. CD30 appears to be expressed only by clones FTC8 clone 1 and FTi31, whereas CD9 is expressed by all clones. C. The relative Nanog expression of the different lines was also evaluated. D. Schematic table demonstrating that only CD30 expression correlates with Nanog expression. [Figure 11D] FIG. 11: CD30 is specific to Nanog expressing clones. A. Representative flow profiles of SSEA4 / Tra181 expression of different hiPSC lines, demonstrating that all clones obtained represent multiple populations expressing both SSEA4 and Tra181. B. Based on the gating found in (A), CD30 and CD9 flow profiles of each cell line were evaluated. CD30 appears to be expressed only by clones FTC8 clone 1 and FTi31, whereas CD9 is expressed by all clones. C. The relative Nanog expression of the different lines was also evaluated. D. Schematic table demonstrating that only CD30 expression correlates with Nanog expression. [Figure 12] Figure 12 shows that depleting non-reprogrammed cells during the reprogramming process enriches reprogrammed cells. During the reprogramming process, there was a mixed population of reprogrammed and non-reprogrammed cells, and only a small population of cells showed cells that were reprogrammed to full pluripotency, as indicated by the expression of SSEA4 and Tra181 (indicated by black dotted arrows). However, when non-reprogrammed cells were removed or depleted from the mixed population, significant enrichment of SSEA4 and Tra181 positive populations was demonstrated. As indicated by gray solid arrows, CD13 negative cell populations showed significantly enriched populations of SSEA4 and Tra181 positive cells compared to CD13 low and high population cells. [Figure 13A]FIG. 13 shows the enhancement of cell reprogramming by single cell sorting and enrichment of pluripotent stem cells from various sources including IMR90 and ASC. A. Cell populations at the beginning of the reprogramming process were enriched by single cell sorting of SSEA4+ cells. The starting cell populations containing reprogrammed and non-reprogrammed cells were enzymatically dissociated into single cells, stained for unique pluripotency markers, and sorted based on surface marker expression by immunoconjugated magnetic beads to obtain a cell population positive for SSEA4. After this enrichment of SSEA4+ cells, 10,000 SSEA4+ cells were seeded on Matrigel™-coated dishes and cultured in FF environment in either conventional or SMC4 medium. AP staining was performed 8 days after sorting. The small inset panels on the far right of these are representative images of cell morphology. No alkaline phosphatase positive colonies were detected in conventional culture, but many alkaline phosphatase positive colonies were obtained in SMC4 medium. B. The number of SSEA4+ / Tra181+ colonies obtained by reprogramming cells in Matrigel™ in the presence or absence of SMC4 medium was scored. C. iPSC clones obtained from either IMR90 fibroblasts or adipose stem cells in feeder-free conditions and single-cell culture with SMC4 medium expressed markers of pluripotency. D. Flow cytometry analysis of the entire population of iPSC clones obtained using the single-cell sorting method revealed that the majority of cells were positive for key pluripotency markers. E. iPSCs generated using single-cell sorting and feeder-free culture on SSEA4 expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts or adipose stem cells. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs generated using single-cell sorting and feeder-free culture, unlike control fibroblasts infected with lentivirus expressing reprogramming factors after 3 days.G. iPSCs generated using single cell sorting based on SSEA4 readily developed into all three somatic cell types upon differentiation: endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III). [Figure 13B]FIG. 13 shows the enhancement of cell reprogramming by single cell sorting and enrichment of pluripotent stem cells from various sources including IMR90 and ASC. A. Cell populations at the beginning of the reprogramming process were enriched by single cell sorting of SSEA4+ cells. The starting cell populations containing reprogrammed and non-reprogrammed cells were enzymatically dissociated into single cells, stained for unique pluripotency markers, and sorted based on surface marker expression by immunoconjugated magnetic beads to obtain a cell population positive for SSEA4. After this enrichment of SSEA4+ cells, 10,000 SSEA4+ cells were seeded on Matrigel™-coated dishes and cultured in FF environment in either conventional or SMC4 medium. AP staining was performed 8 days after sorting. The small inset panels on the far right of these are representative images of cell morphology. No alkaline phosphatase positive colonies were detected in conventional culture, but many alkaline phosphatase positive colonies were obtained in SMC4 medium. B. The number of SSEA4+ / Tra181+ colonies obtained by reprogramming cells in Matrigel™ in the presence or absence of SMC4 medium was scored. C. iPSC clones obtained from either IMR90 fibroblasts or adipose stem cells in feeder-free conditions and single-cell culture with SMC4 medium expressed markers of pluripotency. D. Flow cytometry analysis of the entire population of iPSC clones obtained using the single-cell sorting method revealed that the majority of cells were positive for key pluripotency markers. E. iPSCs generated using single-cell sorting and feeder-free culture on SSEA4 expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts or adipose stem cells. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs generated using single-cell sorting and feeder-free culture, unlike control fibroblasts infected with lentivirus expressing reprogramming factors after 3 days.G. iPSCs generated using single cell sorting based on SSEA4 readily developed into all three somatic cell types upon differentiation: endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III). [Figure 13C]FIG. 13 shows the enhancement of cell reprogramming by single cell sorting and enrichment of pluripotent stem cells from various sources including IMR90 and ASC. A. Cell populations at the beginning of the reprogramming process were enriched by single cell sorting of SSEA4+ cells. The starting cell populations containing reprogrammed and non-reprogrammed cells were enzymatically dissociated into single cells, stained for unique pluripotency markers, and sorted based on surface marker expression by immunoconjugated magnetic beads to obtain a cell population positive for SSEA4. After this enrichment of SSEA4+ cells, 10,000 SSEA4+ cells were seeded on Matrigel™-coated dishes and cultured in FF environment in either conventional or SMC4 medium. AP staining was performed 8 days after sorting. The small inset panels on the far right of these are representative images of cell morphology. No alkaline phosphatase positive colonies were detected in conventional culture, but many alkaline phosphatase positive colonies were obtained in SMC4 medium. B. The number of SSEA4+ / Tra181+ colonies obtained by reprogramming cells in Matrigel™ in the presence or absence of SMC4 medium was scored. C. iPSC clones obtained from either IMR90 fibroblasts or adipose stem cells in feeder-free conditions and single-cell culture with SMC4 medium expressed markers of pluripotency. D. Flow cytometry analysis of the entire population of iPSC clones obtained using the single-cell sorting method revealed that the majority of cells were positive for key pluripotency markers. E. iPSCs generated using single-cell sorting and feeder-free culture on SSEA4 expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts or adipose stem cells. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs generated using single-cell sorting and feeder-free culture, unlike control fibroblasts infected with lentivirus expressing reprogramming factors after 3 days.G. iPSCs generated using single cell sorting based on SSEA4 readily developed into all three somatic cell types upon differentiation: endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III). [Figure 13D]FIG. 13 shows the enhancement of cell reprogramming by single cell sorting and enrichment of pluripotent stem cells from various sources including IMR90 and ASC. A. Cell populations at the beginning of the reprogramming process were enriched by single cell sorting of SSEA4+ cells. The starting cell populations containing reprogrammed and non-reprogrammed cells were enzymatically dissociated into single cells, stained for unique pluripotency markers, and sorted based on surface marker expression by immunoconjugated magnetic beads to obtain a cell population positive for SSEA4. After this enrichment of SSEA4+ cells, 10,000 SSEA4+ cells were seeded on Matrigel™-coated dishes and cultured in FF environment in either conventional or SMC4 medium. AP staining was performed 8 days after sorting. The small inset panels on the far right of these are representative images of cell morphology. No alkaline phosphatase positive colonies were detected in conventional culture, but many alkaline phosphatase positive colonies were obtained in SMC4 medium. B. The number of SSEA4+ / Tra181+ colonies obtained by reprogramming cells in Matrigel™ in the presence or absence of SMC4 medium was scored. C. iPSC clones obtained from either IMR90 fibroblasts or adipose stem cells in feeder-free conditions and single-cell culture with SMC4 medium expressed markers of pluripotency. D. Flow cytometry analysis of the entire population of iPSC clones obtained using the single-cell sorting method revealed that the majority of cells were positive for key pluripotency markers. E. iPSCs generated using single-cell sorting and feeder-free culture on SSEA4 expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts or adipose stem cells. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs generated using single-cell sorting and feeder-free culture, unlike control fibroblasts infected with lentivirus expressing reprogramming factors after 3 days.G. iPSCs generated using single cell sorting based on SSEA4 readily developed into all three somatic cell types upon differentiation: endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III). [Figure 13E]FIG. 13 shows the enhancement of cell reprogramming by single cell sorting and enrichment of pluripotent stem cells from various sources including IMR90 and ASC. A. Cell populations at the beginning of the reprogramming process were enriched by single cell sorting of SSEA4+ cells. The starting cell populations containing reprogrammed and non-reprogrammed cells were enzymatically dissociated into single cells, stained for unique pluripotency markers, and sorted based on surface marker expression by immunoconjugated magnetic beads to obtain a cell population positive for SSEA4. After this enrichment of SSEA4+ cells, 10,000 SSEA4+ cells were seeded on Matrigel™-coated dishes and cultured in FF environment in either conventional or SMC4 medium. AP staining was performed 8 days after sorting. The small inset panels on the far right of these are representative images of cell morphology. No alkaline phosphatase positive colonies were detected in conventional culture, but many alkaline phosphatase positive colonies were obtained in SMC4 medium. B. The number of SSEA4+ / Tra181+ colonies obtained by reprogramming cells in Matrigel™ in the presence or absence of SMC4 medium was scored. C. iPSC clones obtained from either IMR90 fibroblasts or adipose stem cells in feeder-free conditions and single-cell culture with SMC4 medium expressed markers of pluripotency. D. Flow cytometry analysis of the entire population of iPSC clones obtained using the single-cell sorting method revealed that the majority of cells were positive for key pluripotency markers. E. iPSCs generated using single-cell sorting and feeder-free culture on SSEA4 expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts or adipose stem cells. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs generated using single-cell sorting and feeder-free culture, unlike control fibroblasts infected with lentivirus expressing reprogramming factors after 3 days.G. iPSCs generated using single cell sorting based on SSEA4 readily developed into all three somatic cell types upon differentiation: endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III). [Figure 13F]FIG. 13 shows the enhancement of cell reprogramming by single cell sorting and enrichment of pluripotent stem cells from various sources including IMR90 and ASC. A. Cell populations at the beginning of the reprogramming process were enriched by single cell sorting of SSEA4+ cells. The starting cell populations containing reprogrammed and non-reprogrammed cells were enzymatically dissociated into single cells, stained for unique pluripotency markers, and sorted based on surface marker expression by immunoconjugated magnetic beads to obtain a cell population positive for SSEA4. After this enrichment of SSEA4+ cells, 10,000 SSEA4+ cells were seeded on Matrigel™-coated dishes and cultured in FF environment in either conventional or SMC4 medium. AP staining was performed 8 days after sorting. The small inset panels on the far right of these are representative images of cell morphology. No alkaline phosphatase positive colonies were detected in conventional culture, but many alkaline phosphatase positive colonies were obtained in SMC4 medium. B. The number of SSEA4+ / Tra181+ colonies obtained by reprogramming cells in Matrigel™ in the presence or absence of SMC4 medium was scored. C. iPSC clones obtained from either IMR90 fibroblasts or adipose stem cells in feeder-free conditions and single-cell culture with SMC4 medium expressed markers of pluripotency. D. Flow cytometry analysis of the entire population of iPSC clones obtained using the single-cell sorting method revealed that the majority of cells were positive for key pluripotency markers. E. iPSCs generated using single-cell sorting and feeder-free culture on SSEA4 expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts or adipose stem cells. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs generated using single-cell sorting and feeder-free culture, unlike control fibroblasts infected with lentivirus expressing reprogramming factors after 3 days.G. iPSCs generated using single cell sorting based on SSEA4 readily developed into all three somatic cell types upon differentiation: endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III). [Figure 13G]FIG. 13 shows the enhancement of cell reprogramming by single cell sorting and enrichment of pluripotent stem cells from various sources including IMR90 and ASC. A. Cell populations at the beginning of the reprogramming process were enriched by single cell sorting of SSEA4+ cells. The starting cell populations containing reprogrammed and non-reprogrammed cells were enzymatically dissociated into single cells, stained for unique pluripotency markers, and sorted based on surface marker expression by immunoconjugated magnetic beads to obtain a cell population positive for SSEA4. After this enrichment of SSEA4+ cells, 10,000 SSEA4+ cells were seeded on Matrigel™-coated dishes and cultured in FF environment in either conventional or SMC4 medium. AP staining was performed 8 days after sorting. The small inset panels on the far right of these are representative images of cell morphology. No alkaline phosphatase positive colonies were detected in conventional culture, but many alkaline phosphatase positive colonies were obtained in SMC4 medium. B. The number of SSEA4+ / Tra181+ colonies obtained by reprogramming cells in Matrigel™ in the presence or absence of SMC4 medium was scored. C. iPSC clones obtained from either IMR90 fibroblasts or adipose stem cells in feeder-free conditions and single-cell culture with SMC4 medium expressed markers of pluripotency. D. Flow cytometry analysis of the entire population of iPSC clones obtained using the single-cell sorting method revealed that the majority of cells were positive for key pluripotency markers. E. iPSCs generated using single-cell sorting and feeder-free culture on SSEA4 expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9, but not their original source fibroblasts or adipose stem cells. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs generated using single-cell sorting and feeder-free culture, unlike control fibroblasts infected with lentivirus expressing reprogramming factors after 3 days.G. iPSCs generated using single cell sorting based on SSEA4 readily developed into all three somatic cell types upon differentiation: endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, beta tubulin III (β TUB III). [Figure 14A] Figure 14 illustrates single cell sorting to select unique populations of pluripotent stem cells. A. Using FACS sorting, a unique and rare population of cells that were SSEA4+ / Tra181+ early in the reprogramming process was selected and transferred to feeder-free culture supplemented with SMC4+ fibronectin medium, and the above medium was replaced with SMC4 medium after 24-72 hours. After an additional 6 days of culture, SSEA4+ / Tra181+ derived iPSC colonies appeared to grow in feeder-free culture. However, when SSEA4- / Tra181- colonies were transferred and maintained in feeder-free culture, no colonies expressing alkaline phosphatase were detected after 14 days of culture. B. Various SSEA4+ / Tra181+ sorted cells developed into colonies while maintaining their expression of SSEA4 and Tra181. [Figure 14B] Figure 14 illustrates single cell sorting to select unique populations of pluripotent stem cells. A. Using FACS sorting, a unique and rare population of cells that were SSEA4+ / Tra181+ early in the reprogramming process was selected and transferred to feeder-free culture supplemented with SMC4+ fibronectin medium, and the above medium was replaced with SMC4 medium after 24-72 hours. After an additional 6 days of culture, SSEA4+ / Tra181+ derived iPSC colonies appeared to grow in feeder-free culture. However, when SSEA4- / Tra181- colonies were transferred and maintained in feeder-free culture, no colonies expressing alkaline phosphatase were detected after 14 days of culture. B. Various SSEA4+ / Tra181+ sorted cells developed into colonies while maintaining their expression of SSEA4 and Tra181. [Figure 15]Figure 15 shows a high-throughput approach to generate and primarily characterize iPSCs under feeder-free conditions. Three-factor (Oct, Sox, Klf)-induced fibroblasts were sorted in two steps using SMC4 medium (and SMC4+fibronectin medium during sorting) to generate an efficient 96-well plating platform. Wells containing individual colonies were marked and expanded into 4 x 96-well plates. One set was named master plate and expanded, and the other three plates were processed for characterization, including flow cytometry analysis for the expression of surface markers including SSEA4 and Tra181, qRT PCR for the expression of key markers including Nanog and transgene silencing, and immunofluorescence for pluripotency markers including Oct4 and Nanog. Based on the characterization readout, selected hiPSC clones were expanded and banked for further analysis. Data panels show snapshots of clones (represented by wells 31-40 of a 96-well plate) investigated during generation of hiPSCs in the FTC5 high-throughput platform. In the qPCR panel, expression was normalized to Gapdh. Nanog expression is proportional to H1 hESCs, whereas transgene expression is proportional to 4 days post-infection with FTC5 (day 4 of infection). Based on the characterization readouts, selected hiPSC clones were expanded and banked for further analysis. In the highlighted example, well 37 was identified as a candidate for expansion based on its multi-parameter pluripotency profile and was designated FTC5 clone 1. Immunofluorescence images were acquired at 5x magnification. [Figure 16A]FIG. 16: Clonality and FF induction in a high-throughput platform of three-factor (polycistronic OKS) hiPSCs in the presence of SMC4 medium. A. Clones FTi91 and FTi93 obtained with the above strategy were stained for expression of pluripotency markers. B. qRT-PCR of endogenous expression of pluripotency markers for FTC1 (foreskin fibroblasts), H1 and Hues9 (hESC lines), FTi91 and 93 (FTC1-derived hiPSC clones) and Day 4 PI (Day 4 PI). Expression was normalized to Gapdh and compared within each gene group. C. Methylation status of Oct4 promoter. Open circles indicate unmethylated CpG islands and closed circles indicate methylated CpG islands. D. EB formation and differentiation of clones FTi91 and 93 after 28 days of differentiation. Endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, Tuj1. E. Histological sections of teratomas from hiPSC clones generated and maintained in FF culture and SMC4 medium. Black arrows point to regions of interest: mesoderm, white adipose tissue; ectoderm, neurons; endoderm, glands. [Figure 16B]FIG. 16: Clonality and FF induction in a high-throughput platform of three-factor (polycistronic OKS) hiPSCs in the presence of SMC4 medium. A. Clones FTi91 and FTi93 obtained with the above strategy were stained for expression of pluripotency markers. B. qRT-PCR of endogenous expression of pluripotency markers for FTC1 (foreskin fibroblasts), H1 and Hues9 (hESC lines), FTi91 and 93 (FTC1-derived hiPSC clones) and Day 4 PI (Day 4 PI). Expression was normalized to Gapdh and compared within each gene group. C. Methylation status of Oct4 promoter. Open circles indicate unmethylated CpG islands and closed circles indicate methylated CpG islands. D. EB formation and differentiation of clones FTi91 and 93 after 28 days of differentiation. Endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, Tuj1. E. Histological sections of teratomas from hiPSC clones generated and maintained in FF culture and SMC4 medium. Black arrows point to regions of interest: mesoderm, white adipose tissue; ectoderm, neurons; endoderm, glands. [Figure 16C]FIG. 16: Clonality and FF induction in a high-throughput platform of three-factor (polycistronic OKS) hiPSCs in the presence of SMC4 medium. A. Clones FTi91 and FTi93 obtained with the above strategy were stained for expression of pluripotency markers. B. qRT-PCR of endogenous expression of pluripotency markers for FTC1 (foreskin fibroblasts), H1 and Hues9 (hESC lines), FTi91 and 93 (FTC1-derived hiPSC clones) and Day 4 PI (Day 4 PI). Expression was normalized to Gapdh and compared within each gene group. C. Methylation status of Oct4 promoter. Open circles indicate unmethylated CpG islands and closed circles indicate methylated CpG islands. D. EB formation and differentiation of clones FTi91 and 93 after 28 days of differentiation. Endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, Tuj1. E. Histological sections of teratomas from hiPSC clones generated and maintained in FF culture and SMC4 medium. Black arrows point to regions of interest: mesoderm, white adipose tissue; ectoderm, neurons; endoderm, glands. [Figure 16D]FIG. 16: Clonality and FF induction in a high-throughput platform of three-factor (polycistronic OKS) hiPSCs in the presence of SMC4 medium. A. Clones FTi91 and FTi93 obtained with the above strategy were stained for expression of pluripotency markers. B. qRT-PCR of endogenous expression of pluripotency markers for FTC1 (foreskin fibroblasts), H1 and Hues9 (hESC lines), FTi91 and 93 (FTC1-derived hiPSC clones) and Day 4 PI (Day 4 PI). Expression was normalized to Gapdh and compared within each gene group. C. Methylation status of Oct4 promoter. Open circles indicate unmethylated CpG islands and closed circles indicate methylated CpG islands. D. EB formation and differentiation of clones FTi91 and 93 after 28 days of differentiation. Endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, Tuj1. E. Histological sections of teratomas from hiPSC clones generated and maintained in FF culture and SMC4 medium. Black arrows point to regions of interest: mesoderm, white adipose tissue; ectoderm, neurons; endoderm, glands. [Figure 16E]FIG. 16: Clonality and FF induction in a high-throughput platform of three-factor (polycistronic OKS) hiPSCs in the presence of SMC4 medium. A. Clones FTi91 and FTi93 obtained with the above strategy were stained for expression of pluripotency markers. B. qRT-PCR of endogenous expression of pluripotency markers for FTC1 (foreskin fibroblasts), H1 and Hues9 (hESC lines), FTi91 and 93 (FTC1-derived hiPSC clones) and Day 4 PI (Day 4 PI). Expression was normalized to Gapdh and compared within each gene group. C. Methylation status of Oct4 promoter. Open circles indicate unmethylated CpG islands and closed circles indicate methylated CpG islands. D. EB formation and differentiation of clones FTi91 and 93 after 28 days of differentiation. Endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, Tuj1. E. Histological sections of teratomas from hiPSC clones generated and maintained in FF culture and SMC4 medium. Black arrows point to regions of interest: mesoderm, white adipose tissue; ectoderm, neurons; endoderm, glands. [Figure 17A] Figure 17 shows the generation and maintenance of pluripotent stem cells in SMC4 medium, which can maintain genomic integrity and enrich for pluripotent cells. A. Copy number variation assessed by array comparative genomic hybridization. The table below summarizes the interpretation of the data demonstrating minimal copy number variation between human pluripotent stem cells cultured in SMC4 medium and their parental cell lines. Cytogenesis analysis of BG-banded metaphase cells. The table below summarizes the data showing genomic stability after feeder-free culture in SMC4 medium over time. [Figure 17B]Figure 17 shows the generation and maintenance of pluripotent stem cells in SMC4 medium, which can maintain genomic integrity and enrich for pluripotent cells. A. Copy number variation assessed by array comparative genomic hybridization. The table below summarizes the interpretation of the data demonstrating minimal copy number variation between human pluripotent stem cells cultured in SMC4 medium and their parental cell lines. Cytogenesis analysis of BG-banded metaphase cells. The table below summarizes the data showing genomic stability after feeder-free culture in SMC4 medium over time. [Figure 18A] FIG. 18 shows the characterization of FTC5- and FTC7-derived clonal hiPSCs under FF and SMC4 culture. To determine the reproducibility of the high-throughput platform, additional patient consented lines FTC5 and FTC7 were tested for the generation of hiPSCs. A. Representative immunofluorescence staining of pluripotency markers Oct4, Tra181, Nanog, and Tra160 expressed in individual FTC5- and FTC7-derived hiPSC clones induced with OKS and generated using the multiplex platform. B. Representative lineage-specific staining of FTC5- and FTC7-derived hiPSCs 28 days after induction of differentiation. Endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, Tuj1. C. Cytogenetic analysis of G-banded metaphase cells from FTC5- and FTC7-derived hiPSC clones after long-term FF and single-cell culture at various passages. [Figure 18B]FIG. 18 shows the characterization of FTC5- and FTC7-derived clonal hiPSCs under FF and SMC4 culture. To determine the reproducibility of the high-throughput platform, additional patient consented lines FTC5 and FTC7 were tested for the generation of hiPSCs. A. Representative immunofluorescence staining of pluripotency markers Oct4, Tra181, Nanog, and Tra160 expressed in individual FTC5- and FTC7-derived hiPSC clones induced with OKS and generated using the multiplex platform. B. Representative lineage-specific staining of FTC5- and FTC7-derived hiPSCs 28 days after induction of differentiation. Endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, Tuj1. C. Cytogenetic analysis of G-banded metaphase cells from FTC5- and FTC7-derived hiPSC clones after long-term FF and single-cell culture at various passages. [Figure 18C] FIG. 18 shows the characterization of FTC5- and FTC7-derived clonal hiPSCs under FF and SMC4 culture. To determine the reproducibility of the high-throughput platform, additional patient consented lines FTC5 and FTC7 were tested for the generation of hiPSCs. A. Representative immunofluorescence staining of pluripotency markers Oct4, Tra181, Nanog, and Tra160 expressed in individual FTC5- and FTC7-derived hiPSC clones induced with OKS and generated using the multiplex platform. B. Representative lineage-specific staining of FTC5- and FTC7-derived hiPSCs 28 days after induction of differentiation. Endoderm, FoxA2; mesoderm, alpha smooth muscle actin (αSMA); ectoderm, Tuj1. C. Cytogenetic analysis of G-banded metaphase cells from FTC5- and FTC7-derived hiPSC clones after long-term FF and single-cell culture at various passages. [Figure 19A]Figure 19 shows how cell surface markers are used to maintain pluripotent cultures. A. The population of pluripotent stem cells was further enriched during culture as demonstrated by Tra181 enrichment in feeder-free and SMC4 platforms. B. During culture of pluripotent cell populations, some percentage of cells in the population may begin to differentiate and lose their pluripotency. Using the pluripotency selection method, i.e., Tra181 enrichment, a uniform culture of undifferentiated cells was achieved. [Figure 19B] Figure 19 shows how cell surface markers are used to maintain pluripotent cultures. A. The population of pluripotent stem cells was further enriched during culture as demonstrated by Tra181 enrichment in feeder-free and SMC4 platforms. B. During culture of pluripotent cell populations, some percentage of cells in the population may begin to differentiate and lose their pluripotency. Using the pluripotency selection method, i.e., Tra181 enrichment, a uniform culture of undifferentiated cells was achieved. [Figure 20] Figure 20 shows the separation of differentiated cells from heterogeneous populations of non-pluripotent, semi-pluripotent and fully pluripotent stem cells. FACS sorting of a population of reprogrammed human fibroblasts: No alkaline phosphatase (AP) positive colonies were detected when sorting cells double negative for the pluripotency markers SSEA4- / Tra181- (blue box; bottom left box), indicating a loss of pluripotency and potential tumorigenicity after one week of culture, whereas sorting the double positive population SSEA4+ / Tra181+ (orange box; top right box) selects for and enriches for a population of pluripotent cells as evidenced by the formation of AP+ colonies. [Figure 21A]Figure 21 shows cytokine-free culture of human pluripotent stem cells in feeder-free culture. A. Human pluripotent stem cells maintained their growth and morphology when cultured on gelatin-coated surfaces supplemented with SMC4 medium in the absence of any cytokines, including bFGF. B. A magnified image showing individual cells within a human pluripotent stem cell colony. C. Co-expression of SSEA4 and Tra181 at the third passage provided further evidence that human pluripotent stem cells cultured without cytokines maintained their undifferentiated state. [Figure 21B] Figure 21 shows cytokine-free culture of human pluripotent stem cells in feeder-free culture. A. Human pluripotent stem cells maintained their growth and morphology when cultured on gelatin-coated surfaces supplemented with SMC4 medium in the absence of any cytokines, including bFGF. B. A magnified image showing individual cells within a human pluripotent stem cell colony. C. Co-expression of SSEA4 and Tra181 at the third passage provided further evidence that human pluripotent stem cells cultured without cytokines maintained their undifferentiated state. [Figure 21C] Figure 21 shows cytokine-free culture of human pluripotent stem cells in feeder-free culture. A. Human pluripotent stem cells maintained their growth and morphology when cultured on gelatin-coated surfaces supplemented with SMC4 medium in the absence of any cytokines, including bFGF. B. A magnified image showing individual cells within a human pluripotent stem cell colony. C. Co-expression of SSEA4 and Tra181 at the third passage provided further evidence that human pluripotent stem cells cultured without cytokines maintained their undifferentiated state. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] Detailed Description of the Invention The present invention provides a robust culture system for culturing stem cells, including feeder-free conditions for generating and culturing human induced pluripotent stem cells (iPSCs). In particular, the present invention provides a culture platform that allows long-term culture of pluripotent cells in a feeder-free environment; reprogramming of cells in a feeder-free environment; dissociation of pluripotent cells into single cells; cell sorting of pluripotent cells; improving the efficiency of reprogramming; generating naive pluripotent cells; and identifying markers for identifying and selecting pluripotent cells. The media and culture methods of the present invention support the viability and survival of human stem cells dissociated into single cells, maintain the undifferentiated state of stem cells, and allow dissociated single cells to be cultured and passaged without differentiation.

[0084] definition 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 cell into a less differentiated state.For example, a cell with increased cell potential has higher 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.

[0085] As used herein, the term "potency" refers to the sum of all developmental options (i.e., developmental potential) available to a cell. 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 greatest developmental potential, to terminally differentiated cells, which are the least plastic cells with the least 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. In the strictest sense, stem cells are multipotent; thus, they can give rise to any mature cell type. However, multipotent, oligopotent, or unipotent progenitor cells are sometimes referred to as lineage-restricted stem cells (e.g., mesenchymal stem cells, adipose tissue-derived stem cells, etc.) and / or progenitor cells.

[0086] As used herein, the term "pluripotency" refers to the ability of a cell to form all lineages of soma or cell bodies (i.e., embryo proper). 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. Pluripotency is a continuum of developmental potential ranging from incompletely or partially pluripotent cells (e.g., epiblast stem cells or EpiSCs) that cannot give rise to a complete organism, to earlier, more pluripotent cells (e.g., embryonic stem cells) that can give rise to a complete organism. The level of pluripotency of a cell can be determined by assessing the pluripotency characteristics of the cell. Pluripotency characteristics include, but are not limited to, i) pluripotent stem cell morphology; ii) the ability to form a complete organism; expression of pluripotent stem cell markers including SSEA1 (mouse only), SSEA3 / 4; TRA1-60 / 81; TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD105, CD31, CD34, OCT4, Nanog and / or Sox2, and as described in the present invention, CD30 and CD50; iii) the ability of pluripotent mouse stem cells to contribute to germline transmission in mouse chimeras; iv) the ability of pluripotent stem cells to contribute to embryo proper using a tetraploid embryo complementation assay; v) formation of teratomas from pluripotent stem cells; vi) formation of embryoid bodies; and vii) reactivation of the inactive X chromosome.

[0087] 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 round and small shape, a high nuclear-cytoplasmic ratio, prominent presence of nucleoli, and typical intercellular spacing.

[0088] As used herein, the term "multipotency" refers to the ability of adult stem cells to form multiple cell types of one lineage. For example, hematopoietic stem cells can form all cells of blood cell lineages, such as lymphoid and myeloid cells.

[0089] "Adherent" refers to cells that are attached to a container, e.g., a sterile plastic (or coated plastic) cell culture dish or flask in the presence of an appropriate medium. Certain classes of cells are not persistent or will not grow in culture unless attached to a cell culture container. Certain classes of cells ("non-adherent cells") are maintained and / or proliferate in culture without attachment.

[0090] "Culture" or "cell culture" refers to the maintenance, growth, and / or differentiation of cells in an in vitro environment. The terms "culture media" (in each case singular "medium"), "supplement" and "media supplement" refer to a nutritional composition in which a cell culture is cultivated.

[0091] "Culturing" refers to sustaining, propagating (growing), and / or differentiating cells outside of a tissue or body, for example, in a sterile plastic (or coated plastic) cell culture dish or flask. In "culturing," medium can be utilized as a source of nutrients, hormones, and / or other factors that serve to propagate and / or sustain the cells.

[0092] As used herein, "dissociated" cells refer to cells that have been substantially separated or purified from other cells or from a surface (e.g., a culture plate surface). For example, cells can be dissociated from animals or tissues by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be dissociated from each other, for example, by enzymatic or mechanical dissociation 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 disrupting the interaction of cells with the extracellular matrix (ECM) and substrate (e.g., a culture surface), or disrupting the ECM between cells.

[0093] 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, e.g., a cell population, means that the amount of a particular component is proportionally increased compared to the proportion of such component in the cell population prior to enrichment. The term "enriched" refers to a cell population that is enriched for a target cell type (i.e., cells with a particular characteristic). For example, a composition such as a cell population can be enriched for a target cell type (i.e., cells with a particular characteristic), and thus the proportion or percentage of the target cell type is increased compared to the proportion of target cells present in the cell population before enrichment. The cell population can be enriched for a target cell type by cell selection and sorting methods known in the art. In some embodiments of the invention, the cell population is enriched by a sorting or selection process described in the Examples herein. In certain embodiments of the invention, the method of enriching a target cell population enriches the cell population for the target cell population by at least about 20%, i.e., the enriched cell population contains about 20% more target cell types proportionally than in the population before enrichment. In one embodiment, the method of enriching a target cell population results in a cell population that is proportionally enriched with respect to 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 particular embodiment, about 99%.

[0094] In certain aspects of the invention, cell populations are enriched for the amount of pluripotent cells or cells that exhibit pluripotent characteristics. In certain embodiments of the invention, cell populations undergoing reprogramming are enriched for target cells that have pluripotent characteristics, for example, expression of pluripotent markers, including but not limited to SSEA4, TRA1-60, TRA-1-81, CD30 or CD50. In another specific embodiment of the invention, cell populations, such as cell populations undergoing reprogramming, are depleted of non-pluripotent cells using surface markers specific for differentiating or non-pluripotent cells, for example, CD13, CD26, CD34, CD45, CD31, CD46 or CD7. Thus, the resulting cell population can be described as a cell population enriched for pluripotent cells. In one aspect of the invention, cells in the enriched cell population are enriched for target cells that have distinct gene or protein expression profiles, for example, cell surface expression of at least two pluripotent markers, for example, SSEA4, TRA1-60, TRA-1-81, CD30 and CD50. In some embodiments, the cell population is enriched for target cells expressing two or more pluripotency markers. In certain embodiments, the cell population is enriched for target cells expressing SSEA4 in combination with either Tra-181 or Tra-160. In more specific embodiments, the cell population is enriched for target cells expressing SSEA4, Tra181, and CD30. In one embodiment, at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% of the cells in the enriched cell population are of a target cell type, such as a pluripotent cell.

[0095] Thus, in some embodiments, the present invention provides a method for enriching a population of cells for pluripotent cells by sorting the cell population based on cell surface expression of pluripotency markers, such as SSEA4, TRA1-60, TRA-1-81, CD30 and CD50, and harvesting a fraction of cells expressing such markers to obtain a cell population enriched for pluripotent cells. In other embodiments of the present invention, the cell population 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 such cells from the cell population to obtain a cell population enriched for pluripotent cells. In certain embodiments, the cell population is sorted based on expression of CD13, and CD13+ cells are removed from the cell population to obtain a cell population enriched for pluripotent cells.

[0096] 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 and provides an environment in which the second type of cell can grow, because the second cell is not transferred from the feeder cell to the feeder. Feeder cells are suitable for use in culture as they provide growth factors and nutrients to support the species. Feeder cells are optionally derived 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 can generally be inactivated by irradiation or treatment with antimitotic agents such as mitomycin c to prevent them from outgrowing the cells they support when co-cultured with other cells. Without being limited to the above, one particular feeder cell type can be human feeder, for example, human skin fibroblasts or human embryonic stem cells. Another feeder cell type can be mouse embryonic fibroblasts (mEF).

[0097] As used herein, "feeder-free" (FF) environment refers to an environment such as a cell culture or medium that is essentially free of feeder cells and has not been pre-conditioned by culturing feeder cells. "Pre-conditioned" medium refers to a medium that is collected after culturing feeder cells in the medium for a period of time, for example, for at least one day. Pre-conditioned medium contains many mediator substances, such as growth factors and cytokines, secreted from feeder cells cultured in the medium.

[0098] In some embodiments of the invention, the feeder-free environment is essentially free of human feeder cells, including but not limited to human fibroblasts, keratinocytes, and embryonic stem cells, and in certain embodiments, is not preconditioned with feeder cells. In further embodiments of the invention, the feeder-free environment is essentially free of animal feeder cells, and in certain embodiments, is not preconditioned with feeder cells. In certain embodiments of the invention, the feeder-free environment is essentially free of both human and animal feeder cells, and in certain other embodiments, the feeder-free environment is essentially free of both human and animal feeder cells, and is not preconditioned with feeder cells.

[0099] Genomic stability refers to the ability of cell to faithfully replicate DNA and maintain the integrity of DNA replication process.As used herein to describe the cell of the present invention, " genomically stable cell " and " cell with genomic stability " refer to the cell that shows substantially similar mutation and chromosomal abnormality frequency (such as translocation, aneuploidy, copy number variation and duplication) frequency compared with normal human somatic cell.

[0100] "Component" refers to any compound or other material, whether chemical or biological in origin, that can be used in cell culture media to maintain and / or promote cell growth and / or differentiation. The terms "constituents," "nutrients," and "components" can be used interchangeably. Conventional components used for cell culture media may include, but are not limited to, amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins, and the like. Other components that promote and / or maintain the culture of cells ex vivo can be selected by the skilled artisan as required for the desired effect.

[0101] "Isolate" or "isolating" refers to the separation and harvesting of a composition or material from its natural environment, e.g., isolating individual cells or cell cultures from a tissue or body. In one aspect, a cell population or composition of cells is substantially free of cells and materials that may be naturally associated with it. "Isolated" or "purified" or "substantially pure" refers to, with respect to a target cell population, a cell population that is at least about 50%, at least about 75%, at least about 85%, at least about 90%, and in certain embodiments, at least about 95% pure with respect to the target cells that make up the total cell population. The purity of a cell population or composition of cells can be evaluated by any suitable method known in the art. For example, a substantially pure population of pluripotent cells refers to a cell population that is at least about 50%, at least about 75%, at least about 85%, at least about 90%, and in certain embodiments, at least about 95%, and in certain embodiments, 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".

[0102] "Passing" or "passaging" refers to the act of distributing and plating cells onto multiple cell culture surfaces or vessels when the cells have grown to a desired extent. In some embodiments, "passaging" or "passaging" refers to distributing, diluting, and plating cells. Once cells have been 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," or the like. Each act of distributing and plating into a new culture vessel is considered one passage.

[0103] "Plating" refers to placing one or more cells into a culture vessel such that the cells adhere to and spread onto the cell culture vessel.

[0104] "Pluripotency factor" refers to an agent that can increase the developmental potential of a cell, either alone or in combination with other agents. Pluripotency factors include, without limitation, 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. Transcription factors, unless otherwise indicated in the use herein, can refer to proteins (i.e., polypeptides) as well as polynucleotides that code for proteins. Exemplary transcription factors include, for example, Oct, Klf, Myc, and Sox polypeptides, as well as polynucleotides that code for these polypeptides. Additional examples of transcription factors are provided herein.

[0105] As used herein, the terms "polypeptide" and "protein" are used interchangeably according to their conventional meaning, i.e., as a sequence of amino acids, unless otherwise specified. A polypeptide is not limited to a particular length, e.g., a polypeptide may include a full-length protein sequence or a fragment of a full-length protein, and may include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like, as well as other naturally occurring and non-naturally occurring modifications known in the art. The polypeptides used in the methods of the present invention can be prepared using any of a variety of well-known recombinant and / or synthetic methods.

[0106] The methods of the invention, in certain embodiments, employ active fragments of the polypeptides described herein (e.g., Sox-2, c-Myc, Oct3 / 4, Klf4, Lin28, Nanog, etc., or substrates, cofactors and / or downstream effectors thereof), e.g., active fragments comprising at least about 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, etc., contiguous amino acids, or more, of the polypeptides described herein, including all intermediate lengths. In certain embodiments, the fragment or combination of fragments employed retains the ability to regulate, induce and / or maintain pluripotency when used in the methods described herein.

[0107] In another aspect, the invention employs variants of the polypeptide compositions described herein (e.g., Sox-2, c-Myc, Oct3 / 4, Klf4, Lin28, Nanog, etc., or substrates, cofactors and / or downstream effectors thereof). Generally, polypeptide variants encompassed by the invention will generally have a similar structure along their length to the polypeptides described herein. In certain embodiments, the variant or combination of variants used retains the ability to induce pluripotency as described herein.

[0108] In another aspect, the invention employs a polypeptide variant that exhibits at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity along its length (as determined below) to the corresponding region of a wild-type mammalian polypeptide used in accordance with the present disclosure. A polypeptide variant may differ from a naturally occurring polypeptide by one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the above polypeptide sequences used in the methods of the invention, and their effects may be assessed using any of a number of techniques well known in the art.

[0109] "Proliferate" refers to the property of a single cell dividing into two essentially identical cells or of a population of cells increasing in number (eg, to replicate).

[0110] "Propagation" refers to cells growing (e.g., replicating 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.

[0111] "Primary culture" refers to cells, tissues, and / or cultures in which isolated cells are placed into an original culture vessel with medium. The cells, tissues, and / or cultures may be maintained and / or expanded, but as long as the cells, tissues, and / or cultures remain in the original vessel, they are referred to as primary cultures.

[0112] 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 either alone or in combination with other pluripotency factors. "Small molecule" refers to an agent that has 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 .5 kD. Small molecules can be nucleic acids, peptidomimetics, peptoids, carbohydrates, lipids, or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, e.g., fungal, bacterial, or algal extracts, are known in the art and can be screened using any of the assays of the present invention. In certain embodiments, small molecule reprogramming agents used herein have a molecular weight of less than 10,000 daltons, e.g., less than 8000 daltons, less than 6000 daltons, less than 4000 daltons, less than 2000 daltons, e.g., between 50 and 1500 daltons, between 500 and 1500 daltons, between 200 and 2000 daltons, between 500 and 5000 daltons.

[0113] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition, e.g., a cell population or a medium, refer to a composition that is free of a particular substance, e.g., 95% free, 96% free, 97% free, 98% free, 99% free of a particular substance, or undetectable as measured by conventional means. A similar meaning can be applied to the term "absent" when referring to the absence of a particular substance or component of a composition.

[0114] Cells for use in the present invention The starting cell population for use in the present invention may include: The cells may be derived from essentially any suitable source and may be heterogeneous or homogeneous with respect to cell type or pluripotency state. In one embodiment, the cells are mammalian cells, and in certain embodiments, the cells are isolated from a mammal selected from the group consisting of rodents, cats, dogs, pigs, goats, sheep, horses, cows, or primates. In certain embodiments, the mammal is a human. In other certain embodiments, the cells used or treated according to the present invention are adult cells, including essentially any available adult cell type.

[0115] The cell may be a mixed population of cells, including somatic stem cells, non-pluripotent stem cells, incompletely or partially pluripotent stem cells, multipotent cells, micropotent cells, unipotent cells, terminally differentiated cells, or any combination of the above cells. The pluripotent cells used in the method of the present invention may be naturally occurring stem cells, including embryonic stem cells, or may be induced pluripotent stem cells. A "mixed" cell population is a cell population with different degrees of developmental potential. For example, the mixed population of cells may include cells that have undergone reprogramming, and thus the mixed population includes pluripotent cells, partially pluripotent cells, and non-pluripotent cells, such as fully differentiated cells.

[0116] In one embodiment, the starting cell population is selected from adult or neonatal stem / progenitor cells. In certain embodiments, the starting population of stem / progenitor cells is selected from the group consisting of mesodermal stem / progenitor cells, endodermal stem / progenitor cells, and ectodermal stem / progenitor cells.

[0117] In another embodiment, the starting population of stem / progenitor cells are mesodermal stem / progenitor cells. Illustrative 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 derived stem / progenitor cells, hematopoietic stem / progenitor cells (HSC), mesenchymal stem / progenitor cells, muscle stem / progenitor cells, kidney stem / progenitor cells, osteoblast stem / progenitor cells, chondrocyte stem / progenitor cells, and the like.

[0118] In other related embodiments, the starting population of stem / progenitor cells are ectodermal stem / progenitor cells. Illustrative 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.

[0119] In other related embodiments, the starting population of stem / progenitor cells are endodermal stem / progenitor cells. Illustrative 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.

[0120] 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, cardiomyocytes, skeletal muscle cells, smooth muscle cells, hematopoietic cells, bone cells, liver cells, adipocytes, kidney cells, lung cells, chondrocytes, skin cells, follicular cells, vascular cells, epithelial cells, immune cells, endothelial cells, and the like.

[0121] Inducing reprogramming and enhancing cell potency To induce pluripotency or increase potency 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 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, A variety of strategies have been pursued (K., Curr Biol 19:1718-1723 (2009b); Esteban et al., Cell Stem Cell 6:71-79 (2010); and Feng et al., Cell Stem Cell 4:301-312 (2009)).

[0122] Generally, reprogramming techniques involve directly or indirectly modulating specific cellular pathways using polynucleotide-based, polypeptide-based and / or small molecule-based approaches. The developmental potential of a cell can be increased, for example, by contacting the cell with one or more pluripotency factors. "Contacting," as used herein, can include culturing the cell in the presence of a pluripotency factor (such as, for example, a small molecule, protein, peptide, etc.) or introducing a pluripotency factor into the cell. A pluripotency factor can be introduced into a cell by culturing the cell in the presence of a factor, including a transcription factor, such as a protein, under conditions that allow the transcription factor to be introduced into the cell. See, for example, Zhou H et al., Cell Stem Cell. 2009 May 8; vol. 4(5): 381-4; WO / 2009 / 117439. Introduction into a cell can be facilitated, for example, using transient methods, such as protein transduction, microinjection, non-integrative gene delivery, mRNA transduction, etc., or any other suitable technique. In some embodiments, the transcription factor is introduced into the cell by expression from a recombinant vector introduced into the cell, or by incubating the cell in the presence of an exogenous transcription factor polypeptide, allowing the polypeptide to enter the cell.

[0123] In certain embodiments, the pluripotency factor is a transcription factor. Exemplary transcription factors associated with increasing, establishing, or maintaining the potency of a cell include, but are not limited to, Oct-3 / 4, Cdx-2, Gbx2, Gsh1, HesX1, HoxA10, HoxA11, HoxB1, Irx2, Isl1, Meis1, Meox2, Nanog, Nkx2.2, Onecut, Otx1, Oxt2, Pax5, Pax6, Pdx1, Tcf1, Tcf2, Zfhx1b, Klf-4, Atbf1, Esrrb, Gcnf, Jarid2, Jmjd1a, Jmjd2c, Klf-3, Klf-5, Mel-18, Myst3, Nac1, REST, Rex-1, Rybp, Sa ll4, Sall1, Tif1, YY1, Zeb2, Zfp281, Zfp57, Zic3, Coup-Tf1, Coup-Tf2, Bmi1, Rnf2, Mta1, Pias1, Pias2, Pias3, Piasy, Sox2, Lef1, Sox15, Sox6, Tcf-7, Tcf7l1, c-Myc, L-My c, N-Myc, Hand1, Mad1, Mad3, Mad4, Mxi1, Myf5, Neurog2, Ngn3, Olig2, Tcf3, Tcf4, Foxc1, Foxd3, BAF155, C / EBPβ, mafa, Eomes, Tbx-3; Rfx4, Stat3, Stella, and UTF-1. Exemplary transcription factors include Oct4, Sox2, Klf4, c-Myc, and Nanog.

[0124] Small molecule reprogramming agents are also pluripotency factors, which can also be used in the methods of the invention to induce reprogramming and to maintain or increase cellular potency. In some embodiments of the invention, small molecule reprogramming agents are used to induce pluripotency in somatic cells. To enhance or maintain the potency of, or improve the efficiency of, reprogramming, one or more small molecule reprogramming agents are used.

[0125] In some embodiments, small molecule reprogramming agents are used in the methods of the invention to improve the efficiency of reprogramming. Improved efficiency of reprogramming can be measured by (1) a decrease in the time required for reprogramming and generating pluripotent cells (e.g., by shortening the time to generate pluripotent cells by at least one day compared to a similar or same process without small molecules), or alternatively or in combination with (2) an increase in the number of pluripotent cells generated by a particular process (e.g., an increase of at least 10%, 30%, 50%, 100%, 200%, 500%, etc. in the number of cells reprogrammed within a given period compared to a similar or same process without small molecules). 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, an improvement in efficiency of more than 100-fold is observed (e.g., an increase of more than 100-fold in the number of pluripotent cells generated) compared to a method that does not use small molecule reprogramming agents.

[0126] Several classes of small molecule reprogramming agents may be important for increasing, establishing, and / or maintaining the capacity of cells. Exemplary small molecule reprogramming agents include, but are not limited to: agents that inhibit H3K9 methylation or promote H3K9 demethylation; agents that inhibit H3K4 demethylation or promote H3K4 methylation; agents that inhibit histone deacetylation or promote histone acetylation; L-type Ca channel agonists; cAMP pathway activators; DNA methyltransferase (DNMT) inhibitors; nuclear receptor ligands; GSK3 inhibitors, MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; Erk inhibitors; ROCK inhibitors; FGFR inhibitors; and PARP inhibitors. Exemplary small molecule reprogramming agents include GSK3 inhibitors, MEK inhibitors; TGFβ receptor / ALK5 inhibitors; HDAC inhibitors; Erk inhibitors; and ROCK inhibitors. Each of these classes of small molecule agents is described in more detail below.

[0127] In some embodiments of the present invention, small molecule reprogramming agents are used to replace one or more transcription factors of the method of the present invention to induce pluripotency, improve the efficiency of reprogramming, and / or increase or maintain the capacity of the cell. For example, in some embodiments, cells are contacted with one or more small molecule reprogramming agents, where the agents are included in an amount sufficient to improve the efficiency of reprogramming. In other embodiments, one or more small molecule reprogramming agents are used in addition to the transcription factors of the method of the present invention. In one embodiment, cells are contacted with at least one pluripotency transcription factor and at least one small molecule reprogramming agent under conditions to increase, establish, and / or maintain the capacity of the cell or improve the efficiency of the reprogramming process.

[0128] In another embodiment, cells are contacted with at least one pluripotency transcription factor and at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten small molecule reprogramming agents under conditions and for a time sufficient to increase, establish, and / or maintain the potency of the cells or improve the efficiency of reprogramming. The potency or differentiation state of the cells can be assessed by monitoring pluripotency characteristics as described elsewhere herein.

[0129] In one embodiment, the cells are treated with one or more pluripotency factors and / or small molecule reprogramming agents. The cells of the present invention are contacted with a composition comprising a combination of pluripotency factors and small molecules, where the pluripotency of the cells is increased or induced. It is contemplated that the cells of the present invention can be contacted in vitro, ex vivo, or in vivo.

[0130] Characterization of pluripotent cells After inducing reprogramming, reprogrammed cells can be selected based on the relevant detectable morphological, molecular and / or biochemical changes associated with pluripotency. Specific characteristics of cellular pluripotency that can be monitored separately or in combination in assessing cellular competence include, but are not limited to, gene expression, methylation, and in vivo and in vivo cell proliferation. In vitro characteristics include, for example, i) round and flat pluripotent stem cell morphology; ii) expression of pluripotent stem cell markers including SSEA1 (mouse pluripotent stem cells), 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, CD31, CD34, OCT4, Nanog and / or Sox2, as well as CD30 and CD50 provided by the present invention, and combinations of the foregoing; iii) ability of pluripotent stem cells to contribute to germline transmission in mouse chimeras; iv) ability of pluripotent stem cells to contribute to embryo proper using a tetraploid embryo complementation assay; v) formation of teratomas of pluripotent stem cells; vi) formation of embryoid bodies and tri-lineage differentiation in vitro; and vii) reactivation of the inactive X chromosome. In some embodiments, any subset of the above characteristics is used to monitor cell ability. In one embodiment, pluripotent cells are characterized by having flat round colony morphology, expression of SSEA4 and Oct4, and the ability to form chimeras and teratomas.

[0131] As discussed herein, pluripotency exists as a continuum, and induced pluripotent stem cells exist in both "primed" and "naive" states, with cells in the naive state likely having greater differentiation potential. Induced pluripotent stem cells generated in conventional media exist in a primed state and more closely resemble cells derived from post-implantation blastocysts, while naive iPSCs exhibit pluripotent properties more closely resemble cells derived from mouse embryonic stem cells or pre-implantation blastocysts. Primed and naive cell states can be defined by a variety of differences, including differences in colony morphology, cellular response to inhibition or activation of key signaling pathways, gene expression signatures, and ability to reactivate genes associated with extraembryonic cells. For example, conventional iPSCs, which exhibit a primed pluripotent state, exhibit flat colony morphology, while naive iPSCs exhibit a dense, dome-shaped colony morphology similar to mouse embryonic stem cells. Furthermore, conventional iPSCs require exogenous signaling of key cytokines, e.g., TGFβ, activin, and bFGF, to maintain their undifferentiated state, depend on ERK / MEK cell signaling, and differentiate when these pathways are inhibited, e.g., by contacting the cells with TGFβ or MEK inhibitors. In contrast, naive cells do not require exogenous signaling and maintain pluripotency even when treated with inhibitors of the TGFβ and MEK signaling pathways.

[0132] Furthermore, gene expression analysis shows significant differences between naïve and primed pluripotent cells. For example, expression of Xist is significantly suppressed in naïve iPSCs, whereas conventional iPSCs show only a modest suppression of Xist expression; naïve cells show significant X chromosome reactivation and increased expression of genes located on the X chromosome compared to expression found in conventional iPSCs; and naïve cells express extraembryonic stem cell markers, including, but not limited to, Gata6, CDX2, and CGB. In contrast, early markers of differentiation, such as Foxa2, Sox17, and B Lineage-specific genes such as rachyury are more highly expressed in conventional iPSCs compared to naive iPSCs. Additional markers useful for identifying cells in a naive pluripotent state include increased Klf4, Tbx3, Gbx2, Lin28, Soc3 or decreased Otx2, Sox17, Cer1, FoxA2, ​​Zic1, Lhx2, Xist.

[0133] In certain embodiments of the invention, naive cells express at least 2-fold, at least 5-fold, or at least 10-fold less Xist than conventional iPSCs. In some embodiments of the invention, cells in a naive pluripotent state express Xist at 2-fold lower levels than conventional iPSCs and express at least five genes located on the X chromosome at levels 3-fold higher than conventional iPSCs.

[0134] X chromosome reactivation can be indicated by an increase in expression of at least 5 genes, at least 10 genes, or in certain embodiments at least 100 genes located on the X chromosome, by at least 2-fold, 3-fold, 5-fold, or more compared to the levels of such genes in conventional iPSCs.

[0135] In certain embodiments of the invention, the pluripotent cells of the invention retain pluripotent properties over multiple cell passages, e.g., at least 1, 3, 5, 7, 10, 15, 20 or more passages.

[0136] Media Platforms for Use in the Methods of the Invention The medium (i.e., culture platform) of the present invention comprises a chemically defined, preserved basal medium, and Long-term culture of pluripotent cells in a feeder-free environment; Reprogramming cells in a feeder-free environment; Dissociation of pluripotent cells into single cells; Cell sorting for pluripotent cells; Maintenance of undifferentiated state; Improved efficiency of reprogramming; and Generation of naïve pluripotent cells The present invention includes various combinations of small molecules, including small molecule inhibitors, that enable

[0137] Chemically defined storage basal media for use in the media of the present invention may be any predefined basal medium suitable for supporting the maintenance, growth, and / or differentiation of stem cells, such as conventional human embryonic stem cell media. Examples of predefined basal media that can be used in accordance with the present invention include, but are not limited to, Dulbecco's Modified Eagle's Medium ("DMEM"), Eagle's Basal Medium (BME), DMEM / F-12 (1:1 DMEM and F-12, vol:vol); 199 Medium; F-12 (Ham) Nutrient Mix; F-10 (Ham) Nutrient Mix; Minimum Essential Medium (MEM), Williams Medium E; and RPMI 1640, all of which are available from Gibco-BRL / Life Technologies, Inc., Gaithersburg, Md. Several versions of many of these media are available, and media that are particularly useful for constructing the media of the present invention include, but are not limited to: DMEM11966, DMEM10314, MEM11095, Williams' Medium E12251, Ham F12 11059, MEM-alpha 12561, and Medium-199 11151 (all available from Gibco-BRL / Life Technologies (1995-1996 catalog). Media may include, for example, one or more of the following: amino acids, vitamins, organic salts, inorganic salts, trace elements, buffer salts, sugars, ATP, etc. (Suitable basal medium components are available from Sigma-Aldrich of Saint Louis, Maryland, USA). (Available from http: / / www.nature.com / ).

[0138] Small molecules and classes thereof for use in the cell culture medium of the present invention are described in more detail below. In certain embodiments, the medium of the present invention comprises one or more, two or more, or three or more of TGFβ inhibitor, GSK3 inhibitor, MEK inhibitor, and ROCK inhibitor. In some embodiments, the medium of the present invention comprises TGFβ inhibitor, GSK3 inhibitor, MEK inhibitor, and ROCK inhibitor. Exemplary TGFβ inhibitor, GSK3 inhibitor, MEK inhibitor, and ROCK inhibitor for use in the cell culture medium and method of the present invention are described below. The medium may further comprise a PARP inhibitor, such as Olaparib (AZD-2281).

[0139] GSK-3β inhibitors Inhibitors of GSK-3β include, but are not limited to, antibodies that bind to GSK-3β, dominant-negative GSK-3β mutants, and siRNAs and antisense nucleic acids that target GSK-3β.Other exemplary GSK-3β inhibitors include, but are not limited to, Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, AR-A014418, CT99021, CT20026, SB216763, AR-A014418, lithium, SB415286, TDZD-8, BIO, BIO-acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopenadienylruthenium complex, TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, 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; TWSl 19 pyrrolopyrimidine compound, L803 H-KEAPPAPPQSpP-NH2 or its myristoylated form; 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, SB216763, and SB415286. Exemplary GSK3 inhibitors for use in the cell culture medium of the present invention include CHIR99021, BIO, and Kenpaullone, with CHIR99021 being preferred in certain embodiments.

[0140] ERK / MEK Inhibitors Exemplary inhibitors of ERK / MEK pathway include, but are not limited to, antibodies against MEK or ERK, dominant negative MEK or ERK mutants, and siRNA and antisense nucleic acids that suppress the expression of MEK and / or ERK.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.

[0141] Additional MEK / ERK inhibitors include compounds disclosed in international published patent applications WO 99 / 01426, WO 02 / 06213, WO 03 / 077914, WO 05 / 051301 and WO 2007 / 044084.

[0142] Further illustrative examples of MEK / ERK inhibitors include the following compounds: --6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H -benzimidazole-e-5-carboxylic acid (2,3-dihydroxy-propoxy)-amide;6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-ylmethyl(ylm- ethyl))-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 1-[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-e-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-ylmethyl(ylm- ethyl))-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(4-bromo-2-fluoro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-e-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-e-5-carboxylic acid (2-hydroxy-ethoxy)-amide Midazole-e-5-carboxylic acid (2-hydroxy-ethoxy)-amide, hereafter referred to as MEK inhibitor 1; 2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide; hereafter 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 pharma- ceutical acceptable salt thereof. In one embodiment, the MEK / ERK inhibitor for use in the cell culture medium of the present invention is PD98059.

[0143] TGFβ receptor / ALK5 inhibitor Exemplary ALK5 inhibitors include antibodies against ALK5, dominant negative mutants of ALK5, and antisense nucleic acids that suppress the expression of ALK5.Other exemplary ALK5 inhibitors include, but are not limited to, SB431542, A-83-01, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, Wnt3a / BIO, BMP4, GW788388, SM16, IN-1130, GW6604, SB-505124, and pyrimidine derivatives, see, for example, WO2008 / 006583, which is incorporated herein by reference.

[0144] Furthermore, it should be understood that "ALK5 inhibitors" do not include non-specific kinase inhibitors, and that "ALK5 inhibitors" include inhibitors that inhibit ALK4 and / or ALK7 in addition to ALK5, such as, for example, SB-431542 (see, e.g., Inman et al., J MoI. Pharmacol. 62(1):65-74 (2002)).

[0145] Considering the data herein showing the effect of inhibiting ALK5, it is believed that inhibiting TGFβ / activin pathway will have a similar effect.Thus, for example, any inhibitor of the upstream or downstream of TGFβ / activin pathway can be used in combination with or instead of the ALK5 inhibitor described in each paragraph of this specification.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.In addition, the categorization below is merely for organizational purposes, and those skilled in the art will understand that a compound can affect one or more points in the pathway, and therefore a compound can function in more than one defined category.

[0146] TGFβ receptor inhibitors can include antibodies against TGFβ receptors, dominant negative mutants of TGFβ receptors, and siRNA or antisense nucleic acids targeting TGFβ receptors. Specific examples of inhibitors include, but are not limited to, SU5416; 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124); lerdelimumb (CAT-152); metelimumab (CAT-192); GC-1008; IDl 1; AP-12009; AP-11014; LY550410; LY580276; LY364947; LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; 3,5,7,2',4'-pentahydroxyfiavone (Morin); Activin-M108A; P144; soluble TBR2-Fc; and antisense transfected tumor cells targeting the TGFβ receptor (e.g., Wrzesinski et al., Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska et al., Acta Biochimica See Polonica 52(2):329-337 (2005); and Chang et al., Frontiers in Bioscience 12:4393-4401 (2007).

[0147] Exemplary TGFβ receptor inhibitors for use in the cell culture medium of the present invention include SB431542, A-83-01, and RepSox.In certain embodiments, the TGFβ inhibitor is SB431542.

[0148] ROCK inhibitors ROCK is a serine / threonine kinase that acts as a target protein of Rho (there are three isoforms: RhoA, RhoB and RhoC).Exemplary ROCK inhibitors include, but are not limited to, antibodies against ROCK, dominant negative ROCK mutants, and siRNA and antisense nucleic acids that suppress the expression of ROCK. Other exemplary ROCK inhibitors 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.

[0149] Exemplary ROCK inhibitors for use in the cell culture medium of the present invention include thiazovivin, Y27632, pyrintegrin, and blebbistatin. In some embodiments, the ROCK inhibitor is thiazovivin.

[0150] FGFR inhibitors Exemplary FGFR inhibitors include, but are not limited to, antibodies against FGFR, dominant negative FGFR mutants, and siRNA and antisense nucleic acids that suppress the expression of FGFR.Other exemplary FGFR inhibitors include, but are not limited to, RO-4396686, CHIR-258, PD 173074, PD 166866, ENK-834, ENK-835, SU5402, XL-999, SU6668, R04383596, and BIBF-1120.

[0151] PARP inhibitors PARP inhibitors inhibit poly(ADP-ribose) polymerase ("PARP"). PARP protein is a DNA repair enzyme that functions to regulate DNA repair pathways in cells. PARP is involved in base excision repair (BER) pathway, and inhibiting PARP can promote the genomic stability of cells during reprogramming or the maintenance of pluripotent cells. Exemplary PARP inhibitors for use in the cell culture medium of the present invention include, without limitation, iniparib, veliparib, and olaparib (AZD-2281).

[0152] The amount of small molecule in the cell culture medium of the invention can vary and can be optimized according to the particular culture conditions, including the particular molecules and combinations used, the type of cells cultured in the medium, and the particular application of the medium of the invention. In some embodiments, the small molecule is present in the medium at a concentration sufficient to induce pluripotency, improve the efficiency of reprogramming, or increase or maintain the potency of the cells.

[0153] In certain embodiments, preferred small molecule concentrations and combinations in the cell culture media of the invention are shown in Table 1. In certain embodiments of the cell culture media of the invention, the cell culture media is the "SMC4" medium described in Table 1. The SMC4 medium comprises a conventional human ESC medium and certain pathway modifiers and additives as shown in Table 1. The components of the medium may be present in the medium in amounts within the optimal ranges for such components as shown in Table 1, and are present at the optimal concentrations as shown in Table 1. Embodiments of the SMC4 medium may optionally include any one or more of the alternative media and pathway modifiers and activators as shown in Table 2, at concentrations within the optimal ranges as shown in Table 2, and in certain embodiments, at concentrations within the optimal ranges as shown in Table 2. In certain embodiments of the medium, the SMC4 medium comprises soluble fibronectin, and is referred to throughout as "SMC4+fibronectin."

[0154] In some embodiments, the medium of the present invention further comprises one or more of Oct polypeptide, Klf polypeptide, Myc polypeptide, and Sox polypeptide.In some embodiments, the medium does not comprise cells.In some embodiments, the medium further comprises cells, such as a mixed population of cells that contains non-pluripotent cells, partially pluripotent cells, pluripotent cells, or cells in various states of competence.

[0155] Table 1. Cell culture components and additives. The table below lists examples of molecules that can be used to enhance the viability and pluripotency of cells undergoing single cell passaging and cell sorting and enrichment procedures, to enhance the reprogramming process, and to maintain pluripotent stem cells in an undifferentiated state, and the signaling pathways that they affect.

[0156] [Table 1] As described above, SMC4+fibronectin refers to an SMC4 medium containing soluble fibronectin at a concentration of about 5 μg / mL. Fibronectin may be present in the SMC4 medium at a concentration ranging from about 0.5 to 500 μg / mL.

[0157] Table 2. Specific alternative cell culture components and additives to enhance cell pluripotency and viability during single cell passaging, cell sorting and culture in feeder-free systems.

[0158] [Table 2-1]

[0159] [Table 2-2]

[0160] [Table 2-3] Cytokines and Growth Factors In some embodiments of the present invention, the cell culture medium of the present invention is substantially free of cytokines and / or growth factors, and optionally is a feeder-free environment. In other embodiments, the cell culture medium contains supplements, such as serum, extracts, growth factors, hormones, cytokines, etc.

[0161] Various growth factors and their use in media are well known in the art and include, for example, ECM proteins, laminin 1, fibronectin, type IV collagen isotypes, proteases, protease inhibitors, cell surface adhesion proteins, cell-signaling proteins, cadherins, intracellular chloride channel 1, transmembrane receptor PTK7, insulin-like growth factors, inhibin beta A, inducers of the TGFβ / activin / nodal signaling pathway, and activin A. Cytokines used in the culture medium can include, for example, one or more of the following: growth factors such as epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), 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 to IL-18), various colony-stimulating factors (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF)), various interferons (e.g., IFN-γ, etc.), and other cytokines that have an effect on stem cells, such as stem cell factor (SCF) and erythropoietin (Epo). These cytokines can be obtained commercially, for example from R&D Systems, Minneapolis, Minn., and can be either natural or recombinant. In some embodiments, for culturing a wide variety of mammalian cells, the basal medium contains FGF at a concentration of about 0.01-100 ng / ml, about 0.2-20 ng / ml, and in certain embodiments, about 0.5-10 ng / ml. If other cytokines are used, If present, they can be added at concentrations that are empirically determined or as guided by the art of established cytokines.

[0162] Further components that can be included in the medium are insulin (especially insulin Zn ++These additional components are commercially available (e.g., from Sigma-Aldrich, St. Louis, Mo.) and can be formulated in the medium at concentrations ranging from about 0.1 to about 100 μg / ml or from about 1 to about 10 μg / ml. Additionally, recombinant insulin or a zinc-based salt of insulin can be substituted for insulin of animal or human origin. Other components or substitutes can be added to the supplement composition as known to those of skill in the art.

[0163] Alternatively (or in addition), cytokines and similar supplement components can be included in the basal medium. Such components are typically included with the supplement composition because the supplement composition is conventionally stored at about -20°C rather than the temperature of about 4°C routinely used to store basal medium. Cytokines and similar components may be more convenient at temperatures closer to -20°C.

[0164] Substrates for use in the methods of the invention Any suitable vessel or cell culture vessel can be used as a support for cell culture in basal medium and / or cell culture supplements. Substrate coating on the support is not required. However, coating the surface of the culture vessel with an adhesion-promoting substratum (e.g., collagen, fibronectin, RGD-containing polypeptide, gelatin, etc.) can promote cell attachment, thereby enhancing the effectiveness of the cell culture medium and supplements disclosed herein. Suitable substrates for culturing and passage of cells are known in the art and include, without limitation, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, mixtures of matrices produced by naturally occurring cell lines, such as Matrigel™, and synthetic or artificial surfaces, such as polyamine monolayers and carboxy-terminated monolayers. Feeder-free environment Cells are generally preconditioned on or with feeder cells and cultured in a culture environment containing fetal bovine serum, but such an environment may be inappropriate for producing cells for clinical and therapeutic use.For example, cells cultured in such a contaminated environment are generally considered inappropriate for human cell transplantation, since exposure to animal components may result in immune rejection and significant risk of transmitting unidentified pathogens to treated patients, and may potentially reactivate animal retroviruses.Culture systems using animal-free media, such as the feeder-free environment of the present invention, facilitate the production of clinical grade cell lines, particularly hESC and iPSC cell lines.

[0165] In some embodiments of the invention, the feeder-free environment of the invention is essentially free of human feeder cells, including, but not limited to, human fibroblasts, keratinocytes, and embryonic stem cells, and is not preconditioned with feeder cells. In further embodiments of the invention, the feeder-free environment is essentially free of animal feeder cells, and in some embodiments, is not preconditioned with feeder cells. In particular embodiments of the invention, the feeder-free environment is essentially free of both human and animal feeder cells, and in even more particular embodiments, the feeder-free environment is essentially free of both human and animal feeder cells, and is not preconditioned with feeder cells.

[0166] The feeder-free cell culture medium of the present invention is useful for culturing pluripotent cells, reprogramming cells, and The feeder-free environment is used in the methods of the present invention, including reprogramming, dissociation of pluripotent cells into single cells, cell sorting of pluripotent cells, generation of naive pluripotent cells, and maintenance of the undifferentiated state of cells. In certain methods of the present invention, the feeder-free environment is used in methods for inducing pluripotency, improving the efficiency of reprogramming, and / or increasing or maintaining the potency of cells. In some embodiments, the feeder-free environment is substantially free of cytokines and growth factors, including bFGF.

[0167] dissociation Dissociating cells into single cells, e.g., single cell suspension, can be achieved by enzymatic or mechanical means. Any enzymatic agent known in the art that can dissociate cells into single cells can be used in the method of the present invention. In one embodiment of the present invention, the dissociating agent is selected from trypsin / EDTA, TrypLE-Select, collagenase IV, and dispase.

[0168] Chelating agents, such as EDTA, Accutase, or AccuMax, may also be used alone or in combination with enzymatic agents in dissociating cells according to the methods of the invention. To facilitate dissociation into single cells, the dissociation agent may be dissolved in calcium- and magnesium-free PBS.

[0169] To enhance cell survival during and after dissociation, survival-promoting substances (e.g., growth factors, inhibitors of cell pathways involved in cell death and apoptosis, or conditioned medium) can be added.In some embodiments, cells cultured in conventional medium are dissociated, and single cells are placed in the cell culture of the present invention with one or more small molecule inhibitors, for example, SMC4 medium or SMC4+fibronectin medium.Dissociated single cells can be placed in a feeder-free environment if necessary.In other embodiments, cells can be cultured in a feeder-free environment, then dissociated, and placed in the cell culture of the present invention with one or more small molecule inhibitors, for example, SMC4 medium or SMC4+fibronectin medium, and placed in a feeder-free environment if necessary.

[0170] Enzymatic dissociation into single cells can be supported by mechanical force, or the dissociation agent can be mechanical alone, such as by pulling the cells apart using a mechanical tool such as a pipette or sharp microcapillary.

[0171] Common techniques for cell culture and media collection include Large Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8:148, 1997); Serum-free Media (K. Kitano, Biotechnology 17:73, 1991); Large Reviewed in Scale Mammalian Cell Culture (Curr. Opin. Biotechnol. 2:375, 1991); and Suspension Culture of Mammalian Cells (Birch et al., Bioprocess Technol. 19:251, 1990). Other relevant reading includes Understanding Media (M. McLuhan, Mentor NY, 1964) and The Medium is the Massage (M. McLuhan & Q. Fiore, Bantam NY, 1967).

[0172] For further details of the general techniques useful in the practice of the present invention, the practitioner may refer to standard textbooks and reviews in cell biology, tissue culture, and embryology. , IRL Press Ltd., 1987); "Guide to Techniques in Mouse Development" (PM Wasserman et al., eds., Academic Press, 1993); "Embryonic Stem Cell Differentiation in vitro" (MV Wiles, Meth. Enzymol. vol. 225:900, 1993); and "Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Gene Therapy" (PD Rathjen et al., 1993). Stem cell differentiation is described in Robertson, Meth. Cell Biol. 75:173, 1997; and reviewed in Pedersen, Reprod. Fertil. Dev. 10:31, 1998.

[0173] Enrichment and depletion strategies The present invention also provides a strategy for enriching a cell population for pluripotent cells as a method to increase the efficiency of iPSC generation. The enrichment strategy provides a method to obtain clonal iPSC colonies in a relatively short time and improve the efficiency of iPSC generation. The enrichment method of the present invention includes sorting a cell population induced to be reprogrammed to identify and obtain cells expressing markers of pluripotency, thereby obtaining a cell population enriched for pluripotent cells. The cells to be sorted may have been induced to be reprogrammed and may include a mixed population of cells undergoing reprogramming, such that the population includes pluripotent cells, partially pluripotent cells, and non-pluripotent cells, e.g., fully differentiated cells. In one embodiment, the cell population to be sorted has been induced to be reprogrammed and expresses markers of pluripotency. In some embodiments, the cells are cultured after induction of reprogramming for about 4 to 30 days, about 4 to 24 days, about 6 to 22 days, or about 8 to about 12 days. Further enrichment methodologies involve depleting cells expressing markers of differentiation or non-pluripotency to obtain a population enriched for pluripotent cells.

[0174] The enrichment strategy of the present invention comprises obtaining a single cell suspension of the cell population to be sorted. In one embodiment of the present invention, the single cell suspension is obtained by dissociating the cells in the population and resuspending the cells. The dissociated cells can be resuspended in any suitable solution or medium to maintain the cells or to perform cell sorting. In certain embodiments, the single cell suspension contains one or more of a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor. In certain embodiments, the single cell suspension comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, and in certain embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0175] In the enrichment process of the present invention, cells are sorted to obtain pluripotent cells, or cells are depleted of non-reprogrammed or non-pluripotent cells, thereby obtaining a cell population enriched in pluripotent cells.In one embodiment, single cell suspension is prepared, and then single cells are prepared for sorting, for example, by staining for pluripotency markers, for example, using suitable antibodies.Cells can be sorted by any suitable method of sorting cells, for example, sorting by magnetic beads or flow cytometry (FACS).

[0176] The cells were stained with Oct, Sox, Nanog, SSEA3 / 4; TRA1-60 / 81; TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD105, and CD31. Cells can be selected based on various pluripotency markers, including expression of CD34, OCT4, KLF4, SSEA1 (mouse), and CD30 and CD50 as demonstrated in the present invention. In various embodiments, cells are selected based on at least two, at least three, or at least four pluripotency markers. In an embodiment, cells are selected based on expression of SSEA4, and in a specific embodiment, based on expression of SSEA4 combined with TRA181 or TRA160. In an embodiment, cells are selected based on SSEA4, Tra181 or Tra160 and CD30. In an embodiment, cells are first depleted of non-reprogrammed cells using surface markers of differentiating cells, which may include, but are not limited to, CD13, CD26, CD34, CD45, CD31, CD46, or CD7, and then enriched for pluripotency markers, such as SSEA4, Tra181, and CD30.

[0177] The desired cell fraction after sorting to obtain cells positive for pluripotency markers is a cell population enriched with pluripotent cells. The enriched population of pluripotent cells can 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 can be supplemented with feeder cells or can be a feeder-free environment as needed. In some embodiments, the sorted cells expressing pluripotency markers are placed in a culture system supplemented with feeder cells and then transferred to a feeder-free environment. The cell culture system can be supplemented with one or more of the specific pathway modifiers and additives shown in Table 1. In one embodiment, the cell culture medium is a feeder-free environment and comprises at least one of a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, and in a particular embodiment, the cell culture medium comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, and in an embodiment, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin. In another particular embodiment of the invention, the cell culture system is a feeder-free environment comprising a Matrigel™-coated tissue plate, a conventional hESC medium, and a particular pathway modifier, as shown in Table 1. In one embodiment, the cell culture system comprises the SMC4 medium described in Table 1, optionally in combination with any of the alternative media and pathway modifiers shown in Table 2.

[0178] The enriched cell population can be cultured in the cell culture system described herein to obtain iPSC colonies that typically appear after about 3 to about 25 days of sorting; about 5 to 9 days of sorting, or about 5 to 7 days of sorting. The iPSC colonies can be picked or selected for clonal expansion. Using the enrichment strategy of the present invention, the cell population is enriched 3-fold for pluripotent cells.

[0179] The present invention also provides a method for depleting undesirable cells from a cell population. In some embodiments, differentiated cells are depleted from a cell population, for example, a cell population undergoing reprogramming or a population of pluripotent cells. In the method of the present invention, cells with cell surface markers of differentiated cells can be depleted from a population of pluripotent cells or cells induced to be reprogrammed. The cell population can be sorted based on a surface marker of differentiating cells, for example, CD13, CD26, CD34, CD45, CD31, CD46, or CD7, and cells expressing the markers of differentiating cells can be removed from the cell population to obtain a cell population enriched for pluripotent cells. In a specific embodiment of the present invention, CD13 is used as a surface marker of differentiating cells.

[0180] In other embodiments, a cell population induced to differentiate, e.g., a cell population induced to differentiate into a desired lineage, is depleted of pluripotent cells to obtain differentiated or differentiated cells. In some embodiments, the population of differentiated cells includes a cell population, such as ESCs or iPSCs, that have been induced to differentiate into a particular lineage. The cell population can be depleted of pluripotent cells using the above sorting techniques, for example, sorting the cells in the population based on a marker of pluripotency by magnetic beads or FACs. In some embodiments, the cell population including differentiated cells is sorted by FACs using a pluripotency marker to obtain a fraction depleted of cells expressing the pluripotency marker. In other embodiments, the cell population is sorted by FACs based on a marker of differentiation, such as a lineage-specific marker, for example, CD13, CD26, CD34, CD45, CD31, CD46, or CD7, to obtain a fraction depleted of the marker of pluripotency. In certain embodiments of the invention, CD13 is used as a surface marker for differentiating cells.

[0181] The practice of the present invention employs, unless otherwise indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, 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 for illustrative purposes. Such techniques are fully explained in the literature. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 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, Volumes I and 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 (ed. N. Gait, 1984);Nucleic Acid Hybridization (eds. B. Hames and S. Higgins, 1985);Transcription and Translation (eds. B. Hames and S. Higgins, 1984);Animal Cell Culture (R.Freshney (eds., 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 Sansea. u, microRNA: Biology, Function & Expression (Nuts & Bolts series; DNA Press, 2006); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (eds. J. H. Miller and M. P. Calos, 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, volumes I-IV (DM Weir and CC Blackwell, eds., 1986); Riott, Essential Immunology, 6th ed. (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)(ed. Kurstad Turksen, 2006);Human Embryonic Stem Cell Protocols(Methods in Molecular Biology)(ed. Kursad Turksen, 2006);Mesenchymal Stem Cells: Methods and Protocols(Methods in Molecular Biology)(ed. Darwin J. Prockop, Donald G. Phinney, and Bruce A. Bunnell, 2008);Hematopoietic Stem Cell Protocols(Methods in Molecular Medicine)(ed. Christopher A. Klug and Craig T. Jordan, 2001);Hematopoietic Stem Cell Protocols (Methods in Molecular Biology) (Ed. Kevin D. Bunting, 2008); Neural Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Ed. Leslie P. Weiner, 2008); Hogan et al., Methods of Manipulating See, The Mouse Embryo (2nd ed., 1994); Nagy et al., Methods of Manipulating the Mouse Embryo (3rd ed., 2002); and The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th ed., (Univ. of Oregon Press, Eugene, Oreg., 2000).

[0182] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0183] Throughout this specification, unless the context otherwise requires, the words "comprises" and "includes" are used interchangeably. The words "comprises" and "comprising" are understood to mean the inclusion of a specified 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. "Consisting of" means inclusive and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that no other elements may be present. "Consisting essentially of" means inclusive of any elements recited following the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action set forth in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0184] The above various embodiments can be combined to produce further embodiments.All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referenced herein and / or listed in the data sheets of this application are incorporated herein by reference in their entirety.Aspects of the embodiments can be modified, if necessary, to use the concepts of various patents, applications and publications to produce still further embodiments.

[0185] These and other changes can be made to the embodiments in light of the above detailed description. In general, the terms used in the following claims should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but rather to embrace all possible embodiments, along with the full range of equivalents to which such claims are entitled. Thus, the claims are not limited by this disclosure. EXAMPLES

[0186] Example 1 Experimental Methods A. Cell culture of pluripotent stem cells Before being adapted to feeder-free, human induced pluripotent stem cells (hiPSCs) were maintained on feeder cells (Mitomycin C-treated mouse embryonic fibroblast (MEF) cells (Millipore)) and cultured in conventional media. As used in this application, "conventional media" refers to basic human embryonic stem cell (hESC) medium containing DMEM / F12 (Mediatech), 10 ng / mL bFGF (Invitrogen), 20% v / v Knockout serum replacement (Invitrogen), 1% v / v non-essential amino acids (Mediatech), 2 mM L-glutamine (Mediatech) and 100 μM β-mercaptoethanol (Invitrogen). Conventional media are also listed in the first section of Table 1. hiPSCs were passaged every 5-7 days by mechanically cutting and scraping the colonies into small pieces using a fine-tipped glass pipette (clump passaging), harvesting, and dilution passage 1:3-1:6 onto freshly seeded, mitomycin C-treated MEF cells, with hESC medium added daily. Cell cultures were maintained in a humidified incubator set at 37 °C and 5% CO2. The step of culturing cells in conventional medium with feeder cells and using clump passaging is referred to herein as "conventional culture".

[0187] To dissociate hiPSCs into single cells, they were soaked in phosphate-buffered saline (PBS) (Media Dissociated cells were washed once with 500μM DMSO (Millipore) and treated with Accutase (Millipore) or TrypL (Invitrogen) for 3–5 min at 37°C, followed by disruption into single cells by pipetting. The single-cell suspension was then mixed with an equal volume of conventional medium as described above, spun down at 300g for 5 min, and resuspended in SMC4 medium or SMC4+fibronectin medium. In most cases, dissociated cells were maintained in SMC4 medium, which consists of conventional hESC medium supplemented with various small molecules and additives, including 0.4μM PD0325901, 1μM CHIR99021, 5μM thiazovivin, and 2μM SB431542 (all Biovision). Small molecules were maintained at stock concentrations of 5–25μM in DMSO at -20°C before being added to the medium. All working media were maintained at 4°C for up to 4 weeks. With regard to Rock inhibitors, culturing with thiazovivin was preferred compared to Y27632 for maintaining cells in an undifferentiated state.

[0188] After resuspension in the appropriate medium, cells were transferred to feeder-free tissue culture plates (BD Falcon) previously coated with Matrigel™ (1:25 dilution; BD Biosciences) for 1-2 hours at 37°C. In this format, cells routinely received fresh medium every other day and were passaged when they reached 66-75% confluency, which usually occurred 4-5 days after passaging. At each passage, cells were re-dissociated into single cells and transferred at a dilution passage of 1:5 to 1:10 to new tissue culture plates coated with Matrigel™ (BD Biosciences). For defined, growth factor-free culture, cell suspensions were added to tissue culture plates previously coated with 1% Gelatin (Mediatech). Cells were maintained and passaged as described above, except that SMC4 medium was substantially free of all cytokines and growth factors, including bFGF.

[0189] For freezing, cells were dissociated into single cells, resuspended in SMC4+fibronectin medium supplemented with 10% v / v DMSO (Mediatech), and placed in cryovials (Nalgene). Once capped, the cryovials were placed in Mr. Frosty (Nalgene) and kept at -80°C overnight. The following day, the cryovials were transferred to liquid nitrogen for long-term storage. To thaw, the frozen cryovials were placed in a 37°C water bath for approximately 1-2 minutes until most of the ice had melted. The thawed cell solution was then gently mixed with fresh conventional hESC medium and spun down at 300g for 5 minutes. The cell solution was resuspended in SMC4+fibronectin medium and transferred to tissue culture plates coated with Matrigel™ (BD Biosciences). As with all other cell culture incubations, cells were maintained in a humidified incubator set at 37°C and 5% CO2. B. Induction of Reprogramming To initiate the reprogramming process, ectopic expression of reprogramming factors (human Oct4, Sox2, Klf4, c-Myc, Lin28, and Nanog in various combinations) was achieved using lentiviral transduction or other methods, e.g., protein-only treatment. In most cases, starting cells were cultured at 10% confluency (i.e., 1 × 10 cells per well of a 6-well plate). 5The cells were plated at 1000 x 1000 (1000 x 1000) on a gelatin (Mediatech) coated surface. For the method of viral infection, freshly harvested lentivirus was added to the starting cells at a 1:2 dilution, supplemented with 4 μg / mL polybrene (Millipore) and spin-infected at 650g for 1.5 hours at 32°C. The culture was transferred to 37°C, 5% CO2 for another 7 hours. Once incubation was achieved, the cells were washed 3 times with PBS and fed with fresh medium. As cells, such as IMR90 fibroblasts, are difficult to infect in a feeder-free culture system, this process was repeated once more 48 hours after the first infection. For the use of non-genetic methods of induction of reprogramming, such as applying proteins directly to the cells, a protein mixture, or cocktail, consisting of 8 μg / mL of reprogramming factors was added to the cell solution and maintained for 24 hours. The medium was then changed. This step was repeated 2-4 more times. All starting cells were cultured in their own respective somatic medium until day 4 after the first protein addition, at which point the medium was switched partly to somatic medium and partly to conventional hESC medium. Once confluent (usually 4-6 days), the cells were trypsinized, mixed with an equal volume of medium, spun down at 300g for 5 minutes, resuspended in 1:1 somatic / conventional hESC medium, and expanded 1:4-1:6 into larger culture plates. For example, cells in two wells of a 6-well plate are typically expanded onto a 10 cm dish. The day after expansion, the medium is fully switched to conventional hESC medium. Once the expanded cells reach confluence (usually 8-12 days), the cells are processed for enrichment (see unique population enrichment). In all cases, the medium was routinely changed every other day.

[0190] C. Unique population enrichment Starting cells were induced to be reprogrammed using various strategies, including individual lentiviral constructs or polycistronic vectors containing Oct4 and / or Klf4 and / or Sox2 and / or Myc, and after approximately 8-12 days of culture (see above), the cells were dissociated into single cells (see Cell Culture of Pluripotent Stem Cells) and stained with various surface markers of pluripotency, markers of somatic cells and / or markers of incomplete reprogramming. Briefly, dissociated cells were resuspended in staining solution containing Hanks' Balanced Salt Solution (Invitrogen), 4% fetal bovine serum (Invitrogen) and 10 mM Hepes (Invitrogen) and kept on ice. Conjugated primary antibodies were added to the cell solution according to the manufacturer's recommended dilution, and the solution was incubated on ice for 15 minutes. The cell solution was washed, resuspended in staining buffer and kept on ice. At this point, various enrichment / depletion strategies were undertaken, including Fluorescent Activated Cell Sorting (BD Biosciences, see below) and Magnetic Cell Sorting (Miltenyi Biotec, see below).

[0191] Flow cytometric sorting was performed on a FACS Aria (BD Biosciences). Primary antibodies used included SSEA4 (BD Biosciences), Tra-181 (Biosciences), Tra-161 (BD Biosciences), CD30 (BD Biosciences), and CD50 (BD Biosciences) as specified. Sorted cells were then spun down, resuspended in SMC4+fibronectin medium, and transferred to Matrigel™-coated tissue culture plates. When sorted into microwells, i.e., 96-well plates, plates were spun down at 300 g for 2 min. SMC4+fibronectin medium was replaced every other day for 3-4 days. After 3-4 days, typically, SMC4+fibronectin medium was replaced with SMC4 medium for the remainder of the culture. Colony formation was typically observed 7-9 days after sorting. Flow cytometric analysis was performed on a Guava EasyCyte 8HT (Millipore).

[0192] MACS Microbead (Miltenyi Biotec) separation was performed according to the protocol. Briefly, cells were dissociated into single cells (see Cell Culture of Pluripotent Stem Cells) and stained with appropriate FITC-conjugated primary antibodies, including SSEA4 (BD Biosciences), Tra-1-81 (BD Biosciences), Tra-160 (BD Biosciences), CD30 (BD Biosciences), and CD50 (BD Biosciences), as specified. Cells were then magnetically labeled with Anti-FITC Microbeads (Miltenyi Biotec). The labeled cell suspension was then loaded onto a LS MACS Column (Miltenyi Biotec). Cells from either the positively or negatively selected fractions were spun down at 300g for 5 min, resuspended in SMC4+fibronectin medium, and transferred to tissue culture plates coated with Matrigel™ (BD Biosciences). Fresh medium was added to the cultures the next day and changed every other day thereafter. After 3-4 days, typically the SMC4+fibronectin medium was replaced with SMC4 medium for the remainder of the culture. Colonies typically appeared after 5-7 days of sorting.

[0193] D. Alkaline phosphatase staining Cells were fixed in 4% v / v paraformaldehyde (Alfa Aesar) for 30 seconds, washed three times with PBS, and stained with Alkaline Phosphatase Staining Kit (Sigma-Aldrich). Briefly, 1 mL of Sodium Nitrite Solution was added to 1 mL of FRV-Alkaline Solution, mixed, and incubated at 25°C for 2 minutes. This solution was then mixed with 45 mL of H2O, after which 1 mL of Naphthol AS-BI Alkaline Solution was added. The alkaline dye mixture was added to the fixed cells and incubated at 25°C for 15 minutes, followed by a PBS wash. Cells were then scored for the presence of alkaline phosphatase.

[0194] E. Immunofluorescence staining Cells were fixed using 4% v / v paraformaldehyde (Alfa Aesar) for 15 min, washed three times with PBS containing 0.2% v / v Tween (PBST) (Fisher Scientific) and permeabilized with 0.15% v / v TritonX-100 (Sigma-Aldrich) in PBS for 1 h at 25° C. After permeabilization, cells were blocked with 1% v / v BSA (Invitrogen) in PBST (Fisher Scientific) (PBSTB) for 30 min at 25° C. After gentle removal of PBSTB, cells were incubated with primary antibodies in PBSTB overnight at 4° C. Primary antibodies used in this study included Nanog (Abcam), Tra-1-60 (BD Biosciences), Tra-181 (BD Biosciences), SSEA4 (BD Biosciences), β-III Tubulin (R&D Systems), α-Smooth Muscle Actin (Sigma) and Sox17 (R&D Systems). After overnight incubation, cells were washed three times with PBST and stained with secondary antibodies (Alexa 488 or 555; Invitrogen) diluted 1:200 in PBSTB for 1 h at 25°C. Cells were washed three times in PBST and stained with Hoechst dye (Invitrogen). Images of stained cells were captured using fluorescence microscopy and a CCD camera.

[0195] F. Induction of Differentiation and Teratoma Formation Feeder-free iPSCs were differentiated both as monolayers and as embryoid bodies. For monolayer differentiation, iPSCs were allowed to reach near confluence before switching to differentiation medium, as cells normally reduce their proliferation upon differentiation. Briefly, once confluent, SMC4 medium was switched to differentiation medium containing DMEM / F12 (Mediatech), 20% fetal bovine serum (Invitrogen), 1% non-essential amino acids (Mediatech), 2 mM L-glutamine (Mediatech) and 100 μM β-mercaptoethanol. Once the medium was switched, iPSCs were allowed to differentiate for 14 days. Medium was changed every 2-3 days. For embryoid body ("EB") formation and differentiation, hiPSCs were dissociated into single cells using Accutase (Millipore) and resuspended in differentiation medium to a final concentration of 75,000 cells per mL and 5 uM thiazovivin (Thi azovivan) was added. Cells were seeded at 100 μL per well in V-bottom 96-well non-tissue culture plates (Nunc) and centrifuged at 950 g for 5 min. The next day, dense "sphere-like aggregates" were transferred to ultra-low binding 6-well plates (Corning) with a P1000 at approximately 30-40 EBs per well. After 7 days, the transferred EBs were transferred 1:1 to Matrigel-coated 6-well plates. After 3 weeks of culture, cells were fixed and stained.

[0196] Teratoma implantation and analysis were performed by Applied Stem Cells (Menlo Park, CA). Briefly, 1–2 million dissociated hiPSCs were mixed in medium supplemented with 100 microliters (uL) of SMC4 medium and 100uL of Matrigel and introduced into the kidney capsule and testis of Beige SCID mice. The resulting teratomas were harvested, sectioned, and analyzed for various differentiated cell types and structures.

[0197] G. RT-qPCR and qPCR Analysis RNA was isolated using a PicoPure RNA Isolation kit (MDS Analytical Technologies) and 0.5 μg of RNA was used to generate first strand cDNA using an iScript cDNA Synthesis Kit (Bio-Rad). Relative gene expression levels were determined using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and the FAM-labeled TaqMan probes listed below in Table 3.

[0198] Table 3: ABI primers and probes

[0199] [Table 3-1]

[0200] [Table 3-2] H. Gene Expression Analysis Total RNA was isolated from cells using the Pico Pure RNA Isolation Kit (Molecular Devices, Sunnyvale, CA). Briefly, biotinylated aRNA was prepared using the standard protocol for the MessageAmp II aRNA Amplification Kit (Applied Biosystems / Ambion, Austin, TX) with optional Second Round Amplification, and then purified using the Messa The geAmp II Biotin Enhanced Kit (Applied Biosystems / Ambion, Austin, TX) was used to transcribe biotinylated aRNA using standard protocols. Biotinylated aRNA was purified and fragmented as recommended by Affymetrix. 20 μg of fragmented aRNA was used to hybridize to Human Genome U133 Plus2.0 chips (Affymetrix Inc. Santa Clara, CA) for 16 hours at 45°C. Arrays were washed, stained on an Affymetrix Fluidics Station 450, and scanned using an Affymetrix GeneChip Scanner 3000 7G. Image data was analyzed using the Affymetrix Expression Console software with default analysis settings. Arrays were normalized by log scale robust multi-array analysis (RMA) and visualized in Spotfire for Genomics 3.1 (Tibco Spotfire, Palo Alto, CA).

[0201] I. Karyotyping and copy number variation analysis Cytogenetic analysis on 20 G-banded metaphase cells was performed by Cell Line Genetics, Madison, WI.

[0202] High-resolution comparative genomic hybridization (NimbleGen 12x135k) and subsequent copy number variation analysis was performed by WiCell (Madison, WI).

[0203] Example 2 Cell culture conditions and methods to enable a feeder-free culture environment and enzymatic dissociation and passaging of pluripotent stem cells into single cells - Patent Application 20070233334 This example relates to the culture and dissociation of pluripotent cell populations. Such cell populations include, but are not limited to, embryonic stem cells (ESCs) and induced pluripotent cells, such as those generated by somatic cell nuclear transfer (SCNT) or by introducing pluripotency factors - induced pluripotent stem cells (iPSCs). Culture conditions for pluripotent stem cells have traditionally included the use of feeder cells, which are mitotically inactive by irradiation or mitomycin C treatment, but provide the growth factors and nutrients necessary to support the culture of stem cells. Culturing stem cell populations without the use of feeder cells would be advantageous for research and industrial applications where a homogenous population of stem cells is required, or where expanded industrial activities require xeno-free defined culture conditions for stem cell products. In this example, several small molecule modulators of specific cell signaling pathways were tested to establish whether individual small molecules or combinations of small molecules ("cocktails") could be used to enhance the culture of pluripotent cells in feeder-free systems.

[0204] When pluripotent cell populations were cultured without feeders but instead with more defined extracellular matrix, such as Matrigel™, in conventional hESC cell culture media (e.g., conventional / basal media formulations described in the first section of Table 1), cell viability and pluripotency were not supported (Figure 1). However, the use of small molecule inhibitors of Rock kinase improved cell viability under these conditions. Furthermore, the use of small molecule inhibitors of MAP kinase, TGFβ and Wnt / β-catenin pathways maintained cell pluripotency in the absence of feeders, but changes in cell viability could be observed. The combination of Rock kinase inhibitors with MEK inhibitors, TGFβ inhibitors and GSK3 inhibitors maintained both viability and pluripotency of pluripotent stem cells when cultured in a feeder-free environment.

[0205] Pluripotent cells, such as ESCs or iPSCs, generally grow as clumps. Traditionally, these cells have been expanded and passaged by manually picking colonies that have morphology recognized by researchers in the art. Such a procedure is described in Example 1 of this document (described as clump passage). The picked colonies are then mechanically broken and dissociated cells are re-plated. For rapid expansion of pluripotent cell populations, the use of enzymatic single-cell passaging is effective. Enzymes such as trypsin and accutase are commonly used to dissociate cells into single cells during passaging.

[0206] In a particular demonstration, iPSC cells, when enzymatically passaged and seeded as single cells in a feeder-free environment, showed a significant drop in viability, as can be seen by the incorporation of 7AAD in FIG. 3B. By using the culture platform of the present invention, including media compositions such as those listed in Table 1, pluripotent cell populations were cultured in feeder-free culture and enzymatically dissociated for single cell passaging, with much improved maintenance of cell viability and pluripotency. More specifically, by using a combination of conventional / basal hESC media formulations and modulators of cell signaling pathways such as MAP kinase pathway, TGFβ pathway, Wnt / β-catenin pathway and Rho / Rock pathway, the viability and pluripotency of pluripotent stem cells were maintained during applications requiring dissociation into single cells and feeder-free culture.

[0207] As shown in Figure 2, iPSCs generated using clump passaging in feeder cell culture were dissociated into single cells using enzymes such as Accutase and plated as single cells in a feeder-free system, for example on Matrigel™, using the SMC4 medium composition or SMC4+fibronectin medium composition described in Table 1. Cell viability was maintained only when the dissociated cells were placed in media containing the combination of small molecules described in these media. By using conventional media, a large amount of cells were lost when pluripotent cells were enzymatically dissociated into single cells and plated in a feeder-free environment. Once the cells were adapted to feeder-free culture and single-cell passaging, they were continuously maintained in this manner using SMC4 medium. A complete characterization of hiPSC cells adapted to feeder-free, single-cell passaging conditions is shown in Figure 3. The pluripotent state of these cells was maintained after multiple passaging: pluripotency markers were identified by immunofluorescence and gene expression. Furthermore, approximately 99.8% of the cells maintained SSEA4 and Tra1-81 positive staining as measured by flow cytometry. The gene expression profile of these cells was similar to human ESCs cultured on feeders. Transgene silencing of hiPSCs was maintained, and the karyotype was normal and identical to the same clones grown in feeder cell culture. Furthermore, hiPSCs passaged and maintained in this manner differentiated into the three germ layers, as demonstrated by both in vitro differentiation and teratoma formation (Figures 3I and 3J).

[0208] Example 3 Methods and culture conditions for enabling single cell sorting from pluripotent cells while maintaining pluripotency and cell viability As described in Example 2, stem cell cultures of ESCs or iPSCs are routinely cultured on feeder cells and passaged by manually selecting cell colonies, which are then mechanically dissociated and then replated. A skilled researcher can recognize stem cell colonies with pluripotent, non-differentiated properties based on colony morphology, which can be used as a method to select pluripotent cells. Thus, pluripotent populations or populations with desired properties can be manually selected and enriched from cell populations where some cells have less desirable properties, e.g., cells showing signs of differentiation in cultures or clumps of dead cells. This process is laborious and relies on a skilled researcher to select the desired cell population. Therefore, the use of cell enrichment or sorting techniques where cells are individually selected based on desired properties would be of great benefit to the field. Such enrichment steps using currently available techniques, e.g., magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS), require the enzymatic passaging of pluripotent cell populations into a single-cell format prior to enrichment and seeding in culture. Furthermore, the use of feeder-supported cultures would be less desirable for these techniques, necessitating the use of feeder-free culture systems.

[0209] In certain embodiments, using the media compositions of the invention described in Table 1 and the methods described in Examples 1 and 2, pluripotent cell populations were dissociated into single cells, enriched using Magnetic Activated Cell Sorting (MACS) or Fluorescence Activated Cell Sorting (FACS), and plated onto feeder-free cultures without loss of pluripotency or cell viability.

[0210] In a particular example, as shown in FIG. 4, a population of dissociated pluripotent cells maintained in SMC4 medium (Table 1) was selected and cells positive for the cell surface marker SSEA4 were identified by FACS. + / Tra181 +These surface antigens are commonly used as markers for pluripotency. The sorted double positive populations were then transferred to feeder-free cultures (Matrigel™ ECM coated) and grown in either SMC4 medium (Table 1) or SMC4 + fibronectin medium for 2-4 days before being switched back to SMC4 medium (Table 1). Cell division was observed within 24 hours of sorting, and cells were nearly confluent by 5 days after sorting. The sorted cells were cultured in single cell culture for an additional 5 passages, and the SSEA4 + / Tra181 + As judged by dual staining flow cytometry, the cells were shown to not only express markers of an undifferentiated state, but also to display a substantially pure population of pluripotent cells (Figures 4B and 4C).

[0211] In an example of enriching and selecting pluripotent stem cells during culture, a culture containing both pluripotent and differentiated cells is enriched for cells positive for Tra181 and then cultured using a 50% ... + Cells continued to be passaged to maintain a pure population of pluripotent stem cells (Figure 17C).

[0212] The efficiency of single-cell sorting of pluripotent cells was investigated. As can be seen in Figure 4D and E, various numbers of post-sorting events measured by FACS instrument were plated in a feeder-free culture system using either SMC4 medium or SMC4+fibronectin medium and the number of alkaline phosphatase positive cells was scored. Alkaline phosphatase is an early indicator of pluripotent colony formation. The quantified post-sorting seeding efficiency, indicated by the number of alkaline positive colonies per number of cells plated, was found to be approximately 8-10% in this system (Figure 4E). In a further demonstration of the potential of single-cell sorting in maintaining pluripotent cell populations, iPSC cultures were dissociated into single cells, labeled with fluorescently conjugated antibodies specific for the pluripotency markers SSEA4 and Tra181, and applied to FACS and selected based on these markers. SSEA4 was selected. + / Tra181 +Cells were plated directly from the FACS machine into 96-well plates at 1-9 events per well. As few as one event per well gave rise to a clonal alkaline phosphatase-positive colony (Figures 4F and 4G). Thus, such a single-cell sorting system was used to perform clonal selection of pluripotent cells based on favorable properties associated with specific cell surface markers.

[0213] Example 4 Methods and conditions for efficiently reprogramming cells to a pluripotent state in a feeder-free culture system The use of iPSCs for industrial and / or clinical applications requires fully defined It is necessary to generate, select and maintain cells in culture conditions, particularly in xeno-free conditions.Therefore, it is highly desirable to reprogram cells in feeder-free culture conditions.However, although fibroblasts and keratinocytes are the most commonly used cell types for reprogramming, as they are obtained through skin biopsy or hair follicles, the efficiency of reprogramming these cell types is very low, and an efficient method for reprogramming these cells in feeder-free culture has not yet been demonstrated.

[0214] As described in Example 2, the use of conventional hESC stem cell medium in the presence of inhibitors of cell signaling pathways, specifically MAP kinase pathway, TGFβ pathway, Wnt / β-catenin pathway and Rho / Rock pathway, allowed for the growth and maintenance of pluripotent cultures in the absence of feeder cells and for these cultures to be expanded using enzymatic single cell passaging. In this example, iPSC cells were generated in a culture system lacking feeder cells. Specifically, human fibroblasts were infected with viruses expressing the pluripotency factors Oct4, KLF4, Sox2 and C-myc. The reprogramming protocol was performed as described in Example 1, using cells plated on Matrigel™ rather than feeder cells.

[0215] Comparisons of reprogramming of feeder-free cells were performed using either conventional hESC stem cell medium or conventional hESC medium supplemented with specific pathway modifiers listed in SMC4 medium (Table 1). As can be seen in Figure 5, 20 days after inducing reprogramming with individual lentiviruses expressing Oct4, Klf4, Sox2 and Myc, many iPSC-like colonies were observed in feeder-free cultures supplemented with SMC4 medium, whereas few or no colonies were observed in cultures maintained in conventional hESC medium alone. Subsequent characterization of colonies generated in SMC4 medium showed that many of them were true iPSC colonies. These cells were determined to be pluripotent by expression of pluripotency markers Oct4, Nanog, Sox2, KLF4, SSEA4 and TRA 1-81 using immunofluorescence, flow cytometry and gene expression profiling (Figure 13C-E). Furthermore, the cells were shown to differentiate effectively into all three germ layers when cultured in differentiation medium. Thus, the efficient generation of iPSCs in a feeder-free culture system has been demonstrated by the present invention. The efficiency of this approach compared to other reprogramming methods is described in Example 8 and Table 4.

[0216] Example 5 Cell culture compositions for use in generating and maintaining naïve pluripotent cells - Patents.com Recent studies have demonstrated that through epigenetic reprogramming, terminally differentiated cells have the ability to recourse back to a precursor-like state (Xie, H., Ye, M., Feng, R., and Graf, T. 2004), a different differentiated cell state (Szabo, Bhatia 2010), or even the original embryonic-like state (Takahashi et al., 2006), e.g., iPSCs. Although the generation of iPSCs has become more routine, in a given experiment, only a very low percentage of somatic cells are reprogrammed into iPSCs. This low efficiency is attributed to several parameters, including the proliferative state of the somatic cells, further mutagenesis leading to gene activation or repression, the mode of gene delivery, and environmental factors. It has also been reported that not all cells identified as iPSCs behave similarly to ESCs. For example, gene expression profiling has demonstrated that many iPSCs exhibit significant differences in expression profiles relative to their ESC counterparts. Furthermore, testing for Xist activity and analysis of X chromosome reactivation demonstrated that while some ESCs were in a naïve state (i.e., a ground state of pluripotency), most, but not all, of the resulting iPSCs were in a primed state (i.e., primed to differentiate). Taken together, these differences may contribute to the reduced pluripotency and inefficient differentiation of iPSCs into specific cell types, reducing their value in regenerative medicine.

[0217] By targeting key cellular pathways involved in the mechanisms behind the naive and primed states, we have demonstrated the ability to transform pluripotent stem cells present in a primed state, including conventional iPSCs, into a naive state. Using the medium compositions listed in Table 1, it was possible to further reprogram such primed pluripotent cells and conventional iPSCs into a naive state. More specifically, by reprogramming somatic cells or culturing iPSCs in a medium containing modulators of cell signaling pathways, such as the MAP kinase pathway, the TGFβ pathway, and / or the Wnt / β-catenin pathway, the gene expression signature of the generated or cultured iPSCs becomes more ESC-like than conventional iPSCs (iPSCs generated and / or cultured in conventional medium and not contacted with modulators of cell signaling pathways). As demonstrated in hierarchical clustering, hiPSCs obtained in conventional medium and SMC4 medium were similar to each other and both were different from their parental lineage, IMR90 (Figure 6B). However, when iPSCs cultured in SMC4 medium were plated back onto feeder cells, they more closely resembled mouse ESCs than conventionally cultured iPSCs, another demonstration of a naïve state (Figure 6E). Furthermore, iPSCs cultured in SMC4 medium had significantly reduced Xist activity and enhanced expression of genes on the X chromosome (Figures 6C and 6D). Finally, iPSCs cultured in SMC4 medium not only differentiated into all three germ layers, but also demonstrated the ability to reactivate genes associated with extraembryonic cells, a capacity that only cells in a naïve state possess (Figure 3H). Thus, culturing pluripotent stem cells, including iPSCs existing in a primed state, in SMC4 medium promoted the naïve state and enhanced their differentiation potential.

[0218] To determine the pluripotent status of hiPSC clones generated using SMC4-supplemented medium and cell sorting platform (Figure 7), hiPSCs were derived from the same starting fibroblast cell line and a three-factor polycistronic vector expressing Oct4, Klf4 and Myc, but maintained under conventional culture including clump passaging and feeder cells (FTi99). Affymetrix global gene expression analysis of the different clones derived from hESCs as well as mRNA was performed (Figure 7B). It was found that all pluripotent lines had a different profile from the control fibroblast cell line (FTC1, Pearson score 0.886) and essentially similar expression profiles to each other (Pearson score 0.969). A heatmap signature was created to show 1,739 probes that were differentially expressed 4-fold between the hESC / FTi99 (hESCs or hiPSCs generated and maintained in feeder cells and conventional medium) and FTi91 / FTi93 (hiPSCs generated and maintained in feeder-free and SMC4 medium) groups (Figure 7C). A deep analysis of genes within the 1,739 differentially expressed transcripts identified an interesting trend: several pluripotency genes, generally associated with a naive state, were upregulated in the FTi91 / FTi93 group, but more significantly, within this group, many differentiation genes associated with a primed state were repressed (Figure 7D). Our comprehensive analysis of hiPSCs generated in SMC4 medium suggests that culture conditions play a more influential role in determining the undifferentiated state compared to the starting cell line or derivation strategy. The data also suggest that clones obtained in or adapted to SMC4 culture conditions demonstrate favorable qualities, e.g., naïve characteristics with a highly undifferentiated state accompanied by reduced expression of early lineage markers (Figure 7E).

[0219] Example 6: Identification of antibody cocktails to identify authentic hiPSCs Pluripotent stem cell surface markers were investigated. In addition to the expression of SSEA4 and Tra181, the expression of CD30 and CD50 was also identified and considered to represent additional surface markers of pluripotency (Figure 10). Cells reprogrammed with polycistronic lentivirus expressing Oct4, Klf4 and Sox2 show various states of competence and are identified with a few cells carrying the expression of true pluripotent markers, such as Nanog. To date, there is no reliable method to identify truly pluripotent hiPSCs based on the expression of surface markers. For example, as can be seen in Figure 11, some clonal populations identified as positive for SSEA4, Tra181 and CD9 do not express Nanog, resulting in incorrect identification as hiPSCs.

[0220] The present invention provides a combination of cell surface markers that identify a population of cells expressing Nanog, a marker of truly pluripotent cells. In particular, cells expressing Nanog are identified by cells positive for the following surface markers: CD30, SSEA4 and Tra181 (Figure 11).

[0221] In another example of additional enrichment, the cell population undergoing reprogramming was sorted to identify cells positive for the expression of CD13 surface marker, and these CD13+ cells were removed from the reprogrammed cell population. The CD13+ population correlated with somatic cells and non-reprogrammed cells, and depleting CD13+ cells from the reprogrammed cell population enhanced the enrichment of SSEA4 / Tra181 positive cells (Figure 12). As demonstrated in Figure 12, when somatic cells were reprogrammed for 21 days with polycistronic vectors expressing Oct4, Klf4 and Sox2, various cell populations with diverse surface marker expression patterns were generated, and only a small subset of cells showed SSEA4 and Tra181 positive cells (dotted arrows, Figure 12). However, when the same populations were first assessed based on CD13 expression (solid arrows, FIG. 12), the population subset negative for CD13 (i.e., depleted of CD13 cells) showed a significantly enriched population for cells expressing SSEA4 and Tra181 (11.04%, FIG. 12). Conversely, the subset of cells that expressed high levels of CD13 showed few cells that expressed both SSEA4 and Tra181 (1.14%, FIG. 12).

[0222] Example 7 Use of single cell sorting in the generation of induced pluripotent stem cells from differentiated cells As can be seen in FIG. 8A, fibroblast cultures early in the reprogramming process induced with individual lentiviruses expressing Oct4, Klf4, Sox2, and myc contained colonies of morphologically distinct cells. Some of these cells stained positive for pluripotency markers, while others were simply transformed, fast-growing cells. At this stage of the reprogramming process, cell morphology alone does not reveal which cell colonies will go on to form iPSCs. Faster growing, transformed but not pluripotent cells quickly take over the culture. It would therefore be advantageous to have an enrichment step to select for iPSCs early in the reprogramming process. Furthermore, as shown in FIG. 8, some colonies expressed some pluripotency markers during the reprogramming process and were true iPSCs, while some colonies were not fully reprogrammed and expressed only some pluripotency markers. Colony 1 in FIG. 8B was positive for both SSEA4 and Tra181, while colonies 4 and 5 were only positive for one or the other marker. Therefore, it may be more efficient and less technically challenging to select pluripotent cells using cell surface markers of pluripotency or several markers simultaneously, rather than by colony morphology.

[0223] The cell culture compositions described herein can also be used for enriching and culturing the cells provided herein. When used in combination with the in vitro and / or in vivo sorting methodologies, it was possible to obtain larger numbers of iPSCs in a shorter period of time by selecting individual cells that displayed surface markers of pluripotency during the reprogramming process. In more detail, as shown diagrammatically in Figures 9A and B, two routes for generating iPSCs using single cell sorting and enrichment based on markers of pluripotency during reprogramming were implemented using the methods of the present invention.

[0224] In Pathway A, after initiation of reprogramming, a mixed population of cells in various states of competence was generated. The mixed population of cells contained differentiated cells, partially reprogrammed cells, reprogrammed cells, and cells undergoing reprogramming. Using methods such as magnetic bead sorting or flow cytometry sorting (see Example 1 for methodology), the cell population was enriched for cells that expressed pluripotency markers, e.g., SSEA4. Once enriched, the cells were cultured in SMC4 medium (Table 1), or, in certain embodiments, SMC4+fibronectin medium for approximately 3 days, then replaced with SMC4 medium, and after a culture period of approximately 6-10 days, iPSC colonies were identified based on live culture staining of markers, e.g., SSEA4 and TRA181, and picked or selected for clonal expansion (Figure 9).

[0225] Under the schematic of Pathway B (Figure 9), early after the initiation of reprogramming, a mixed population of cells was sorted to obtain a rare population of cells positive for two or more pluripotency markers. Such markers include, but are not limited to, SSEA4 and TRA181. The selected cells expressing the combination of pluripotency markers were transferred to a culture system supplemented with feeder cells or a feeder-free culture system, specifically, a culture system supplemented with the cell culture medium composition listed in Table 1. In this way, iPSC colonies were generated with significantly reduced timelines and technical barriers compared to the methodology described above.

[0226] In a specific demonstration of this technique, IMR90 fibroblasts were infected with lentivirus expressing Oct4 and Sox2 and Klf4 and c-Myc (OSKM). After several days of feeder-free culture, the reprogrammed cells were switched from their somatic medium to feeder-free culture supplemented with SMC4 medium (Table 1). Eight days after the start of reprogramming, flow cytometry analysis showed that the infected cell population contained a modest subpopulation of cells that expressed the pluripotency marker SSEA4 (Figure 13A). Using magnetic activated cell sorting as described in Example 1, the pluripotent population was enriched three-fold for cells expressing SSEA4 (Figure 13A). After the cells were enriched for cells expressing SSEA4, the sorted cells were transferred to feeder-free culture containing Matrigel™ and supplemented with either conventional hESC medium or SMC4 medium, or in a specific embodiment, SMC4+fibronectin medium. Alkaline phosphatase staining of the cultures showed that the use of small molecule inhibitors of MEK, GSK3, Rock kinase and TGFβ supported enrichment of pluripotent cells using single-cell sorting, but not conventional basal medium (Figure 13A).

[0227] Quantification of three independent experiments clearly demonstrated the selection of pluripotent cells by single-cell sorting only in the presence of small molecule inhibitors (Figure 13B). Colonies derived from such enriched cells were further cultured in SMC4 medium and characterized as true iPSCs: they stained positive for pluripotency markers SSEA4 and Tra181 in immunofluorescence and flow cytometry (Figure 13C and D); showed a gene expression profile similar to human ESCs (Figure 13E); showed significant silencing of exogenous transgenes (Figure 13F) and were able to differentiate into all three germ layers (Figure 13G). Using the same protocol but under conventional culture conditions (containing no feeder cells), iPSCs were cultured in SMC4 medium and characterized as true iPSCs: they stained positive for pluripotency markers SSEA4 and Tra181 in immunofluorescence and flow cytometry (Figure 13C and D); showed a gene expression profile similar to human ESCs (Figure 13E); showed significant silencing of exogenous transgenes (Figure 13F) and were able to differentiate into all three germ layers (Figure 13G). Pluripotent cell colonies were rarely observed when single-cell sorting was used (consisting of conventional medium lacking SMC4). Thus, the ability to robustly enrich for pluripotent cell populations based on cell surface markers of pluripotency using single-cell sorting during cell reprogramming was dependent on the use of cell signaling pathway inhibitors and additives listed in Table 1. Furthermore, improved post-sort seeding was observed when fibronectin was used as an additive in the culture system for 3 days. This reprogramming process was completed for other starting cell types as well, including human adipose-derived stem cells.

[0228] In a further example of the technique, FACS was used to enrich pluripotent cell populations from a mixture of non-reprogrammed, partially reprogrammed and fully reprogrammed cells. As in the previous example, IMR90 fibroblasts were infected with lentivirus expressing Oct4 and Sox2 and Klf4 / c and Myc (OSKM). A few days after infecting the fibroblasts, the cell culture was switched to feeder-free culture in SMC4 medium. The infected cell population contained a modest population of cells positive for both pluripotency markers SSEA4 and Tra181 (Figure 14A). Cells positive for both pluripotency markers were selected and sorted away from cells negative for both markers or positive for only one marker. Comparison of subsequent feeder-free cultures using medium supplemented as described in Table 1 and, in some embodiments, medium supplemented as described in Table 1 containing fibronectin, showed that cultures from cells positive for both markers formed colonies that were subsequently characterized as pluripotent iPSCs, while cells gated as negative for pluripotency markers by FACS did not produce alkaline phosphatase positive colonies (Figure 14A, B). More specifically, inhibitors of signaling molecules MEK, GSK, Rock and TGFβ as additives to the basal medium formulation facilitated the use of FACS in sorting and enrichment of pluripotent cells early in the reprogramming process. Furthermore, improved post-sort seeding was observed when fibronectin was used as an additive in the culture system for 3 days.

[0229] Next, we developed a high-throughput method to generate feeder-free and cloning-derived hiPSCs using the advantages of SMC4 medium and culture system. The scheme was devised to treat cells induced to be reprogrammed with SMC4 medium and select rare individual cells that were faithfully reprogrammed, as indicated by a combination of pluripotency markers. Furthermore, we combined the reprogramming process with a multiplex platform to effectively select the top clones based on dual-marker flow cytometry, qRTPCR and immunofluorescence selection assays (Figure 15).

[0230] In our optimized multiplex protocol, we use the three-factor (OKS) polycistronic virus to initiate reprogramming and SSEA4 + / Tra181 + An initial bulk FACS sort of the population was completed 20 days post-infection, followed by SSEA4+ / -100 cells / ml in 96-well plates on day 30. + / Tra181 + Cells were FACS re-sorted (Figure 15 and B). This strategy allowed obtaining multiple clones, generating FTC1 clones 1 and 2, clones that expressed pluripotency markers and showed attenuation of exogenous gene activity and demethylation of the Oct4 promoter (Figure 15A-C). FTC1 clones 1 and 2 were also shown to be pluripotent by differentiation into three somatic lineages in vitro and in vivo (Figure 15D, E). Selected clones were also assessed for chromosomal integrity, demonstrating that copy number variation from their parental lineages and normal karyotype was minimal even after 20 consecutive single-cell passages in FF medium, a notable improvement over other reprogramming strategies (Figure 17).

[0231] To determine the reproducibility of the platform, additional fibroblast cell lines, FTC5 and FTC7, were induced to be reprogrammed with a polycistronic vector expressing Oct4, Klf4 and Sox2 and applied to the high-throughput platform described in FIG. 15. As described in FIG. 18, colonies derived from FTC5 and FTC7 fibroblast cell lines were shown to maintain their undifferentiated state and retain their pluripotency and genomic stability. Thus, the combination of three pluripotency factors, SMC4 medium and a multiplex characterization platform significantly enhances the kinetics of feeder-free reprogramming, while allowing for the identification, selection and expansion of clonally derived and genomically stable hiPSCs in a high-throughput manner.

[0232] Example 8 Methods and culture compositions for the rapid generation of multiple iPSC clones Generating human iPSCs by ectopic expression of pluripotency genes, e.g., Oct4, Sox2, Klf4, c-myc, Lin28 and Nanog, is an inefficient and technically challenging process. Strategies resulting in lentiviral or retroviral integration of pluripotency factor transgenes into the genome of host cells in combination with a culture system containing feeder cell support have traditionally been the most efficient methods for generating iPSCs. A literature review of historical studies using viral and feeder cell methodologies to generate human iPSCs has shown that infected cells become iPSCs with an efficiency of 0.001%-0.01%, with potentially pluripotent cells being observed in a period of 21-30 days post-infection, and these are clonally obtained by manual "clump" passaging between 30-45 days post-infection (Table 4).

[0233] Other methods for introducing pluripotency genes include episomal vector systems and transduction of modified proteins. Such methods are considered to be important developments toward the eventual clinical application of iPSC technology. However, these methodologies are even less efficient than reprogramming using viral systems. Furthermore, even when feeder-free systems are used in combination with traditional stem cell media formulations, the efficiency of the reprogramming process is reduced or impossible in some conditions, which has hindered the development of iPSCs for industrial and therapeutic use. Somatic cell reprogramming has been characterized as a stochastic process, with the majority of cells eventually reprogramming over time. However, a robust, technically easy, efficient, and scalable method for generating multiple iPSC clones in a single reprogramming has not yet been described.

[0234] The present invention provides cell culture conditions and methodology for obtaining clonal iPSC colonies in a relatively short time and with lower technical barriers than current methods.In particular, as can be seen in Figure 13B, by using small molecule inhibitors of specific signaling pathways as additives in SMC4 medium, it is possible to generate iPSC colonies in the environment of feeder-free culture with efficiency far exceeding that when SMC4 medium is not used.Inhibitors of MEK, GSK, Rock and TGFβ signaling pathways are used to enable efficient reprogramming in feeder-free environment.

[0235] As can be seen from Figure 13 and Table 4, three independent reprogramming experiments showed that while no or one colony was observed when using conventional hESC medium in a feeder-free environment during the reprogramming process, small molecule culture additives (i.e., SMC4 medium) enhanced the feeder-free reprogramming event to an efficiency of 0.035%, resulting in colonies 14-21 days after infection and clonal iPSCs by 28-35 days. Thus, small molecule inhibitors The use of increased efficiency of reprogramming in terms of time to reprogramming and percentage of reprogrammed cells. Colonies generated using this approach were characterized as pluripotent using standard procedures such as immunofluorescence and gene expression profiling.

[0236] In further demonstration of this technique, differentiated cells were infected with viruses expressing individual pluripotency genes Oct4, Klf4, Sox2, and Myc and cultured in SMC4 medium and feeder-free culture environment (Table 1) for 8-12 days. At this point, and as described in Example 6 and Figures 13 and 14, cells were enriched using FACS or MACS single cell sorting to obtain a small population of pluripotent cells defined by positive staining for pluripotency markers, e.g., SSEA4 and Tra181. Using this approach, the timeline for generating iPSCs was significantly reduced, demonstrating an efficiency of 0.22% in terms of percent of reprogrammed cells, with colonies appearing within days (10-16 days) shortly after sorting. This efficiency of reprogramming allowed for clonal iPSCs to be obtained by 21-28 days after infection.

[0237] Further demonstration of improved reprogramming efficiency used a polycistronic vector system expressing three (Oct4, Klf4, and Sox2) or four (Oct4, Klf4, Sox2, and myc) pluripotency factors from the same promoter element in combination with optimized SMC4 medium, feeder-free culture, and a single-cell sorting system using SSEA4 and TRA181, resulting in a reprogramming efficiency of 0.756%, with colonies first visible 6-8 days after infection. This method was so efficient that iPSC colonies were present just 4 days after infection. These techniques represent a significant improvement over traditional methods of generating iPSCs.

[0238] Table 4: Kinetics of reprogramming in different strategies. NI, not identified; *morphological appearance of iPSC-like colonies; **calculated as the number of SSEA4+ / Tra181+ per plated cells; ***time required to expand iPSC colonies and maintain them as clonal lines (based on Tra181 / SSEA4 staining). Grey boxes show data from literature search. Individual factor viruses; infections were performed by combining individual viruses, each expressing one of the key transcription factors, 3 represents the Oct4 / Klf4 / Sox2 combination and 4 represents the Oct4 / Klf4 / Sox2 / cMyc combination.

[0239] [Table 4-1]

[0240] [Table 4-2] Example 9 Methods for depleting pluripotent cell populations from differentiated cell populations using single cell sorting and enrichment Some clinical applications of drug screening and stem cell biology require the generation of homogeneous cell populations differentiated to specific lineages from pluripotent cells such as ESCs or iPSCs. Contamination of differentiated cell populations with pluripotent cells may lead to erroneous screening results or even to tumor / teratomas formation in vivo. Methods for either enriching a cell population for differentiated cells or depleting pluripotent cells from a cell population may include sorting techniques as described in Examples 3 and 6 herein. The use of small molecule additives in cell culture media, particularly as provided by the present invention, prevents pluripotent cells from differentiating or partially differentiating during the single cell sorting process, which allows for negative selection of pluripotent cells from a population of fully differentiated cells. Conversely, positive selection of differentiated cells from a cell population by cell sorting may be more effective under culture conditions where pluripotent cells remain fully positive for surface markers of pluripotency. As can be seen in Figure 20, a mixed population of fully differentiated fibroblasts and pluripotent cells was effectively separated using FACS when the cells were pre-cultured in the culture environment described in Table 1. Small molecule culture additives can also be used during the sorting procedure to stabilize the single cell suspension. From Figure 20, it can be seen that the cells selected as negative for pluripotency markers were confirmed to be completely free of pluripotent cells upon subsequent culture and staining for alkaline phosphatase.

[0241] Example 10 Feeder-free culture of pluripotent stem cells without cytokines or growth factors As discussed in Example 2, conventional human pluripotent stem cell culture systems include feeder cells and cytokines such as bFGF, which serve as exogenous stimuli to maintain human pluripotent stem cells in an undifferentiated state. However, the process for producing feeder cells and recombinant cytokines serves as a source of xenogenic contaminants. Furthermore, the key factor(s) secreted from feeder cells and the exact cellular pathways stimulated by cytokines have yet to be identified. Thus, conventional culture of human pluripotent stem cells represents an unclear system, which may hinder the transition to clinical grade manufacturing.

[0242] To address this issue, the present invention encompasses further embodiments in which the feeder-free and single cell passage system discussed in Example 2 is further modified by removing bFGF and other cytokines and growth factors from the SMC4 medium formulation. Furthermore, in one embodiment of the present invention, Matrigel™ is replaced with gelatin, since Matrigel™ represents an extracellular matrix of animal origin and has not been fully characterized. These embodiments of the present invention provide a fully defined and cytokine-free culture system that allows for the endogenous self-renewal and maintenance of pluripotent stem cells, including iPSCs.

[0243] As demonstrated in Figures 21A and 21B, a polycistronic vector system containing Oct4, Klf4, and Sox2 was used to generate and grow the cells in a feeder-free environment. Human pluripotent stem cells such as iPSCs maintained in gelatin were easily single-cell passaged on gelatin-coated culture surfaces with SMC4 medium lacking additional cytokines or growth factors, such as bFGF. After several passages in this fully defined system, iPSCs maintained their pluripotent state, as demonstrated by the co-expression of Tra181 and SSEA4 (Figure 21C). Furthermore, the inventors have demonstrated the generation of iPSCs in this fully defined and cytokine-free system. Thus, in one embodiment, the present invention provides a fully defined culture system that is growth factor and cytokine-free, comprising a combination of SMC4 medium and gelatin.

[0244] Example 11 Genomic stability in the generation and maintenance of pluripotent stem cells Several studies suggest that the reprogramming process and subsequent culture of pluripotent stem cells may be more prone to genomic abnormalities.Furthermore, feeder-free culture has been shown to result in clonal growth of karyotypically abnormal cells.As demonstrated in Figures 17A, 17B and 18C, the present invention provides a method for reprogramming to obtain cells with genomic stability, and a method for maintaining reprogrammed cells with genomic stability.In the method of the present invention, when cells are reprogrammed using our three-factor polycistronic construct containing Oct4, Klf4 and Sox2 and cultured in SMC4 medium, genomic stability is maintained both during the reprogramming process and during long-term feeder-free culture.As seen in Figure 17A, high-resolution comparative genomic hybridization demonstrated that minimal copy number variation was detected when hiPSCs were generated and maintained using SMC4 medium in long-term feeder-free culture.Furthermore, as demonstrated in Figures 17B and 18C, genomic stability was maintained during routine long-term feeder-free culture and single-cell culture.

[0245] Example 12 Maintenance of pluripotent cell populations during culture using cell surface markers In many cases, it is useful to remove differentiated cells from pluripotent cell cultures in order to maintain the pluripotency of the stem cell culture. To date, this process requires either manually picking differentiated cells from the cell culture or harvesting undifferentiated cells from a substantially differentiated population. Both processes are labor intensive, require technical training, and rely on the selection of cells based on morphology, which does not always indicate the true pluripotent state of the cells in culture (Figure 8).

[0246] In an improved process, the present invention provides the ability to efficiently and accurately select undifferentiated cells during routine culture.As demonstrated in Figure 19A, the population of hiPSCs maintained in SMC4 medium and FF medium was easily enriched or selected to maintain cell culture of undifferentiated pluripotent cells.In another example, as shown in Figure 19B, the population of mostly differentiated cells that would normally be discarded can be removed from cell culture to obtain mostly undifferentiated pluripotent cells, as indicated by the expression of Tra181.

Claims

1. A method for increasing the pluripotency of one or more human cells, comprising: The method comprises culturing one or more human cells in a culture medium; (a) the culture medium comprises a mixture of a ROCK inhibitor, a GSK3 inhibitor, a TGFβ receptor inhibitor, and a MEK inhibitor; (b) prior to said culturing, said one or more human cells lack ground state pluripotency; (c) the culturing is effective to generate a population of cultured human induced pluripotent stem cells (iPSCs) having ground state pluripotency from the one or more human cells; and (d) said ground state pluripotency is characterized by the ability to form cell colonies having a dome-shaped morphology in the absence of non-human feeder cells.

2. The method of claim 1, wherein the one or more human cells lacking ground state pluripotency comprise one or more iPSCs that do not have ground state pluripotency.

3. The method of claim 2, wherein the one or more human iPSCs that do not have ground-state pluripotency are a single human iPSC, and the population of cultured iPSCs comprises a clonal population generated from the single human iPSC.

4. The method of claim 1, wherein (i) the one or more human cells comprise one or more non-pluripotent cells, and (ii) the one or more non-pluripotent cells comprise one or more exogenous polynucleotides encoding an Oct4 polypeptide.

5. The method of claim 4, wherein the one or more exogenous polynucleotides further comprise polynucleotides encoding one or more Sox2 polypeptides and Klf4 polypeptides.

6. The method of claim 4, further comprising the step of introducing the one or more exogenous polynucleotides into the one or more non-pluripotent cells.

7. The method of claim 1, wherein the ground state pluripotency is further characterized by one or more of: (i) reactivation of the X chromosome; (ii) having a normal karyotype and normal chromosome copy number; and (iii) the ability to reactivate one or more of GATA6, CDX2, or CGB.

8. The method of claim 1, wherein the ground state pluripotency is further characterized by an ability to reactivate one or more of GATA6, CDX2, or CGB.

9. The method described in claim 1, wherein the ground state pluripotency is further characterized by the ability to reactivate all of GATA6, CDX2 and CGB.

10. The method of claim 1, further characterized in that the ground-state pluripotency replicates and maintains pluripotency in the absence of exogenous stimuli including one or more of the following exogenous stimuli: TFGβ receptor, activin, MEK signaling pathway, or bFGF pathway.

11. The method of claim 1, wherein the culture medium lacks exogenous cytokines and exogenous growth factors.

12. The method of claim 1, further comprising sorting the cultured population of iPSCs for cells expressing one or more pluripotency markers to obtain an enriched population of iPSCs.

13. The method described in claim 12, wherein the enriched iPSC population is enriched for at least 20% iPSCs.

14. The method of claim 12, wherein the one or more pluripotency markers include SSEA4, TRA160, TRA181, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD30, CD50, CD133 / prominin, CD140a, CD56, CD73, CD105, CD31, CD34, OCT4, Nanog, or Sox2.

15. (i) The ROCK inhibitor is thiazovivin or Y27632; (ii) the TGFβ receptor inhibitor is A-83-01 or SB431542; (iii) the GSK3 inhibitor is CHIR99021 or BIO; (iv) the MEK inhibitor is PD98059 or PD032901; or (v) the TGFβ receptor inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the ROCK inhibitor is thiazovivin; The method of claim 1.

16. The method of claim 1, further comprising passaging iPSCs in the cultured population of iPSCs to generate passaged iPSCs, wherein the passaged iPSCs have ground state pluripotency.

17. The method of claim 16, wherein passaging comprises dissociating the cultured population of iPSCs into single cells.

18. The method of claim 17, wherein dissociating comprises treating with one or more dissociating enzymes.

19. The method described in claim 16, wherein the passaging comprises at least five passagings.

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