Cell culture platform for single cell sorting and enhanced iPSC reprogramming

Abstract

The present invention provides cell culture conditions for culturing stem cells, including feeder-free conditions for producing and culturing human induced pluripotent stem cells (iPSCs). More specifically, 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; single cell dissociation of pluripotent cells; cell sorting of pluripotent cells; maintenance of an undifferentiated state; increased efficiency of reprogramming; and the generation of original pluripotent cells.

Description

[0001] This application is a divisional application of the invention patent application with application number 201180068009.0 filed on December 19, 2011, and claims priority to U.S. patent application No. 61 / 426,369 filed on December 22, 2010 and U.S. patent application No. 61 / 496,991 filed on June 14, 2011.

[0002] Statement regarding sequence listing

[0003] The sequence table associated with this application is provided in a plain text format in lieu of paper form and is incorporated herein by reference. The name of the text file containing the sequence table is FATE_094_00WO_ST25.txt. The size of the text file is 4KB, created on December 15, 2011, and submitted via EFS-Web.

[0004] Related Applications Cited

[0005] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61 / 429,369, filed on December 22, 2010, and U.S. Provisional Patent Application No. 61 / 496,991, filed on June 14, 2011, each of which is hereby incorporated by reference in its entirety. Background of the Invention Technical Field

[0007] The present invention generally relates to cell culture conditions, culture media and culture platforms for culturing stem cells, including feeder-free conditions for regenerating and culturing human induced pluripotent stem cells (iPSCs).

[0008] Description of related fields

[0009] The application of pluripotent stem cell biology has opened new doors for regenerative medicine. The derivation of human embryonic stem cells (hESCs) has led to many promising cell therapy approaches by culturing preimplantation blastocysts in a mixture of growth factors, in which expanded self-renewing cell populations can be differentiated into therapeutically relevant cell lines in vitro or in vivo. In another ESC biological application and by using preimplantation genetic analysis, it is possible to derive ESC lines from several genetic disease backgrounds to simulate these diseases in tissue culture dishes. However, there are some limitations to ESC technology: the genetic background of ESCs is not always known, since the range of genetic backgrounds from which ESCs can be derived is limited by technical and political factors, and ESC-derived cell therapies are essentially allogeneic transplants, risking the same rejection risk as traditional tissue / organ transplants.

[0010] In a major advance, pluripotent cell groups are generated from terminally differentiated cells of adults: such derived cells are called induced pluripotent stem cells (iPSC). iPSC technology allows cells from any donor to be reprogrammed into a pluripotent self-renewal state and thereby allows the expansion of homogeneous cell groups from any genetic background. iPSC overcomes the ethical considerations associated with ESC and can be used to derive models of any hereditary human disease for high-throughput drug screening or drug toxicity screening of hepatocytes and cardiomyocytes xenobiotics. In addition, iPSC can ultimately lead to cell therapy that prevents transplant rejection from being generated in autologous transplants from patients' autologous cells. Expression and differential analysis show that iPSC is very close to ESC at the molecular level, and when compared with multiple ESC lines, the differences between iPSC cultures of clones of these similar magnitudes are seen.

[0011] iPSCs are usually generated by ectopic expression of several key genes, which are required for full reprogramming, i.e., a combination of Oct4, Sox2, Klf4, c-Myc, Lin28, and Nanog. Initially, an integrated viral system was used to express key transcription factors to generate iPSCs. Retroviral and lentiviral systems including polycistronic and inducible systems have been successfully applied to iPSC generation. However, the potential for permanent genomic changes and reactivation of exogenous genes after iPSC differentiation due to insertional mutations may pose potential problems for subsequent drug screening and therapeutic applications of cells generated by these methods. Indeed, significant differences between iPSC clones generated using the same viral system have been reported, with a large proportion of tumor-forming clones when transplanted as differentiated neurospheres in rodents. Studies have shown that iPSCs generated using the same viral method can have different performances once differentiated. Differences in ectopic gene integration sites can lead to different insertional mutations and epigenetic regulation of transgenic expression. For iPSC generation methods including integrated systems, it is necessary to derive and screen many clones to identify those that are stable in both pluripotent and differentiated states. Therefore, the method of rapid derivation of iPSC from clones of a given donor cell source is advantageous. Although the efficiency is relatively low, the use of non-integrated systems for iPSC generation such as adenovirus or episomal transient expression has been demonstrated. These systems can overcome the safety and stability issues in iPSC generation, but when using any DNA-based reprogramming method, there is the potential for genome integration and this needs to be evaluated before they are used to develop iPSC-derived cell therapy.

[0012] Excisable viral systems with whole genome expression profiling show that iPSCs with integrated expression components are less like ESCs than clones of the same viral factors with excision. Moreover, protein-only reprogramming has been demonstrated with the most commonly used transcription factors expressed as fusion proteins with cell-penetrating peptides in Escherichia coli (E. coli). Multiple doses of purified protein applied to mouse fibroblasts resulted in iPSC generation. The efficiency of reprogramming using protein-only systems is very low. This can be attributed to the efficiency of protein transduction, the specific activity of the protein, and / or the stability of the protein.

[0013] The process of reprogramming differentiated cells by ectopic expression of pluripotency genes or their introduction via protein transduction or mRNA requires several months and the knowledge of a skilled stem cell biologist. Initially, identification of reprogrammed cells was performed by eye: screening of ESC-like colony morphology. Such colonies must be picked by hand, usually mechanically passaged and amplified. The introduction of pluripotency factors also produces transformed cell colonies and incompletely reprogrammed cells. Researchers can identify true iPSC colonies from the background of transformed cells, but this is not an efficient process. Further features and confirmation as true pluripotent populations are then required and usually include immunohistochemical staining for pluripotency markers, gene expression and epigenetic analysis, and the ability of pluripotent populations to differentiate into three germ layers (ectoderm, mesoderm and endoderm). Once pluripotent cells are identified and selected, such cells usually grow into colonies and need to be manually passaged to maintain cells for a long time by selecting and mechanically dissociating cells before re-plating.

[0014] Embryonic stem cells derived from before and after the various implantation periods have shown different pluripotency states.For example, the cell derived from the inner cell mass of the blastocyst is considered to be more "original" and has a major characteristic that is completely different from the post-implantation derived cell, which is considered to be more "mature" and has a higher random differentiation tendency.Original cells appear more "ground state" and do not need external signal transduction to keep their undifferentiated state.On the other hand, activated cells need to include the external signal transduction of the key cytokine of TGF β, activin and bFGF and rely heavily on ERK / MAPK cell pathway to keep their undifferentiated state.

[0015] Improvements in the iPSC generation process can significantly reduce technical barriers, accelerate the process and enable subsequent cell scaling and differentiation for industrial applications of the technology such as drug screening and cell therapy. There is a need for more efficient iPSC production without the use of exogenous substances and more efficient methods for identification and screening of reprogrammed cells. Methods that promote the generation of iPSCs in the naive state of human pluripotent stem cells will greatly benefit future applications in regenerative medicine, such as disease correction, directed differentiation, and manufacturing scale-up. Moreover, there is a need for more efficient methods and scalability for generating iPSCs in a specific culture state that enables single cells to be passaged. SUMMARY OF THE INVENTION

[0016] One embodiment of the present invention provides a method for 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 culture medium comprising at least one agent that maintains the pluripotency of the cells while maintaining the pluripotency of the cells during the culture period, wherein the agent is selected from: i) a TFGβ inhibitor; ii) a GSK3 inhibitor; iii) a MEK inhibitor and iv) a Rock inhibitor.

[0017] In another embodiment, the culture medium comprises a sufficient amount of the agent to allow at least one cell division while maintaining the pluripotency of the cells. In a specific embodiment, the culture medium comprises at least three or four agents that maintain the pluripotency of the cells.

[0018] In still another embodiment, the agent for maintaining cell pluripotency comprises a Rock inhibitor. In a specific embodiment, the Rock inhibitor is thiazovivin or Y27632.

[0019] In one embodiment, the agent that maintains cell pluripotency comprises a TFGβ inhibitor, and in a specific embodiment, the TFGβ inhibitor is A-83-01 or SB431542.

[0020] In another embodiment, the agent that maintains cellular pluripotency comprises a GSK3 inhibitor, and in specific embodiments, the GSK3 inhibitor is CHIR99021 or BIO.

[0021] In one embodiment, the agent for maintaining cell pluripotency comprises a MEK inhibitor. In a specific embodiment, the MEK inhibitor is PD98059 or PD0325901.

[0022] In a specific embodiment, the culture 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.

[0023] In some embodiments of the invention, the pluripotency of the cell is maintained for at least five cell divisions. In other embodiments, the pluripotency of the cell is maintained for at least ten cell divisions.

[0024] In some embodiments, cells are cultured in the absence of growth factors and cytokines, optionally in the presence of soluble fibronectin. In another embodiment, cells are cultured in the absence of Matrigel TM In yet another embodiment, the cells are cultured in the presence of bFGF.

[0025] In a specific embodiment of the invention, the pluripotent cells are human embryonic stem cells or human induced pluripotent stem cells.

[0026] Another embodiment of the present 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.

[0027] In certain embodiments, the method comprises culturing pluripotent cells in a culture medium comprising at least one agent that maintains cell pluripotency to allow at least one cell division while maintaining cell pluripotency, wherein the agent is selected from: i) a TFGβ inhibitor; ii) a GSK3 inhibitor; iii) a MEK inhibitor and iv) a Rock inhibitor.

[0028] In one embodiment, the pluripotent cells are human embryonic stem cells or human induced pluripotent stem cells.

[0029] In additional embodiments, the culture medium comprises at least two, at least three or four agents selected from: i) a TFGβ inhibitor; ii) a GSK3 inhibitor; iii) a MEK inhibitor and iv) a Rock inhibitor; in specific embodiments, the culture medium comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor and a Rock inhibitor; in other specific embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0030] Another embodiment of the present invention also provides a method for obtaining dissociated human pluripotent cells, comprising dissociating human pluripotent cells to obtain dissociated cells and contacting the dissociated cells with a culture medium, wherein the culture medium contains at least one agent that enhances the viability of the dissociated cells, wherein the agent is selected from: 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 specific 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%.

[0031] In other embodiments, the methods of the invention further comprise culturing the dissociated cells in culture for at least one, at least two, at least five, or at least ten passages while maintaining the pluripotency of the dissociated cells.

[0032] In certain embodiments, the karyotype of the dissociated cells after culturing is substantially similar to the karyotype of the cell population prior to dissociation.

[0033] In some embodiments, the method includes dissociating in the presence of a formulation. In still other embodiments, the method includes contacting the pluripotent cells with a formulation before dissociating. In specific embodiments, contacting the dissociated cells with a culture medium includes suspending the dissociated cells in a culture medium.

[0034] In other embodiments, the present invention provides a method for increasing cell potency in a feeder-free environment, comprising contacting cells with a culture medium containing at least one small molecule agent in a feeder-free environment to obtain cells with increased potency compared to cells before contacting the culture medium, wherein the small molecule agent is selected from: i) TFGβ inhibitor; ii) GSK3 inhibitor; iii) MEK inhibitor and iv) Rock inhibitor. In a specific embodiment, the culture medium 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.

[0035] In one embodiment, contacting comprises culturing the cell under conditions sufficient to increase the potency of the cell.

[0036] 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.

[0037] In specific embodiments, the cell is not a mouse embryonic stem cell. In other specific embodiments, the cell is a human cell, and in certain embodiments, the cell is an induced pluripotent stem cell.

[0038] 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 specific 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.

[0039] Still in other embodiments, cells with increased potency are characterized by one or more of the following: expression of at least one pluripotent stem cell marker selected from Oct4, Nanog, KLF4, SSEA4 and TRA 1-81; pluripotent stem cell morphology; ability to cause germline transmission; formation of teratomas; ability to differentiate or transdifferentiate into a lineage different from the starting lineage, and in vitro tri-lineage differentiation. In some embodiments, cells with increased potency express at least twice as high Oct4 levels as cells before contact with the culture medium. Still in other embodiments, cells with increased potency have at least twice as low Xist activity as compared to conventionally cultured iPSCs. In additional embodiments, cells with increased potency have a compact, hemispherical colony morphology.

[0040] In some embodiments, the cells with increased potency replicate and remain pluripotent 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.

[0041] 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 pluripotency. In a specific embodiment, 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 thiazovivin.

[0042] The present invention also provides cells with increased potency produced by any of the above embodiments.

[0043] The present invention also provides a method for 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 i) a TFGβ inhibitor; ii) a GSK3 inhibitor; iii) a MEK inhibitor, and iv) a Rock inhibitor under conditions sufficient to induce reprogramming, whereby the reprogramming efficiency is increased by at least 10%, at least 50%, at least 100%, at least 300%, or at least 500% compared to the reprogramming efficiency of the cell population not contacted with the small molecule agent.

[0044] In specific 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.

[0045] In some embodiments, the cell population comprises non-pluripotent cells before reprogramming. In other embodiments, the efficiency of reprogramming is measured by the time required for reprogramming or the number of cells reprogrammed.

[0046] In still other embodiments, conditions sufficient to induce reprogramming include contacting the cell population with at least one exogenous transcription factor selected from Oct4, Sox, Klf, Myc, Lin28 or Nanog polypeptides or polynucleotides encoding Oct4, Sox, Klf, Myc, Lin28 or Nanog. In specific embodiments, the conditions include contacting the cell population with Oct4, Sox2 and Klf4 polypeptides or polynucleotides encoding Oct4, Sox2 and Klf4.

[0047] Other embodiments of the present invention also provide a method for sorting a cell population in a feeder-free environment to obtain a cell population rich in pluripotent cells, including obtaining a suspension of dissociated cells containing a mixed cell population in a feeder-free environment, and sorting the cells in the suspension to obtain cells expressing one or more pluripotency markers, thereby obtaining an enriched cell population rich in pluripotent cells. In some embodiments, the suspension comprises at least one of a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor, or a Rock inhibitor. In other embodiments, sorting is by magnetic beads or flow cytometry. In specific embodiments, sorting is by magnetic beads. In other embodiments, sorting is by flow cytometry.

[0048] 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.

[0049] In a specific embodiment, the mixed cell population in the 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 before sorting. In certain embodiments, the mixed cell population is contacted with a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor and a Rock inhibitor; in certain other embodiments, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0050] In a specific embodiment, the mixed cell population comprises cells expressing one or more pluripotency markers. In a specific embodiment, the one or more pluripotency markers 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 a specific embodiment, the pluripotency marker is selected from SSEA4, TRA181, TRA160 and CD30.

[0051] In a specific embodiment, the method includes contacting a mixed cell population with one or more pluripotency factors to induce reprogramming. In some embodiments, contacting includes introducing one or more pluripotency factors into cells in a mixed cell population. In certain embodiments, the pluripotency factors include Oct4, Sox, Klf, Myc, Lin28 or Nanog polypeptides or polynucleotides encoding Oct4, Sox, Klf, Myc, Lin28 or Nanog. In other specific embodiments, the pluripotency factors include Oct4, Sox2 and Klf4 polypeptides or polynucleotides encoding Oct4, Sox2 and Klf4 polypeptides.

[0052] In certain specific embodiments, the pluripotent cells are induced pluripotent cells.

[0053] In some embodiments, the enriched population of cells is enriched for expression of one or more pluripotency markers by at least 20%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0054] In other embodiments, the present invention provides a method for obtaining induced pluripotent stem cells, comprising treating a cell population to induce reprogramming; preparing a suspension of dissociated cells of the cell population; sorting the cells in the suspension to obtain sorted cells expressing one or more pluripotency markers; culturing the sorted cells expressing one or more pluripotency markers, wherein iPSCs are obtained. In a specific embodiment, 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.

[0055] In some embodiments, the cell population is contacted with at least one of i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor. In specific embodiments, the cell population is 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.

[0056] In other embodiments, treating the cell population to induce reprogramming comprises contacting the cell population with one or more pluripotency factors. In specific 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, Sox2 and Klf4 polypeptides or polynucleotides encoding Oct4, Sox2 and Klf4 polypeptides.

[0057] 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, and iv) a Rock inhibitor. In specific embodiments, the cell population is 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.

[0058] In other embodiments, the suspension comprises at least one of i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor. In specific 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.

[0059] In some embodiments, sorting is by flow cytometry or magnetic beads. In specific embodiments, cells are sorted to obtain cells expressing at least one, two, three, four, or multiple pluripotency markers. In other specific embodiments, 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. In other specific embodiments, one or more pluripotency markers are selected from SSEA4, TRA160, TRA181 and CD30. In some embodiments, one or more pluripotency markers are SSEA4, CD30 and TRA160 or TRA181. In other embodiments, cells are sorted using specific markers to remove non-reprogrammed cells from the reprogrammed population.

[0060] In some embodiments, the culturing comprises culturing cells in a medium comprising at least one small molecule agent selected from i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor. In specific embodiments, the culture medium 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.

[0061] In specific embodiments, the cells are cultured in a feeder-free environment. In certain embodiments, the cells are processed, suspended, sorted, and cultured in a feeder-free environment.

[0062] In some embodiments, the induced pluripotent stem cells are obtained in about 2 days to 22 days. In a specific embodiment, the induced pluripotent stem cells are obtained in about 4 days to about 18 days.

[0063] In other embodiments, the induced pluripotent stem cells are obtained within about 4 days to about 22 days after treating the cell population to induce reprogramming. In certain embodiments, the induced pluripotent stem cells are obtained within about 6 days to about 18 days after treating the cell population to induce reprogramming, and in certain other embodiments, the induced pluripotent stem cells are obtained within about 10 days to about 16 days after treating the cell population to induce reprogramming.

[0064] The present invention also provides, in other embodiments, induced pluripotent stem cells obtained by any of the above methods.

[0065] The present invention also provides a method of depleting pluripotent cells from a cell population, comprising obtaining a suspension of dissociated cells comprising a mixed cell population having pluripotent cells, and sorting the cells in the suspension to remove cells expressing one or more pluripotency markers, thereby depleting pluripotent cells from the cell population.

[0066] In some embodiments, the mixed cell population comprises multipotent cells or (mature) somatic cells.

[0067] In other embodiments, before obtaining the suspension, the mixed cell population in the suspension is cultured in a culture medium containing at least one small molecule agent selected from i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor. In a specific embodiment, the culture medium contains 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.

[0068] In another embodiment of the present invention, the suspension comprises at least one small molecule agent selected from i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor. In a specific embodiment, 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.

[0069] In some embodiments, the sorting is by flow cytometry. In other embodiments, the sorting is by antibody-coated magnetic bead enrichment.

[0070] Some embodiments of the present invention provide a method for obtaining pluripotent cells with genomic stability, comprising contacting the cells with at least one small molecule agent selected from i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor in a feeder-free environment, in the absence of c-myc, under conditions sufficient to obtain pluripotent cells with genomic stability.

[0071] 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.

[0072] In certain embodiments, the conditions include contacting the cell with at least one pluripotency factor. In some embodiments, the pluripotency factor is an exogenous transcription factor selected from Oct4, Sox, Klf, Myc, Lin28 or Nanog polypeptides or polynucleotides encoding Oct4, Sox, Klf, Myc, Lin28 or Nanog. In specific embodiments, the pluripotency factor comprises Oct4, Sox2 and Klf4 polynucleotides.

[0073] In some embodiments of the present invention, the small molecule agent comprises a Rock inhibitor. In a specific embodiment, the Rock inhibitor is thiazovivin or Y27632, and in a more specific embodiment, the Rock inhibitor is thiazovivin.

[0074] In other embodiments, the small molecule agent comprises a TFG inhibitor. In some embodiments, the TFG inhibitor is A-83-01 or SB431542.

[0075] In some embodiments, the small molecule agent comprises a GSK3 inhibitor, and in specific embodiments, the GSK3 inhibitor is CHIR99021 or BIO.

[0076] In other embodiments of the present invention, the small molecule agent comprises a MEK inhibitor. In some specific embodiments, the MEK inhibitor is PD98059 or PD0325901.

[0077] 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 a specific embodiment, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0078] The present invention additionally comprises a method of culturing pluripotent cells to maintain the genomic stability of the cells, comprising culturing the pluripotent cells in a feeder-free environment in a culture medium comprising at least one agent that maintains the genomic stability of the pluripotent cells, while maintaining the genomic stability of the pluripotent cells during the culture, wherein the agent is selected from i) a TFGβ inhibitor, ii) a GSK3 inhibitor, iii) a MEK inhibitor, and iv) a Rock inhibitor.

[0079] In certain embodiments, the conditions include contacting the cell with at least one pluripotency factor. In some embodiments, the pluripotency factor is an exogenous transcription factor selected from Oct4, Sox, Klf, Myc, Lin28 or Nanog polypeptides or polynucleotides encoding Oct4, Sox, Klf, Myc, Lin28 or Nanog. In specific embodiments, the pluripotency factor comprises Oct4, Sox2 and Klf4 polynucleotides.

[0080] In some embodiments, the culture medium comprises at least two, at least three, or four formulations.

[0081] In some embodiments of the invention, the formulation comprises a Rock inhibitor. In a specific embodiment, the Rock inhibitor is thiazovivin or Y27632, and in a more specific embodiment, the Rock inhibitor is thiazovivin.

[0082] In other embodiments of the invention, the formulation comprises a TFG inhibitor. In some embodiments, the TFG inhibitor is A-83-01 or SB431542.

[0083] In some embodiments, the formulation comprises a GSK3 inhibitor, and in specific embodiments, the GSK3 inhibitor is CHIR99021 or BIO.

[0084] In other embodiments of the present invention, the formulation comprises a MEK inhibitor. In some specific embodiments, the MEK inhibitor is PD98059 or PD0325901.

[0085] In some embodiments of the present invention, the formulation comprises a TFGβ inhibitor, a GSK3 inhibitor, a MEK inhibitor and a Rock inhibitor. In a specific embodiment, the TFGβ inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

[0086] In some embodiments of the invention, the pluripotent cells are cultured in the culture medium for at least one, at least two, at least five, at least ten, or at least 15 passages while maintaining the genomic stability of the pluripotent cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1. Effects of various small molecule cocktails on single-cell culture of human pluripotent stem cells on feeder-free matrices. A. Different morphologies are evident when single-cell dissociated human pluripotent stem cells are treated with various combinations of small molecules in a feeder-free environment. Culture media containing the ROCKi / MEKi / TGFβi / GSKi combination conferred robust growth and viability of human pluripotent stem cells. B. While some small molecules, MEKi / TGFβi / GSKi, support maintenance of pluripotent stem cells and others, Rocki, promote single-cell dissociation of pluripotent stem cells, the unique combination of these small molecules, ROCKi / MEKi / TGFβi / GSKi, supports single-cell dissociation while maintaining an undifferentiated state, as shown by highly proliferative colonies that react positively to the pluripotency marker Tra181. Tra181, red; DAPI, blue. C. When hiPSCs were cultured at 1×10 5 When cells were plated on artificial matrigel, the combination of MEKi / TFGβi / GSKi / ROCKi best supported viability and proliferation and scored after 4 days for viable cell number and percent viability measured by trypan blue. D. When combined with MEKi / TFGβi / GSKi, colonies cultured with Thiazovivin appeared more compact in morphology relative to cultures supported by MEKi / TFGβi / GSKi and Y27632. E. Flow cytometric analysis of surface expression of SSEA4 and Tra181 of hiPSCs cultured in Y27632 or Thiazovivin in combination with MEKi / TFGβi / GSKi showed that Thiazovivin better supported cells to remain in an undifferentiated state compared to the combination with Y27632.

[0088] Figure 2. Adaptation of human pluripotent stem cells to feeder-free and single-cell culture. A. iPSCs generated on feeder cells readily adapt to feeder-free and single-cell culture conditions using SMC4 medium. However, single-cell dissociation and culture under conventional culture conditions can result in disrupted cell survival and limited cell adhesion and growth. B. Once adapted to a feeder-free environment, iPSCs are routinely cultured as single cells on feeder-free surfaces while being continuously maintained in SMC4 medium.

[0089] Figure 3. Long-term maintenance of single cells and feeder-free culture of human pluripotent stem cells. A. Single-cell dissociated human pluripotent stem cells were maintained in feeder-free SMC4 medium for 10 passages, and the expression of pluripotency markers Nanog and Tra181 was analyzed by immunofluorescence. B. Figure 3B The percentage of 7-aminoactinomycin D (7AAD) stained cells 4 hours after human pluripotent stem cells were dissociated into single cells and placed in conventional culture medium or SMC4 medium. When cultured in SMC4 medium instead of conventional culture medium, single-cell dissociated human pluripotent stem cells showed significantly increased viability, as seen by 7AAD staining and flow cytometry analysis. C. Quantitative population analysis by flow cytometry revealed that almost all cells dissociated into single cells and maintained in SMC4 medium expressed multiple markers of undifferentiated states. D. Overall expression analysis revealed that iPSCs passaged as single cells and maintained in SMC4 medium without feeder cells were highly correlated with human ESCs, including H1 cells, cultured on feeder cells and maintained as cell clumps during passage. E. iPSCs cultured in a feeder-free cell system and passaged as single cells using SMC4 medium, rather than their original source fibroblasts, expressed an array of pluripotency markers similar to human ESCs, including H1 and HuES9. F. The exogenous expression of reprogramming factors in iPSCs maintained in a feeder-free environment was effectively suppressed, unlike the exogenous factor positive control (fibroblasts expressing reprogramming factors 4 days after lentiviral infection). The endogenous expression of reprogramming factors in feeder-free iPSCs was similar to that of ESCs, including H1 and HuES9. G. After 11 passages as single cells in a feeder-free environment, human pluripotent stem cells maintained a normal karyotype. H. After 14 days of replacing SMC4 medium with conventional differentiation medium, cells maintained in a feeder-free environment showed gene expression indicating ectoderm, endoderm, and mesoderm germ layers in addition to extra-embryonic markers, which are usually only expressed by primary and omnipotent stem cells. I. Immunofluorescence analysis: iPSCs cultured in a feeder-free cell system using SMC4 medium and passaged as single cells, once transferred to conventional differentiation medium, are prone to develop into all three somatic cell types. Mesoderm, α-smooth muscle actin (αSMA); ectoderm, β-tubulin III (βTUBIII); endoderm, Sox17. J. Teratoma formation assays demonstrate that human pluripotent stem cells cultured in SMC4 medium generate cell types representing the three germ layers and thus maintain their pluripotent potential.

[0090] 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. Before sorting, human pluripotent stem cells were dissociated into single cells and labeled with antibody markers to SSEA4 and Tra-181. Using FACs technology, cells were sorted based on surface marker expression of pluripotency markers, SSEA4 and Tra-181. After sorting, cells were initially plated in SMC4+fibronectin medium and switched to SMC4 medium after 24-72 hours. On day 1 after sorting, the sorted cells were separated and by day 5, large colonies consisting of hundreds of cells appeared. B. The sorted cells retained their expression of the pluripotency marker Tra160. Tra160, red; DAPI, blue. C. After 5 passages and before sorting, approximately 1 month in culture, quantitative flow cytometric analysis based on SSEA4 and Tra181 staining revealed that most sorted cells retained their pluripotent state. D. Colonies derived from SSEA4+ and Tra181+ human pluripotent stem cells were plated on feeder-free surfaces in SMC4+fibronectin medium at various densities, including 500 events per well of a 6-well plate, approximately 52 events / cm 2 . 7 days after sorting, many alkaline phosphatase-positive colonies appeared in each plated well. E. Scoring of the resulting alkaline phosphatase-positive colonies. Approximately 8-10% of the plated events produced colonies with 16,000 events per well, resulting in nearly confluent wells. NCs were not counted as fusions prevented accurate counting. F. SSEA4+ and Tra181+ human pluripotent stem cells were sorted directly into 96-well plates at various densities, including 1 event / well, approximately 3 events / cm 2 . 8 days after sorting, the wells were stained with alkaline phosphatase. G. On day 8, alkaline phosphatase colonies were scored in each well.

[0091] Figure 5. Reprogramming kinetics in the absence and presence of SMC4 medium. A. 7 days after initiation of reprogramming by ectopic expression of Oct4, Sox2, Klf4, and cMyc, cells were either maintained in regular medium or transferred to SMC4 medium. After 20 days of reprogramming, few iPSC-like colonies formed in regular medium culture, while many iPSC-like colonies appeared in cells transferred to SMC4 medium. B. Many iPSC-like colonies in SMC4 medium expressed alkaline phosphatase while few colonies expressed alkaline phosphatase in regular culture.

[0092] Figure 6. Cultivation in SMC4 medium enhances naive characteristics. A. Schematic representation of human pluripotent stem cells adapted to SMC4 medium and its comparison to conventionally maintained human pluripotent stem cells. B. Pearson correlation coefficients between hiPSCs derived from IMR90 parental cells (conventionally derived hiPSCs (Conv) and hiPSCs adapted to SMC4 medium (SMC4 medium)) based on Affymetrix global gene expression and cultured in the respective media for 11 passages, both demonstrating that the hiPSCs are similar to each other and distinct from their parental IMR90 cells. C. While Xist expression is moderately suppressed in hiPSCs cultured in the conventional format compared to their parental IMR90 cell line, Xist expression is significantly suppressed in hiPSCs cultured in SMC4 medium, to levels similar to those of female hESCs, HUES9. D. Genes located on the X chromosome are expressed to a higher degree in hiPSCs cultured in SMC4 medium compared to conventionally cultured hiPSCs. E. When transferred back to feeder cells, hiPSCs maintained in SMC4 medium displayed a morphology more similar to mouse ESCs, as seen by a more three-dimensional shape and less planar morphology compared to hiPSCs cultured in conventional medium.

[0093] Figure 7. Generation and maintenance in SMC4 medium improves the undifferentiated state of human pluripotent stem cells. A. Schematic diagram of the parallel generation of FTi91 and 93 clones, generated in SMC4 medium and maintained in SMC4 medium FF culture, and FTi99 clone, generated in conventional medium and maintained on feeder cells. B. Hierarchical clustering based on Affymetrix global gene expression and Pearson correlation coefficients describe the close relationship between the derived pluripotent lines and the distant relationship with the starting cell line (FTC1). C. Hierarchical clustering based on all 4-fold differentially expressed genes between Hues9, H1, FTi99, FTi91 and FTi93 demonstrates the separation between pluripotent lines cultured in conventional systems and those cultured in SMC4 medium. D. Affymetrix gene expression of selected genes related to naive and lineage differentiation in SMC4 medium groups (FTi91 and 93) compared to conventional culture groups (H1, Hues9, and FTi99) shows increased expression of many genes related to pluripotency and significantly reduced expression of lineage-specific genes in SMC4 medium. E. Schematic diagram of various methods for obtaining naive hiPSCs. The purple layer describes the advantages of the naive state over the mature state.

[0094] Figure 8. Non-iPSC colonies generated during the reprogramming process. A. Typical cell populations early in the reprogramming process: Immunostaining shows that most of the rapidly growing cells form colonies that are SSEA4 negative and inevitably non-pluripotent cells. These cells are transformed, highly proliferative, grow rapidly and dominate in culture. Colony 1 shows that SSEA4-positive colonies have the potential to become iPSC colonies while colony 2 shows a transformed population with a high proliferation rate but negative staining for pluripotency markers. B. Many non-iPSC colonies show expression of a single pluripotency marker, while true iPSC colonies show expression of multiple pluripotency markers. In the later stages of reprogramming, various colonies are formed, some colonies are double positive for SSEA4 and Tra181, i.e. colony 1, while other colonies are only positive for one of the markers or none at all, such as colonies 4 and 5.

[0095] Fig. 9 . Selection of pluripotent cells such as iPSCs using cell sorting. Schematic representation of a single cell culture system and cell sorting method for enrichment and selection from a mixture of pluripotent and non-pluripotent cells.

[0096] Fig.10 .Identification of pluripotency markers. + / Tra181 + Additional markers specific to cells were evaluated for various surface markers. Detection of Tra160 expression was used as a positive control, as it has been shown to correlate well with Tra181 expression. CD200 and CD90 do not appear to be specific for pluripotent cells, as they stain similarly to SSEA. - / Tra181 - With SSEA + / Tra181 + group, but no CD9 targeting SSEA was seen + / Tra181 + However, CD50 and CD30 appear to cross SSEA - / Tra181 - The group preferably identified SSEA4 + / Tra181 + group.

[0097] Figure 11. CD30 is specific for Nanog expressing genes. A. Representative flow profiles of SSEA4 / Tra181 expression of various hiPSC lines demonstrate that all derived clones represent a majority population expressing SSEA4 and Tra181. B. Based on the gating seen in (A), CD30 and CD9 flow profiles were assessed for each cell line. Only clones FTC8 clone 1 and FTi31 appeared to express CD30 while all clones expressed CD9. C. Relative Nanog expression was also assessed for each lineage. D. Summary table demonstrates that only CD30 expression correlates with Nanog expression.

[0098] Fig.12 . Removal of non-reprogrammed cells during reprogramming enriches reprogrammed cells. During the reprogramming process, there is a mixed population of reprogrammed and non-reprogrammed cells, with only a smaller population representing cells that have been reprogrammed to full pluripotency, as shown by SSEA4 and Tra181 expression (shown by black dashed arrows). However, when non-reprogrammed cells are removed or eliminated from the mixed population, a significant enrichment of SSEA4 and Tra181 positive populations is shown. As shown by the gray solid arrows, the CD13 negative cell population represents a significant enrichment of SSEA4 and Tra181 positive cells over CD13 low and high population cells.

[0099] Figure 13. Enhanced cell reprogramming and enrichment of pluripotent stem cells from various populations including IMR90 and ASC by single cell sorting. A. Enrichment of cell populations early in the reprogramming process by single cell sorting of SSEA4+ cells. The starting population of cells, including reprogrammed and non-reprogrammed cells, was enzymatically dissociated into single cells, stained for unique pluripotency markers, and sorted by immunoligated magnetic beads based on surface marker expression to obtain a population of cells positive for SSEA4. After this SSEA4+ cell enrichment, 10,000 SSEA4+ cells were plated on Matrigel TM Coated dishes were cultured in either conventional culture medium or SMC4 medium in a FF environment. AP staining was performed 8 days after sorting. The smaller inserts in the right corner are images of representative cell morphology. No alkaline phosphatase-positive colonies were detected in conventional culture while many alkaline phosphatase-positive colonies were derived from SMC4 medium. B. Scoring of Matrigel derived from the presence or absence of SMC4 medium TM SSEA4 on reprogramming of cells + / Tra181 +The number of colonies. C. iPSC clones derived from IMR90 fibroblasts or adipose stem cells under feeder-free conditions express pluripotency markers compared to single-cell culture using reprogramming with SMC4 medium. D. Whole-population flow cytometric analysis of iPSC clones derived using the single-cell sorting approach revealed that the majority of cells were positive for key pluripotency markers. E. iPSCs generated using single-cell sorting SSEA4 and feeder-free culture rather than their original source fibroblasts or adipose stem cells expressed an array of pluripotency markers, similar to human ESCs, including H1 and HuES9. F. Exogenous expression of reprogramming factors was effectively silenced in iPSCs generated using single-cell sorting and feeder-free culture, unlike control fibroblasts that expressed reprogramming factors 3 days after lentiviral infection. G. iPSCs generated using SSEA4-based single-cell sorting readily develop into all three somatic cell types once differentiated. Endoderm, FoxA2; mesoderm, α-smooth muscle actin (αSMA); ectoderm, β-tubulin III (βTUB III).

[0100] Figure 14. Single cell sorting for selection of unique pluripotent stem cell populations. A. Using FACS sorting, SSEA4 cells were selected early in the reprogramming process. + / Tra181 + The cells were isolated and transferred to SMC4+fibronectin medium and then switched to SMC4 medium without feeder layer after 24-72 hours. + / Tra181 + The 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 + The sorted cells developed into colonies while maintaining their SSEA4 and Tra181 expression.

[0101] Fig.15.High-throughput generation method and key characterization of iPSCs under feeder-free conditions. Three factor (Oct, Sox, Klf) induced fibroblasts were sorted in a two-step manner using SMC4 medium (SMC4+fibronectin medium when sorting) to deliver an efficient 96-well plate platform. Wells containing individual colonies were marked and expanded to 4×96-well plates. While one set was designed as a master plate and expanded, the other three plates were processed for characterization, including flow cytometry analysis for surface marker expression including SSEA4 and Tra181, qRT PCR for key marker expression including Nanog, transgene silencing, and immunofluorescence for pluripotency markers including Oct4 and Nanog. Based on characterization data interpretation, stacked selected hiPSC clones were expanded for further analysis. The data layers represent snapshots of clones studied during the high-throughput platform for hiPSC generation of FTC5 (marked by wells 31-40 of the 96-well plate). In the qPCR layer, expression was normalized to Gapdh. Nanog expression is associated with H1 hESCs while transgene expression is associated with day 4 post-infection with FTC5 (Day 4 infection). Based on the characterization profile, the hiPSC clones selected for the stack were expanded for further analysis. In a standout example, well 37 was identified as an expansion candidate based on its multi-parameter pluripotency profile and was named FTC5 Clone 1. Immunofluorescence images were acquired at 5× magnification.

[0102] Figure 16. High-throughput platform of 3-factor (polycistronic-OKS) hiPSCs in the presence of SMC4, cloned and FF-derived. A. FTi91 and FTi93 derived from the above strategy were stained for expression of pluripotency markers. B. qRT-PCR for exogenous expression of pluripotency markers for FTC1 (foreskin fibroblasts), H1 and Hues9 (hESC lines), FTi 91 and 93 (FTCI-derived hiPSC clones) and 4 days PI (4 days post infection). Expression was normalized to Gapdh and correlation within each gene set. C. Oct4 promoter methylation status. Open circles represent unmethylated CpG islands and black circles represent methylated CpG islands. D. EB formation and differentiation of FTi91 and 93 clones after 28 days of differentiation. Endoderm, FoxA2; Mesoderm, α-smooth muscle actin (αSMA); Ectoderm, Tuj1. E. Histological sections of teratomas derived from hiPSC clones generated and maintained in FF culture and SMC4. Black arrows point to regions of interest: mesoderm, white adipose tissue; ectoderm, neurons; endoderm, glands.

[0103] Figure 17. Generation and maintenance of pluripotent stem cells during reprogramming. SMC4 medium maintains genomic integrity and can enrich for pluripotent cells. A. Copy number variation as assessed by array comparative genomic hybridization. The bottom table is a summary of the interpretation of the data, showing the minimal copy number variation between human pluripotent stem cells cultured in SMC4 medium and their parental cell lines. BG Cytogenetic analysis of metaphase cells. The bottom table is a summary of the interpretation of the data, describing the stability of the genome after long-term feeder-free culture in SMC4 medium.

[0104] Figure 18. Characterization of hiPSCs derived from FTC5- and FTC7-derived clones under FF and SMC4 culture. To test the reproducibility of the high-throughput platform, additional patient-consented FTC5 and FTC7 were tested for hiPSC generation. A. Immunofluorescence staining of representatives of pluripotency markers Oct4, Tra181, Nanog, and Tra160, which are expressed in individual FTC5- and FTC7-derived hiPSC clones induced by OKS and generated using the multiplex platform. B. Lineage-specific staining of representatives of hiPSC-derived FTC5 and FTC7 28 days after induction of differentiation. Endoderm, FoxA2; Mesoderm, α-smooth muscle actin (αSMA); Ectoderm, Tuj1. C. Cytogenetic analysis of G-banded metaphase cells derived from FTC5- and FTC7-derived hiPSC clones after long-term FF and single-cell culture at various passages.

[0105] Figure 19. Methods for maintaining pluripotent cultures using cell surface markers. A. Further enrichment of pluripotent stem cell populations during culture as shown by enrichment of Tra181 in feeder-free and SMC4 platforms. B. During culture of pluripotent cell populations, a certain proportion of cells within the population may begin to differentiate and lose their pluripotency. Using a selection method for pluripotency, i.e., Tra181 enrichment, a homogenous culture of undifferentiated cells is obtained.

[0106] Fig. 20 . Isolation of differentiated cells from heterogeneous populations of non-, partially and fully pluripotent stem cells. FACS sorting of reprogrammed human fibroblast populations: When sorting cells that were double negative for the pluripotency marker SSEA4- / Tra181- (blue boxes; lower left box), no alkaline phosphatase (AP)-positive colonies were detected, indicating a loss of pluripotency and potential oncogenicity after one week of culture, whereas sorting of the SSEA4+ / Tra181+ double positive population (orange boxes, upper right box) resulted in the selection and enrichment of pluripotent cells, as evidenced by AP+ colony formation.

[0107] Figure 21. Cytokine-free culture of human pluripotent stem cells on feeder-free culture. A. When cultured on gelatin-coated surfaces and supplemented with SMC4 medium in the absence of any cytokines including bFGF, human pluripotent stem cells maintain their growth and morphology. B. Magnified images depict individual cells within a human pluripotent stem cell colony. C. Co-expression of SSEA4 and Tra181 at passage 3 further demonstrates that human pluripotent stem cells cultured without cytokines maintain their undifferentiated state. DETAILED DESCRIPTION OF THE INVENTION

[0108] The present invention provides a powerful culture system for culturing stem cells, including feeder-free conditions for producing and culturing induced pluripotent stem cells (iPSCs). Specifically, 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; single-cell dissociation of pluripotent cells; cell sorting of pluripotent cells; improving reprogramming efficiency; generation of naive pluripotent cells; and identification of markers for identification and selection of pluripotent cells. The culture medium and culture method of the present invention support the viability and survival of single-cell dissociated human stem cells, and maintain the undifferentiated state of stem cells to allow single-cell culture and passage of dissociated cells without differentiation.

[0109] definition

[0110] As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potency" or "increasing developmental capacity" refers to a method of increasing cell potency or dedifferentiating a cell to a less differentiated state. For example, a cell with increased cell potency has greater developmental plasticity (i.e., the ability to differentiate into more cell types) than the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell that is less differentiated than the same cell in a non-reprogrammed state.

[0111] As used herein, the term "potential" refers to the total number of developmental options (i.e., developmental capacity) of all available cells. It will be appreciated by those skilled in the art that cell potential is a continuum, ranging from the most plastic cells with the greatest developmental capacity, omnipotent stem cells to the most non-plastic cells with the smallest developmental capacity, terminally differentiated cells. The continuum of cell potential includes, but is not limited to, omnipotent cells, multipotent cells, specialized cells, oligopotent cells, unipotent cells, and terminally differentiated cells. In a strict sense, stem cells are pluripotent; therefore, any mature cell type can be produced. However, specialized cells, oligopotent cells, 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.

[0112] As used herein, the term "pluripotent" refers to the ability of cells to form the pedigree of all organisms or somatic cells (i.e., embryos). For example, embryonic stem cells are a type of pluripotent stem cells that can form cells derived from three germ layers, each of ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental capacity, ranging from incompletely or partially pluripotent cells (e.g., ectoderm stem cells or EpiSCs) that cannot produce a complete organism to more primitive, more capable cells (e.g., embryonic stem cells) that can produce a complete organism. The level of cellular pluripotency can be determined by assessing the pluripotency characteristics of cells. Pluripotency characteristics include, but are not limited to i) pluripotent stem cell morphology; ii) expression of pluripotent stem cell markers, including but not limited to 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 CD30 and CD50 as described herein; iii) the ability of mouse pluripotent stem cells to promote germline transmission in mouse chimeras; iv) the ability to determine the contribution of pluripotent stem cells to embryoid bodies using tetraploid complementation; v) teratoma formation of pluripotent stem cells; vi) formation of embryoid bodies; and vii) reactivation of inactivated X chromosomes.

[0113] As used herein, the term "pluripotent stem cell morphology" refers to the classic morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a round and small shape, a high nuclear-to-cytoplasmic ratio, the prominent presence of nucleoli, and typical intercellular spacing.

[0114] As used herein, the term "multipotent" refers to the ability of an adult stem cell to form multiple cell types of a lineage. For example, a hematopoietic stem cell can form all cells of the blood cell lineage, e.g., lymphocytes and myeloid cells.

[0115] "Adherence" refers to the attachment of cells to a vessel, e.g., to a sterile plastic (plastic-coated) cell culture dish or flask in the presence of an appropriate culture medium. Certain cell types cannot persist or grow in culture unless they adhere to the cell culture dish. Certain cell types ("non-adherent cells") do not require adhesion to remain and / or proliferate in culture.

[0116] "Cultivation" or "cell culture" refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. "Cell culture medium," "medium" (in each case the singular "medium"), "supplement" and "medium supplement" refer to the nutrient components of a culture of cultured cells.

[0117] "Cultivation" refers to the maintenance, proliferation (growth) and / or differentiation of cells outside of a tissue or in vitro, such as in a sterile plastic (or plastic-coated) cell culture dish or flask. "Culture" can use a culture medium as a source of nutrients, hormones and / or other factors that help to proliferate and / or maintain cells.

[0118] As used herein, "dissociated" cells refer to cells that are substantially separated or purified from other cells or from a surface (e.g., a culture dish surface). For example, cells can be separated from an animal or tissue by mechanical or enzymatic methods. Alternatively, cells that aggregate in vitro can be separated from each other, such as by enzymatically or mechanically dissociating into a suspension of clusters, single cells, or a mixture of single cells and clusters. Still in other optional embodiments, adherent cells are separated from a culture dish or other surface. Therefore, dissociation can include destroying the interaction of cells with the extracellular matrix and the matrix (e.g., the culture surface) or destroying the intercellular ECM.

[0119] As used herein, the terms "enrich" and "enrich" refer to increasing the amount of a specific component in a composition, such as a cellular component, and when used to describe a cell composition such as a cell population, "enriched" refers to a cell population that has a proportion of such components, the amount of a specific component, increased in proportion to the proportion of such components in the cell population before enrichment. For example, for a target cell type (i.e., cells with specific characteristics), a composition such as a cell population is enriched so that 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. Cell population enrichment can be performed for target cell types by cell selection and sorting methods known in the art. In some embodiments of the present invention, a cell population is enriched by a sorting or selection process described in the examples herein. In a specific embodiment of the present invention, for a method for enriching a target cell population, the cell population is enriched by at least about 20% for the target cell population, meaning that the enriched cell population contains about 20% more target cell types than the target cell types in the population before enrichment of the population. In one embodiment, the method for enriching a target cell population is such that the cell population is enriched by at least about 30+%, 40+%, 50+%, 60+%, 70+%, 80%, 85%, 90%, 95%, 97%, 98% or 99%, or at least about 98% or in specific embodiments, about 99% for the target cell population.

[0120] In certain aspects of the invention, the cell population is enriched for the amount of pluripotent cells or the pluripotency characteristics displayed by the cells. In a specific embodiment of the invention, the cell population to be reprogrammed is enriched for target cells with pluripotency characteristics such as expression of pluripotency markers, and the pluripotency markers include but are not limited to SSEA4, TRA 1-60, TRA-1-81, CD30 or CD50. In other specific embodiments of the invention, cell populations are consumed using surface markers specific for differentiation or non-pluripotent cells, such as non-pluripotent cells in the cell population to be reprogrammed, for example, surface markers specific for differentiation or non-pluripotent cells include CD13, CD26, CD34, CD45, CD31, CD46 or CD7. Therefore, the resulting cell population is described as a cell population enriched for pluripotent cells. In certain aspects of the invention, cells in the enriched cell population enriched for target cells have unique gene or protein expression profiles, for example, cell surface expression of at least two pluripotency markers such as SSEA4, TRA 1-60, TRA-1-81, CD30, and CD50. In some embodiments, the cell population is enriched for target cells expressing two or more pluripotency markers. In a specific embodiment, the cell population is enriched for target cells expressing SSEA4 in combination with Tra181 or Tra160. In a more specific embodiment, 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 target cell types, such as pluripotent cells.

[0121] Therefore, in some embodiments, the present invention provides a method for enriching a cell population for pluripotent cells, by sorting a cell population based on the cell surface expression of pluripotency markers, such as SSEA4, TRA 1-60, TRA-1-81, CD30 and CD50, and collecting cells expressing such markers to obtain a cell population enriched for pluripotent cells. In other embodiments of the present invention, a cell population is enriched for pluripotent cells, by sorting a cell population based on the cell surface expression of differentiation or differentiation cell markers, such as CD13, CD26, CD34, CD45, CD31, CD46 and CD7, and consuming a cell population of such cells to obtain a cell population enriched for pluripotent cells. In a specific embodiment, a cell population is sorted based on the expression of CD13, and CD13+ cells are removed from the cell population to obtain a cell population enriched for pluripotent cells.

[0122] As used herein, "feeder cells" or "feeder layers" are terms used to describe a type of cell co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, because the feeder cells provide growth factors and nutrients for the support of the second cell type. Feeder cells are optionally derived from different species along with the cells they support. For example, certain human cell types, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts. When co-cultured with other cells, feeder cells are usually inactivated, by radiation or with anti-mitotic dissociation agents such as mitomycin C to prevent them from being too large and unsuitable for the cells they support. Without limiting the foregoing, a specific feeder cell type can be a human feeder cell, such as human skin fibroblasts or human embryonic stem cells. Another feeder cell type can be mouse embryonic fibroblasts (mEF).

[0123] As used herein, a "feeder-free" (FF) environment refers to an environment such as a cell culture or culture medium that is substantially free of feeder cells and is not preconditioned by feeder cell culture. "Preconditioned" culture medium refers to culture medium in which feeder cells have been cultured for a period of time, such as culture medium that has been harvested after at least one day. Preconditioned culture medium contains a number of mediating substances, such as growth factors and cytokines secreted by feeder cells cultured in the culture medium.

[0124] In some embodiments of the present invention, the feeder-free environment is substantially free of human feeder cells and in specific embodiments, is additionally not preconditioned by feeder cells, which include, but are not limited to, human fibroblasts, keratinocytes, and embryonic stem cells. In other embodiments of the present invention, the feeder-free environment is substantially free of animal feeder cells, and further, in specific embodiments, is not preconditioned by feeder cells. In certain embodiments of the present invention, the feeder-free environment is substantially free of human and animal feeder cells and is not preconditioned by feeder cells.

[0125] Genomic stability refers to the ability of a cell to faithfully replicate DNA and maintain the integrity of the DNA replication process. As used herein to describe the cells of the present invention, "genome-stable cells" and "cells with genome stability" refer to cells that show mutations and chromosomal aberrations (such as translocations, aneuploidy, copy number variations, and duplications) at frequencies that are substantially similar to those of normal human cells.

[0126] "Ingredient" refers to any compound or other material, whether of chemical or biological origin, that can be used in a cell culture medium to maintain and / or promote growth and / or cell differentiation. The terms "component," "nutrient," and "ingredient" are used interchangeably. Conventional ingredients used in 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 ingredients that promote and / or maintain cell culture in vitro are selected by those skilled in the art as needed for the desired effect.

[0127] "Isolation" or "separation" refers to separation and collection of a composition or material from a natural environment, such as separation of individual cells or cell cultures from a tissue or body. In one aspect, a cell population or cell composition is substantially free of cells and materials associated with its nature. "Isolated" or "purified" or "substantially pure" 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 specific embodiments, at least about 95% pure, with respect to a target cell population, with respect to the target cells constituting the entire cell population. The purity of a cell population or composition can be assessed by appropriate methods well known in the art. For example, a substantially pure pluripotent cell population 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 specific embodiments, at least about 95%, and in certain embodiments, about 98% pure, with respect to the target cells constituting the entire cell population. The terms "substantially pure" and "substantially pure" are used interchangeably herein.

[0128] "Passaging" or "passaging" refers to the act of subdividing and plating cells onto multiple cell culture surfaces or vessels when the cells have proliferated to a desired extent. In some embodiments, "passaging" or "passaging" refers to subdividing, diluting, and plating cells. Because cells are passed from a primary culture surface or vessel to a subsequent set of surfaces or vessels, subsequent cultures may be referred to as "subcultures" or "first passages," etc. Each subdivision and plating onto a new culture vessel is considered a passaging.

[0129] "Plating" refers to placing a cell or cells in a culture dish so that the cells adhere and spread out on the cell culture dish.

[0130] "Pluripotency factor" refers to a preparation that can increase the developmental capacity of a cell, either alone or in combination with other preparations. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental capacity of a cell. For example, exemplary pluripotency factors include transcription factors and small molecule reprogramming agents. Transcription factors may refer to proteins (i.e., polypeptides) and polynucleotides encoding proteins, unless otherwise indicated in the usage herein. For example, exemplary transcription factors include Oct, Klf, Myc, and Sox polypeptides, and polynucleotides encoding these polypeptides. Examples of additional transcription factors are provided herein.

[0131] As used herein, the terms "polypeptide" and "protein" are used interchangeably unless otherwise specified and in the conventional sense, i.e., an amino acid sequence. Polypeptides are not limited to a particular length, e.g., they may comprise 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, etc., as well as other modifications known in the art, both naturally occurring and non-naturally occurring. The polypeptides used in the methods of the present invention are prepared using any of a variety of well-known recombinant and / or synthetic techniques.

[0132] The methods of the present invention, in certain embodiments, use active fragments of polypeptides described herein (e.g., Sox-2, c-Myc, Oct3 / 4, Klf4, Lin28, Nanog, etc., or substrates, cofactors and / or downstream effectors thereof), for example, 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 a specific embodiment, the fragment or combination of fragments used retains the ability to regulate, induce and / or maintain pluripotency when used in the methods described herein.

[0133] In other aspects, the invention uses 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). The polypeptide variants generally included in the invention typically exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity (e.g., as determined as described below) to the polypeptide sequences set forth herein along their length. In specific embodiments, the variants used or combinations of variants thereof retain the ability to induce pluripotency as described herein.

[0134] In other aspects, the invention employs polypeptide variants that exhibit at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity along their length to the corresponding region of a wild-type mammalian polypeptide used in the disclosure (e.g., as determined as described below).

[0135] Polypeptide variants differ from naturally occurring polypeptides in one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be produced synthetically, for example, by modifying one or more of the above polypeptide sequences used in the methods of the invention and evaluating their effects using any of the many techniques well known in the art.

[0136] "Proliferative" refers to the property of a cell to divide into two essentially identical cells or a population of cells to increase in number (eg, to reproduce).

[0137] "Proliferate" refers to the growth of cells (eg, regeneration via cell proliferation) outside a tissue or organism, for example, in a sterile container such as a plastic (plastic-coated) cell culture dish or flask.

[0138] "Primary culture" refers to cells, tissues and / or cultures in which isolated cells and culture medium are placed in a primary culture vessel. Cells, tissues and / or cultures may be maintained and / or proliferated, however, as long as the cells, tissues and / or cultures remain in the primary culture vessel, the cells, tissues and / or cultures are referred to as primary cultures.

[0139] The term "small molecule reprogramming agent" or "small molecule reprogramming compound" is used interchangeably herein and refers to a small molecule that can increase the developmental capacity of a cell alone or in combination with other pluripotency factors. "Small molecule" refers to a preparation having a molecular weight of less than about 5KD, less than about 4KD, less than about 3KD, less than about 2KD, less than about 1KD or less than about 5KD. Small molecules can be nucleic acids, mimetic peptides, peptoids, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and / or biological mixtures, such as fungal, bacterial, or seaweed extracts, are known in the art and can be screened with any assay of the present invention. In a specific embodiment, the small molecule reprogramming agent used herein has a molecular weight of less than 10,000 Daltons, for example, less than 8,000 Daltons, 6,000 Daltons, 4,000 Daltons, 2,000 Daltons, for example, 50-1,500 Daltons, 500-1,500 Daltons, 200-2,000 Daltons, 500-5,000 Daltons.

[0140] As used herein, the term "substantially free" is used interchangeably with "essentially free" and, when used to describe a composition, such as a cell population or culture medium, refers to a composition that is free of a specified substance, such as 95% free, 96% free, 97% free, 98% free, 99% free of a specified substance or undetectable by conventional assays. A similar meaning can be applied to the term "lack", where reference is made to the lack of a particular substance or component of a composition.

[0141] Cells used in the present invention

[0142] The starting cell population used in the present invention can be from substantially any suitable source and can be heterologous or homologous with respect to cell type or pluripotency state. In one embodiment, the cells are mammalian cells, and in a specific embodiment, the cells are isolated from a mammal selected from a rodent, a cat, a dog, a pig, a goat, a sheep, a horse, a cow, or a primate. In certain embodiments, the mammal is a human. In other embodiments, the cells to be used or treated in the present invention are adult cells, including substantially any available adult cell type.

[0143] Cells can be somatic, non-pluripotent, incompletely or partially pluripotent stem cells, multipotent cells, oligopluripotent cells, unipotent cells, terminally differentiated cells, or mixed cell populations comprising any of the foregoing combinations. The pluripotent cells used in the method of the present invention can be naturally occurring stem cells, including embryonic stem cells, or can be induced pluripotent stem cells. Human embryonic stem cells disclosed herein refer to cells obtained from pre-implantation blastocysts in vitro. "Mixed" cell populations are cell populations with varying degrees of developmental capacity. For example, a mixed cell population can include cells that are reprogrammed so that the mixed population includes pluripotent cells, partially pluripotent cells, and non-pluripotent cells, such as fully differentiated cells.

[0144] In one embodiment, the initial cell population is selected from adult or newborn stem / progenitor cells. In a specific embodiment, the initial stem / progenitor cell population is selected from mesodermal stem / progenitor cells, endodermal stem / progenitor cells and ectodermal stem / progenitor cells.

[0145] In other embodiments, the stem / progenitor cell group started is a mesodermal stem / progenitor cell. The 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, stem / progenitor cells in adipose tissue origin, hematopoietic stem / progenitor cells (HSCs), mesenchymal stem / progenitor cells, muscle stem / progenitor cells, kidney stem / progenitor cells, osteoblast stem / progenitor cells, chondrocyte stem / progenitor cells etc.

[0146] In other related embodiments, the starting stem / progenitor cell population is 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.

[0147] In other related embodiments, the starting stem / progenitor cell population is endodermal stem / progenitor cells. Illustrative examples of endodermal stem / progenitor cells include, but are not limited to, liver stem / progenitor cells, pancreatic stem / progenitor cells, epithelial stem / progenitor cells, and the like.

[0148] In certain embodiments, the starting cell population can be a heterologous or homologous cell population selected from 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, follicle cells, vascular cells, epithelial cells, immune cells, endothelial cells, etc.

[0149] Inducing cell reprogramming and increasing cell potential

[0150] Various strategies continue to be pursued in cells to induce pluripotency or increase potency (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 the efficiency of reprogramming (Shi et al., Cell Stem Cell 2, 525-528 (2008a); Shi et al., Cell Stem Cell 3, 534-535 (2008b); Shi et al., Cell Stem Cell 3, 545-556 (2008c); Shi et al., Cell Stem Cell 3, 557-558 (2008b); Stem Cell 3, 568-574 (2008b); Hua ngfu et al., Nat Biotechnol 26, 795-797 (2008a); Hua ngfu et al., Nat Biotechnol 26, 1269-1275 (2008b); Silva et al., Plos Bio 6, e253.doi:10.1371 / journal.pbio.0060253 (2008); Lyssiotis et al., PNAS 106, 8912-8917 (2009); Ichida et al., Cell Stem Cell 5, 491-503 (2009); Maherali, N., Hochedlinger, K., Curr Biol 19, 1718-1723 (2009b); Esteban et al., Cell Stem Cell 6, 71-79 (2010); Fe ng et al., Cell Stem Cell 4, 301-312 (2009)).

[0151] In general, techniques for reprogramming include directly or indirectly regulating specific cell pathways, using polynucleotide-, polypeptide- and / or small molecule-based methods. For example, by contacting cells with one or more pluripotency factors to increase cell potential. As used herein, "contact" can include culturing cells in the presence of pluripotency factors (such as, for example, small molecules, proteins, peptides, etc.) or introducing pluripotency factors into cells. By including factors such as transcription factors of proteins, under conditions that allow the introduction of transcription factors into cells, pluripotency factors can be introduced into cells. For example, see Zhou H et al., Cell Stem Cell. 2009 May 8; 4 (5): 381-4; WO / 2009 / 117439. For example, transient methods such as protein transduction, microinjection, non-integrating gene delivery, mRNA transduction, etc. or any other suitable technology can be used to promote introduction into cells. In some embodiments, transcription factors are introduced into cells by expression of a recombinant vector introduced into the cell or by incubating the cell in the presence of an exogenous transcription factor polypeptide so that the polypeptide enters the cell.

[0152] In a specific embodiment, the pluripotency factor is a transcription factor. Exemplary transcription factors associated with increasing, establishing, or maintaining cell potential 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, Sall4, Sall1, T if1, 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-Myc, 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.

[0153] Small molecule reprogramming agents are also pluripotency factors and can also be used in the methods of the invention to induce reprogramming and maintain or increase cell potency. In some embodiments of the invention, one or more small molecule reprogramming agents are used to induce pluripotency of somatic cells, increase or maintain cell potency or improve the efficiency of reprogramming.

[0154] In some embodiments, small molecule reprogramming agents are used in the methods of the present invention to increase the efficiency of reprogramming. The increase in reprogramming efficiency can be measured by (1) a reduction in the time required for reprogramming and pluripotent cell generation (e.g., by shortening the time to generate pluripotent cells by at least one day compared to a similar or identical process without a small molecule) or, alternatively or in combination, (2) an increase in the number of pluripotent cells generated by a particular process (e.g., by increasing the number of cells reprogrammed within a certain period of time by at least 10%, 30%, 50%, 100%, 200%, 500%, etc. compared to a similar or identical process without a small molecule). In some embodiments, a 2-fold or 20-fold increase in reprogramming efficiency is observed. In some embodiments, the reprogramming efficiency is increased by more than 20 times. In some embodiments, an efficiency greater than 100 times greater than that of a method without a small molecule reprogramming agent is observed (e.g., the number of pluripotent cells generated is increased by more than 100 times).

[0155] Several classes of small molecule reprogramming agents are important for increasing, establishing and / or maintaining cell potential. 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 calcium channel agonists; activators of the cAMP pathway; 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 detail below.

[0156] In some embodiments of the present invention, small molecule reprogramming agents are used to replace one or more transcription factors in the method of the present invention to induce pluripotency, improve the efficiency of reprogramming, and / or increase or maintain cell potential. For example, in some embodiments, cells are contacted with one or more small molecule reprogramming agents, including a dose sufficient to improve the efficiency of reprogramming. In other embodiments, in addition to transcription factors, one or more small molecule reprogramming agents are also used in the method of the present invention. In one embodiment, under conditions, cells are contacted with at least one pluripotency transcription factor and at least one small molecule reprogramming agent to increase, establish and / or maintain cell potential or improve the efficiency of the reprogramming process.

[0157] In other embodiments, 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 for a period of time and under conditions sufficient to increase, establish, and / or maintain the potency of the cells or to improve the efficiency of the reprogramming process. The pluripotency state or cell differentiation can be assessed by monitoring the pluripotency characteristics described elsewhere herein.

[0158] In one embodiment, the cell is contacted with a composition comprising one or more pluripotency factors and / or a combination of small molecule reprogramming agents, wherein the pluripotency factors and the small molecules increase or induce pluripotency of the cell. It is contemplated that the cells of the invention may be contacted in vitro, ex vivo, or in vivo.

[0159] Characterization of pluripotent cells

[0160] Following induction of reprogramming, reprogrammed cells can be selected based on relevant detectable morphological, molecular and / or biochemical changes associated with pluripotency. Specific features of cellular pluripotency, which can be monitored individually or in combination in assessing cell potential, include but are not limited to gene expression, methylation, and in vivo and in vitro characteristics such as: 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 / pro minin, CD140a, CD56, CD73, CD105, CD31, CD34, OCT4, Nanog and / or Sox2, and as provided herein, CD30 and CD50, and combinations of the foregoing; iii) the ability of pluripotent stem cells to promote germline transmission in mouse chimeras; iv) the ability of pluripotent stem cells to contribute to embryoid bodies using tetraploid compensation; v) teratoma formation of pluripotent stem cells; vi) embryoid body formation and in vitro tri-lineage differentiation; and vii) reactivation of inactivated X chromosomes. In certain embodiments, subtypes of any of the above characteristics are used to monitor cell pluripotency. 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.

[0161] As discussed herein, pluripotency exists as a continuum and induced pluripotent stem cells seem to exist with "mature" state and "primary" state, and the cells in the primary state may have greater differentiation potential.The induced pluripotent stem cells produced in conventional culture medium exist with mature state and are more closely similar to the cell derived from the blastocyst after implantation, and original iPSC has shown the pluripotency characteristics of the cell more closely similar to mouse embryonic stem cell or derived from the blastocyst before implantation.Can define mature and original cell state by various differences, comprise the difference of colony morphology, to suppress or activate the cell response of key signal transduction, gene expression signal and the ability of reactivating the gene relevant with embryonic extracellular cell.For example, conventional iPSC, represents mature pluripotent state, has shown flat colony morphology, and original iPSC has shown the tight hemispherical colony morphology similar to mouse embryonic stem cell. Furthermore, conventional iPSCs require extrinsic signaling of key cytokines such as TGFβ, activin, and bFGF, and rely on ERK / MEK cell signaling to maintain an undifferentiated state, and differentiate when these pathways are inhibited, for example, by contacting the cells with TGFβ or MEK inhibitors. In contrast, naive cells do not require extrinsic signaling and remain pluripotent even when treated with inhibitors of the TGFβ and MEK signaling pathways.

[0162] Additionally, gene expression analysis revealed significant differences between naive and mature pluripotent cells. For example, naive iPSCs significantly suppressed Xist expression while conventional iPSCs showed only moderate suppression of Xist expression; naive cells showed significant X chromosome reactivation and increased gene expression on the X chromosome, exceeding that seen in conventional iPSCs; naive cells expressed extraembryonic stem cell markers, including but not limited to Gata6, CDX2, and CGB. In contrast, early differentiation markers, such as lineage-specific genes, such as Foxa2, Sox17, and Brachyury, exceeded naive iPSCs and were more highly expressed in conventional iPSCs. Additional markers for identifying cells in the naive state of pluripotency include increases in Klf4, Tbx3, Gbx2, Lin28, Soc3 or decreases in Otx2, Sox17, Cer1, FoxA2, ​​Zic1, Lhx2, and Xist.

[0163] In specific embodiments of the invention, the naive cells show at least two-fold, at least five-fold, or at least ten-fold reduction in Xist expression compared to conventional iPSCs. In some embodiments of the invention, the naive state of pluripotency has twice as much Xist expression as conventional iPSCs and three-fold higher expression levels of at least five genes located on chromosome X than conventional iPSCs.

[0164] X chromosome reactivation can be demonstrated by expression levels of at least 5 genes, at least 10 genes, or in specific embodiments, at least 100 genes located on the X chromosome that exceed the levels of such genes in conventional iPSCs by at least two-fold, three-fold, five-fold or more.

[0165] In specific embodiments of the invention, the pluripotent cells of the invention retain characteristics of pluripotency for multiple cell passages, for example, for at least 1, 3, 5, 7, 10, 15, 20 or more passages.

[0166] Culture medium platform used in the method of the present invention

[0167] The culture medium (i.e., culture platform) of the present invention comprises a chemically defined standing basal medium in combination with various small molecules, including small molecule inhibitors, which allow:

[0168] Long-term culture of pluripotent cells in a feeder-free environment;

[0169] Cells are reprogrammed in a feeder-free environment;

[0170] Single-cell dissociation of pluripotent cells;

[0171] Cell sorting of pluripotent cells;

[0172] Maintenance of the undifferentiated state;

[0173] Improved reprogramming efficiency; and

[0174] Generation of naive pluripotent cells.

[0175] The chemically defined stock basal medium used in the culture medium of the present invention can be any defined basal medium suitable for supporting the maintenance, growth and / or differentiation of stem cells, such as a conventional human embryonic stem cell culture medium. Examples of defined basal medium used in the present invention include, but are not limited to, Dulbecco's Modified Eagle Medium ("DMEM"), Basal Medium Eagle (BME), DMEM / F-12 (1:1 DMEM to F-12 volume:volume), Medium 199, F-12 (Ham) Nutrient Mixture, F-10 (Ham) Nutrient Mixture, Minimal Essential Medium (MEM), Williams' Medium E, and RPMI1640, all of which are available from Gibco-BRL / Life Technologies, Gaithersburg, Maryland, among others. Several versions of many of these media may be used, and those particularly useful in constructing the media of the present invention include, but are not limited to, DMEM 11966, DMEM 10314, MEM 11095, Williams' Medium E 12251, Ham F12 11059, αMEM 12561, and Medium-19911151 (all available from Gibco-BRL / Life Technologies, Inc. (1995-1996 catalog). For example, the culture medium may include one or more of the following: amino acids, vitamins, organic salts, inorganic salts, trace components, buffer salts, sugars, ATP, etc. (suitable basal media components are available from Sigma-Aldrich, St. Louis, MO).

[0176] The small molecules used in the cell culture medium of the present invention and their types are described in more detail below. In specific embodiments, the culture medium of the present invention comprises one or more, two or more or three or more TGFβ, GSK3 inhibitors, MEK inhibitors and ROCK inhibitors. In certain embodiments, the culture medium of the present invention comprises TGFβ, GSK3 inhibitors, MEK inhibitors and ROCK inhibitors. Exemplary TGFβ, GSK3 inhibitors, MEK inhibitors and ROCK inhibitors used in the cell culture medium and methods of the present invention are described below. The culture medium may additionally comprise a PARP inhibitor, such as Olaparib (AZD-2281).

[0177] GSK-3β inhibitors

[0178] GSK-3β inhibitors include, but are not limited to, antibodies that bind to GSK-3β, dominant negative GSK-3β variants and siRNA, 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, CT 99021, CT 20026, SB216763, AR-A014418, lithium, SB 415286, TDZD-8, BIO, BIO-acetone oxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridinecarbazole-cyclopentadiene ruthenium complex, TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazosin-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridinyl)-[1,3,4]-oxadiazole, OTDZT, α-4-p-bromoacetophenone, AR-AO 144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazine-2-yl-pyrrole-2,5-dione; TWS119 pyrrolopyrimidine compound, L803 H-KEAPPAPPQSpP-NH2 or its octadecyl form; 2-chloro-l-(4,5-dibromo-thiophen-2-yl)-ethanone, SB216763, and SB415286. Exemplary GSK3 inhibitors used in the cell culture medium of the present invention include CHIR99021, BIO and Kenpaullone, and CHIR99021 is preferred in a specific embodiment.

[0179] ERK / MEK inhibitors

[0180] Exemplary ERK / MEK pathway inhibitors include but are not limited to MEK or ERK antibodies, dominant negative MEK or ERK variants, and siRNA and antisense nucleic acids that inhibit MEK and / or ERK expression. Other exemplary ERK / MEK inhibitors include but are not limited to PD0325901, PD98059, UO126, SL327, ARRY-162, PD184161, PD184352, sunitinib, sorafenib, vandetanib, pazopanib, axitinib, GSK1120212, ARRY-438162, RO5126766, XL518, AZD8330, RDEA119, AZD6244, FR180204 and PTK787.

[0181] Additional ERK / MEK inhibitors include those compounds disclosed in International Published Patent Applications WO 99 / 01426, WO 02 / 06213, WO 03 / 077914, WO 05 / 051301 and WO 2007 / 044084.

[0182] Further exemplary ERK / MEK inhibitors include the following compounds: 6-(4-bromo-2-chloro-aniline)-7-fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2,3-dihydroxy-propoxy)-amine; 6-(4-bromo-2-chloro-aniline)-7-fluoro-3-(tetrahydro-pyran-2-ylm-ethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amine, 1-[6-(4-bromo-2-chloro-aniline)-7-fluoro-3-(tetrahydro-pyran-2-ylm-ethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amine, -2-chloro-aniline)-7-fluoro-3-methyl-3H-benzimidazole-oxazol-5-yl]-2-hydroxy-ethanone, 6-(4-bromo-2-chloro-aniline)-7-fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amine, 6-(4-bromo-2-chloro-aniline)-7-fluoro-3-(tetrahydrofuran-2-ylm-ethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amine -ethoxy)-amine, 6-(4-bromo-2-fluoro-aniline)-7-fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2-hydroxy-ethoxy)-amine, 6-(2,4-dichloro-aniline)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amine, 6-(4-bromo-2-chloro-aniline)-7-fluoro-3-methyl-3H-benzimidazole-e-5-carboxylic acid (2-hydroxy-ethoxy)-amine oxy)-amine, hereinafter referred to as MEK inhibitor 1; 2-[(2-fluoro-4-iodophenyl)amine]-N-(2-hydroxyethoxy)-1,5-dimethyl-6--oxo-1,6-dihydropyridine-3-carboxylic acid amine; hereinafter referred to as MEK inhibitor 2; and 4-(4-bromo-2-fluorophenylamine)-N-(2-hydroxyethoxy)-1,5-dimethyl-6--oxo-1,6-dihydropyridine-3-carboxylic acid amine or a pharmaceutically acceptable salt thereof. In certain embodiments, the MEK / ERK inhibitor used in the cell culture medium of the present invention is PD98059.

[0183] TGFβ receptor / ALK5 inhibitors

[0184] Exemplary ALK5 inhibitors include ALK5 antibodies, dominant negative variants of ALK5, and antisense nucleic acids that inhibit ALK5 expression. 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, for example, see, WO2008 / 006583, incorporated herein by reference.

[0185] Additionally, while "ALK5 inhibitors" are not intended to include non-specific kinase inhibitors, it is understood 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. Phamacol. 62(1):65-74 (2002).

[0186] In view of the data herein showing the effects of inhibiting ALK5, it is believed that inhibiting the TGFβ / activator pathway has a similar effect. Therefore, any inhibitor, such as upstream or downstream of the TGFβ / activator pathway can be combined with ALK5 or used in place of the ALK5 inhibitor described in each paragraph herein. Exemplary TGFβ / activator pathways include, but are not limited to, TGFβ receptor inhibitors, inhibitors of SMAD 2 / 3 phosphorylation, inhibitors of SMAD 2 / 3 and SMAD 4 interactions, and SMAD 6 and SMAD 7 activators / agonists. Moreover, the classifications described below are for organizational purposes only, 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 classification.

[0187] TGFβ receptor inhibitors can include antibodies, dominant negative variants and siRNA or antisense nucleic acids targeting TGFβ receptor. Specific examples of inhibitors include, but are not limited to, SU5416; 2-(5-benzo[l,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinium hydrochloride (SB-505124); lerdelimμMb (CAT-152); metelimμMab (CAT-192); GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580 276; LY364947; LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; 3,5,7,2',4'-pentylhydroxyflavone (molin); activin-M108A; P144; soluble TBR2-Fc; and tumor cells transfected with antisense targeting the TGFβ receptor. (See, e.g., Wrzesinski et al., Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska et al., Acta Biochimica Polonica 52(2):329-337 (2005); and Chang et al., Frontiers in Bioscience 12:4393-4401 (2007).

[0188] Exemplary TGFβ receptor inhibitors used in the cell culture medium of the present invention include SB431542, A-83-01 and RepSox. In a specific embodiment, the TGFβ inhibitor is SB431542.

[0189] ROCK inhibitors

[0190] ROCK is a serine / threonine kinase that acts as a target protein Rho (of which there are three subtypes - RhoA, RhoB and RhoC). Exemplary ROCK inhibitors include, but are not limited to, ROCK antibodies, dominant negative ROCK variants, and siRNA and antisense nucleic acids that inhibit ROCK expression. Other exemplary ROCK inhibitors include, but are not limited to, thiazovivin, Y27632, fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxy-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)-cyclohexylamide.

[0191] Exemplary ROCK inhibitors for use in the cell culture medium of the present invention include thiazovivin, Y27632, pyrintegrin, and Blebbistatin. In certain embodiments, the ROCK inhibitor is thiazovivin.

[0192] FGFR inhibitors

[0193] Exemplary FGFR inhibitors include but are not limited to FGFR antibodies, dominant negative FGFR variants and siRNA and antisense nucleic acids that inhibit FGFR expression.Other exemplary FGFR inhibitors include but are not limited to RO-4396686, CHIR-258, PD173074, PD 166866, ENK-834, ENK-835, SU5402, XL-999, SU6668, CHIR-258, R04383596 and BIBF-1120.

[0194] PARP inhibitors

[0195] PARP inhibitors inhibit poly ADP-ribose polymerase ("PARP"). PARP proteins are DNA repair enzymes that function to regulate DNA repair pathways in cells. PARP is involved in the clip excision repair (BER) pathway, and PARP inhibition can promote the stability of the cell genome during reprogramming or maintenance of pluripotent cells. Exemplary PARP inhibitors used in the cell culture media of the present invention include, but are not limited to, iniparib, veliparib, and olaparib (AZD-2281).

[0196] The amount of small molecules in the cell culture medium of the present invention can vary and be optimized according to the specific culture conditions, including the specific molecules and combinations used, the cell types cultured in the culture medium, and the specific application for which the culture medium of the present invention is used. In some embodiments, the small molecule is present in the culture medium at a concentration sufficient to induce pluripotency, increase the efficiency of reprogramming, or increase or maintain the potency of the cell.

[0197] In specific embodiments, preferred concentrations and combinations of small molecules in the cell culture medium of the present invention are shown in Table 1. In specific embodiments of the cell culture medium of the present invention, the cell culture medium is "SMC4" medium, as shown in Table 1. SMC4 medium comprises conventional human ESC culture medium and specific pathway regulators and additives as shown in Table 1. The culture medium components are present in the culture medium in amounts within the optimal ranges of such components as shown in Table 1, and are present at the optimal concentrations shown in Table 1. Embodiments of the SMC4 culture medium may optionally comprise any one or more of the optional culture media and pathway regulators and activators shown in Table 2, at concentrations within the optimal ranges shown in Table 2, and in certain embodiments, at concentrations within the optimal concentrations shown in Table 2. In specific embodiments of the culture medium, the SMC4 culture medium comprises soluble fibronectin, and is referred to herein as "SMC4+fibronectin".

[0198] In some embodiments, the culture medium of the present invention further comprises one or more Oct polypeptides, Klf polypeptides, Myc polypeptides and Sox polypeptides. In some embodiments, the culture medium does not comprise cells. In some embodiments, the culture medium further comprises cells, for example, non-pluripotent cells, partially pluripotent cells, pluripotent cells or a mixed cell population comprising cells of various potential states.

[0199] Table 1. Cell culture components and additives. The following table lists examples of molecules and the signaling pathways they affect that can be used to enhance the reprogramming process and maintain pluripotent stem cells in an undifferentiated state.

[0200]

[0201]

[0202] SMC4+fibronectin refers to the above-mentioned SMC4 medium to which soluble fibronectin is added at a concentration of about 5 μg / mL. Fibronectin may be present in the SMC4 medium at a concentration of about 0.05-500 μg / mL.

[0203] Table 2. Specific cell culture components and additives that can be selected to enhance the pluripotency and viability of cells cultured in single cell passaging, cell sorting, and feeder-free systems

[0204]

[0205]

[0206] Cytokines and Growth Factors

[0207] In some embodiments of the invention, the cell culture medium of the invention is substantially free of cytokines and / or growth factors, and is optionally a feeder-free environment. In other embodiments, the cell culture medium comprises additives such as serum, extracts, growth factors, hormones, cytokines, etc.

[0208] Various growth factors and their use in culture media are well known in the art and include, for example, ECM proteins, laminin 1, fibronectin, type IV collagen isoforms, protease inhibitors, cell surface adhesion proteins, cell signaling proteins, cadherins, intracellular chloride channel protein 1, transmembrane receptor PTK7, insulin-like growth factor, inhibin βA, inducers of the TGFβ / activin / nodal signaling pathway, and activin A. For example, the cytokines used in the culture medium may include 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 β (TGF-β), vascular endothelial growth factor (VEGF), transferrin, various interleukins (such as IL-1 to IL-18), various colony stimulating factors (such as granulocyte / macrophage colony stimulating factor (GM-CSF)), various interferons (such as gamma interferon) and other cytokines with effects on stem cells, such as stem cell factor (SCF) and erythropoietin (Epo). These cytokines are commercially available, for example, from R&D Systems, Minneapolis, Minnesota, and may be natural or recombinant. In some embodiments, for the culture of a variety of mammalian cells, the basal medium will contain FGF at a concentration of about 0.01-100 ng / ml, about 0.2-20 ng / ml, and in specific embodiments about 0.5-10 ng / ml. Other cytokines, if used, will be added at concentrations determined empirically or as directed by established cytokine technology.

[0209] Additional components that may be included in the culture medium of the present invention are insulin (particularly insulin Zn ++) and transferrin. These additional ingredients, commercially available (e.g., from Sigma-Aldrich, St. Louis, Missouri), can be formulated into the culture medium of the present invention at a concentration range of about 0.1-about 100 μg / ml or about 1 μg / ml-about 10 μg / ml. Additionally, recombinant insulin or insulin basic zinc salts can be replaced with animal-derived or human-derived insulin. Other ingredients or substitutes can be added to those added components known to those of ordinary skill in the art.

[0210] Cytokines and similar supplemental components may be included in the basal medium in the place of (or in addition to) this type of component. Such components are often included with the supplemental components because the supplemental components are routinely stored at about -20°C rather than the about 4°C temperature routinely used to store the basal medium. Cytokines and similar components generally work better at temperatures closer to -20°C.

[0211] Substrate used in the method of the present invention

[0212] Any suitable vessel or cell culture dish can be used as a support for cell culture and / or cell culture additives in the basal medium. No matrix coating is required on the support. However, coating the surface of the culture dish with an adhesion-promoting matrix (e.g., collagen, fibronectin, polypeptides containing RGD, gelatin, etc.) promotes cell attachment and thus enhances the effect of the cell culture medium and the additives disclosed herein. Suitable matrices for culturing and passage cells are known in the art and include, but are not limited to, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, such as Matrigel TM A mixture of naturally occurring cell line-produced matrices as well as synthetic or artificial surfaces, such as polyamine monolayers with carboxyl-terminated monolayers.

[0213] Feeder-free environment

[0214] Although cells are usually cultured on feeder cells or in a culture environment preconditioned with feeder cells and containing fetal bovine serum, such an environment is not suitable for the production of cells for clinical and therapeutic use. For example, cells cultured in such an exogenously contaminated environment are generally considered unsuitable for human cell transplantation because exposure to animal components may present a serious risk of immune rejection and transmission of unidentified pathogens to the treated patient, and may potentially reactivate the animal's retroviruses. The use of a culture system without an animal culture medium, such as the feeder-free environment of the present invention, promotes the production of clinical-grade cell lines, particularly hESC and iPSC cell lines.

[0215] In some embodiments of the present invention, the feeder-free environment of the present invention is substantially free of human feeder cells, including but not limited to human fibroblasts, keratinocytes and embryonic stem cells, and is not preconditioned by feeder cells. In other embodiments of the present invention, the feeder-free environment is substantially free of animal feeder cells, and in addition, in some embodiments, the feeder-free environment is not preconditioned with feeder cells. In specific embodiments of the present invention, the feeder-free environment is substantially free of human and animal feeder cells, and in more specific embodiments, the feeder-free environment is substantially free of human and animal feeder cells and is not preconditioned with feeder cells.

[0216] The feeder-free cell culture medium of the present invention is used in the methods of the present invention, including the culture of pluripotent cells, the reprogramming of cells, the single cell dissociation of pluripotent cells, the cell sorting of pluripotent cells, the generation of naive pluripotent cells and the maintenance of the undifferentiated state of cells. In a specific method of the present invention, the feeder-free environment is used in a method for inducing pluripotency, improving the efficiency of reprogramming, and / or increasing or maintaining cell potential. In certain embodiments, the feeder-free environment is substantially free of cytokines and growth factors, including bFGF.

[0217] Dissociation

[0218] Cell dissociation into single cells can be achieved by enzymatic or mechanical methods, such as dissociation into a single cell suspension. Any enzymatic agent known in the art that allows cell dissociation 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 selection agent, type IV collagenase and neutral protease.

[0219] Chelating agents, such as EDTA, Accutase or AccuMax, can also be used alone or in combination with enzymatic agents to dissociate cells in the methods of the present invention. Dissociation agents can be dissolved in PBS without calcium and magnesium to facilitate dissociation into single cells.

[0220] To enhance the survival of cells 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 culture medium are dissociated and single cells are placed in cell culture of the present invention with one or more small molecule inhibitors, such as SMC4 culture medium or SMC4+ fibronectin. Optionally, the dissociated single cells are placed in a feeder-free environment. In other embodiments, cells are cultured in a feeder-free environment before dissociation and placed in cell culture of the present invention with one or more small molecule inhibitors, such as SMC4 culture medium or SMC4+ fibronectin, which may optionally be a feeder-free environment.

[0221] Enzymatic dissociation into single cells is supported by mechanical forces. Alternatively, the dissociation agent may be mechanical force only, such as by using a mechanical tool, such as a pipette or a pointed microcapillary to separate the cells.

[0222] General techniques in cell culture and media collection are summarized in: Large-Scale Mammalian Cell Culture (Hu et al., Curr. Opin. Biotechnol. 8: 148, 1997); Serum-Free Media (K. Kitano, Biotechnology 17: 73, 1991); Large-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 interesting readings include Understanding Media (M. McLuhan, Mentor NY, 1964) and Media as Information (M. McLuhan & Q. Fiore, Bantam NY, 1967).

[0223] For a detailed description of the general techniques used in the practice of the present invention, practitioners can refer to standard textbooks and reviews in cell biology, tissue culture, and embryology. These include: "Teratocarcinoma and Embryonic Stem Cells: A Practical Approach" (EJ Robertson, ed., IRL Press Ltd. 1987); "Technical Guidelines in Mouse Development" (PM Wasserman et al. eds., Academic Press 1993); "In Vitro Differentiation of Embryonic Stem Cells" (MV Wiles, Meth. Enzymol. 225:900, 1993); "Properties and Uses of Embryonic Stem Cells" (PD Rathjen et al., 1993). Stem cell differentiation is reviewed in Robertson, Meth. Cell Biol. 75:173, 1997 and Pedersen, Reprod. Fertil. Dev. 10:31, 1998.

[0224] Enrichment and depletion strategies

[0225] The present invention also provides a strategy for enriching a cell population of pluripotent cells as a method for increasing the efficiency of producing iPSC. The enrichment strategy provides a method for deriving cloned iPSC colonies in a relatively short period of time, which improves the efficiency of iPSC production. The enrichment method of the present invention includes sorting cell populations, which have been induced to reprogram to identify and obtain cells expressing pluripotency markers, thereby obtaining a cell population of enriched pluripotent cells. The cells to be sorted have been induced to reprogram and may include a mixed cell population for reprogramming, so that the population includes pluripotent cells, partial pluripotent cells, and non-pluripotent cells, such as terminally differentiated cells. In one embodiment, the cell population to be sorted has been induced to reprogram and express pluripotency markers. In some embodiments, cells are cultured after inducing reprogramming for about 4 days to 30 days, about 4 days to 24 days, about 6 days to 22 days, or about 8 days to about 12 days. Additional enrichment methods involve the consumption of expressing differentiation markers or non-pluripotent cells to obtain enriched pluripotent cell populations.

[0226] The enrichment strategy of the present invention includes obtaining a single cell suspension of a cell population to be sorted. In one embodiment of the present invention, a single cell suspension is obtained by dissociating cells in a population and resuspending the cells. The dissociated cells can be resuspended in any suitable solution or culture medium for maintaining cells or performing cell sorting. In a specific embodiment, the single cell suspension comprises one or more TFGβ inhibitors, GSK3 inhibitors, MEK inhibitors and Rock inhibitors. 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.

[0227] In the enrichment process of the present invention, cells are sorted to obtain pluripotent cells or to deplete non-reprogrammed or non-pluripotent cells, thereby obtaining an enriched population of pluripotent cells. In one embodiment, a single cell suspension is prepared and then single cells are prepared for sorting, such as by staining with pluripotency markers, for example using appropriate antibodies. Cells can be sorted by any suitable method for sorting cells, such as by magnetic beads or flow cytometry (FACS) sorting.

[0228] Cells are sorted based on various pluripotency markers, including Oct, Sox, Nanog, SSEA3 / 4, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD105, CD31, CD34, OCT4, KLF4, SSEA1 (mouse), and CD30 and CD50 as shown in the present invention. In various embodiments, cells are sorted based on at least two, at least three, or at least four pluripotency markers. In certain embodiments, cells are sorted based on the expression of SSEA4, and in certain specific embodiments, cells are sorted based on the expression of SSEA4 in combination with TRA181 or TRA160. In certain embodiments, cells are sorted based on SSEA4, Tra181 or TRA160 and CD30. In certain embodiments, initially, cells are depleted for non-reprogrammed cells using surface markers of differentiated 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.

[0229] After sorting to obtain cells that are positive for pluripotency markers, the ideal cell fraction is a cell population rich in pluripotent cells. The population rich in pluripotent cells can be placed in a cell culture system, such as a conventional hESC culture medium or a cell culture medium of the present invention. The cell culture system can be supplemented with feeder cells or optionally be a feeder-free environment. 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 specific pathway regulators and additives shown in Tables 1 and 2. In one embodiment, the cell culture medium is a feeder-free environment and contains one of 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. In other specific embodiments of the present invention, the cell culture system is a culture system containing Matrigel TM Coated tissue plates, conventional hESC culture medium, and specific pathway modulators 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 optional culture media and pathway modulators shown in Table 2.

[0230] The enriched cell population can be cultured in the cell culture system described herein to obtain iPSC colonies, which usually appear about 3 days to about 25 days after sorting; about 5-9 days after sorting or about 5-7 days after sorting. iPSC colonies can be selected or sorted for clonal expansion. Using the enrichment strategy of the present invention, a cell population enriched for 3 times pluripotent cells is obtained.

[0231] The present invention also provides a method for consuming a cell population of undesirable cells. In some embodiments, a cell population, such as a cell population or a pluripotent cell population that is reprogrammed, consumes differentiated cells. In the method of the present invention, a pluripotent cell population or cell that is induced to reprogram can consume cells with cell surface markers of differentiated cells. Based on surface markers of differentiated cells, such as CD13, CD26, CD34, CD45, CD31, CD46, or CD7, cell populations can be sorted, and cells expressing markers of differentiated cells can be removed from the cell population to obtain a cell population rich in pluripotent cells. In a specific embodiment of the present invention, CD13 is used as a surface marker of differentiated cells.

[0232] In other embodiments, a cell group to be differentiated is induced, such as a cell group to be differentiated into a desired lineage, and pluripotent cells are consumed to obtain a differentiation group or differentiated cells. In some embodiments, a differentiated cell group includes a cell group that has been induced to differentiate into a specific lineage, such as ESC or iPSC. Using the sorting techniques described above, such as sorting cells in a population according to magnetic beads or FACs based on pluripotency markers, a cell group can consume pluripotent cells. In some embodiments, pluripotency markers are used by FACs to sort a cell group comprising differentiated cells and obtain a small portion of cells expressing pluripotency markers. In other embodiments, a cell group is sorted based on differentiation markers, such as lineage-specific markers, by FACs to obtain a small portion of pluripotency markers consumed. CD13 is used as a surface marker for differentiated cells in a specific embodiment of the present invention.

[0233] The practice of the present invention will employ, unless expressly indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, cell biology, stem cell techniques, cell culture and transgenic biology, which are within the skill of the art. Many of these are described below for illustrative purposes. Such techniques are fully explained in the literature, for example, see 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 Techniques in Molecular Biology (John Wiley and Sons, updated July 2008); Brief Techniques in Molecular Biology: An Overview of Methods Derived from Current Techniques in Molecular Biology, Greene Pub. Associates and Wiley-interscience; Glover, DNA Cloning: A Practical Approach, Vol. I & II (IRL =Press, Oxford, 1985); Anand, Techniques for Complex Genomic Analysis (Academic Press, New York, 1992); Guthrie & Fink, A Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, Eds., 1985); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Animal Cell Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Fire et al., RNA Interference Technology: From Basic Science to Drug Discovery (Cambridge University Press, Cambridge, 2005); Schepers, RNA Interference in Practice (Wiley-VCH, 2005); E. Ngelke, RNA Interference (RNAi): The Nuts and Bolts of siRNA Technology (DNA Press, 2003); Gott, RNA Interference, ed. and Revised: Methods and Techniques (Methods in Molecular Biology; Man Press, Totowa, NJ, 2004); Sohail, Gene Silencing by RNA Interference: Techniques and Applications (CRC, 2004); Clarke and Sanseau, MicroRNAs: Biology, Function & Expression (Nuts & Bolts Series; DNA Press, 2006); Immobilized Cells and Enzymes (IRL Press, 1986); Speech, Methods in Enzymology (Academic Press, Inc., New York); Gene Transfer Vectors for Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Riott, Basic Immunology, 6th ed., (Blackwell Scientific Publishers, Oxford, 1988); Embryonic Stem Cells: Methods and Techniques (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2002); Embryonic Stem Cell Technology: Volume I: Isolation and Characterization (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Embryonic Stem Cell Technology: Volume II: Differentiation Models (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Turksen, Ed., 2006); Human Embryonic Stem Cell Technology (Methods in Molecular Biology) (Kursad Turksen Ed., 2006); Mesenchymal Stem Cells: Methods and Techniques (Methods in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney, and Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell Technology (Methods in Molecular Medicine) (Christopher A. Klug and Craig T. Jordan Eds., 2001); Hematopoietic Stem Cell Technology (Methods in Molecular Medicine) (Kevin D. Bunting Ed., 2008) Neural Stem Cells: Methods and Techniques (Methods in Molecular Biology) (Leslie P. Weiner Ed., 2008); Hogan et al., Methods for Manipulating Mouse Embryos (2nd ed., 1994); Nagy et al., Methods for Manipulating Mouse Embryos (3rd ed., 2002), and the zebrafish book, A Laboratory Guide to Working with Zebrafish (Danio rerio), 4th ed., (University of Oregon Press, Eugene, OR, 2000).

[0234] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0235] Throughout this specification, unless the context requires otherwise, it should be understood that the words "comprise", "comprises" and "comprising" imply the inclusion of a specified step or ingredient or group of steps or ingredients, but do not exclude any other step or ingredient or group of steps or ingredients. By "consisting of" is meant to include and be limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed ingredients are required or mandatory, and no other ingredients are present. By "consisting essentially of" is meant to include any ingredient listed after the phrase, and be limited to other ingredients that do not interfere with or contribute to the activity or activity specified in the disclosure of the listed ingredients. Thus, the phrase "consisting essentially of" indicates that the listed ingredients are required or mandatory, but other ingredients are optional and may or may not be present, depending on whether they affect the activity or activity of the listed ingredients.

[0236] The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to in this specification and / or listed in the application data page are incorporated by reference in their entirety. If necessary, various aspects of the embodiments can be modified to provide other embodiments applying the concepts of the various patents, applications, and publications.

[0237] These and other changes may be made to the embodiments in light of the above detailed description. In summary, in the appended claims, the terms used should not be interpreted as limiting the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments, and the full range of equivalents of such claims to which rights are granted. Therefore, the claims are not limited to the disclosed contents. Example

[0238] Example 1: Experimental method

[0239] A. Cell Culture of Pluripotent Stem Cells

[0240] Before adapting to the feeder-free layer, human induced pluripotent stem cells (hiPSC) were maintained on feeder cells (mouse embryonic fibroblasts (MEF) cells (Millipore) treated with mitomycin C) and cultured with conventional culture medium. As used in this application, "conventional culture medium" refers to a basic human embryonic stem cell (hESC) culture medium containing DMEM / F12 (Mediatech), 10ng / mL bFGF (Invitrogen), 20% v / v knockout serum replacement (Invitrogen), 1% v / v non-essential amino acids (Mediatech), 2mM L-glutamine (Mediatech) and 100μM β-mercaptoethanol (Invitrogen). Conventional culture medium is also described in the first part of Table 1. Using a fine-tipped glass pipette, the colonies were disintegrated into small pieces by mechanically cutting. HiPSCs were passaged every 5-7 days (clump passage), collected and passaged to newly seeded mitomycin C-treated MEF cells at a dilution of 1:3-1:6, and hESC culture medium was added daily. The cell cultures were maintained in an incubator set at 37°C, humidified with 5% CO2. Culturing cells in conventional culture medium with feeder cells and using pellet passaging is referred to herein as "conventional culture".

[0241] For single cell dissociation, hiPSC was washed once with phosphate buffered saline (PBS) (Mediatech) and treated with Accutase (Millipore) or TrypL (Invitrogen) at 37°C for 3-5 minutes and then broken into single cells by pipette. The single cell suspension was then mixed with the conventional culture medium described above with equal volume, spun down at 300g for 5 minutes and resuspended in SCM4 culture medium or SMC4+ fibronectin culture medium. In most cases, the cells of single cell dissociation were maintained in SMC4 culture medium, which consisted of conventional hESC culture medium with various small molecules and additives including 0.4 μM PD0325901, 1 μM CHIR99021, 5 μM Thiazovivin and 2 μM SB431542 (all Biovision). At 20°C, small molecules were maintained in DMSO with a reserve concentration of 5-25 μM before being added to the culture medium. All working culture mediums were maintained at 4°C for up to 4 weeks. Culture with ROCK inhibitor, together with Thiazovivin, was superior to Y27632 for maintaining cells in an undifferentiated state.

[0242] After resuspension in appropriate culture medium, cells were transferred to a pre-warmed plate containing Matrigel at 37°C. TM(1:25 dilution; BD Biosciences) coated with feeder-free tissue culture plates (BD Falcon) for 1-2 hours. In this format, cells routinely receive fresh culture medium every other day and are passaged when confluence reaches 66-75%, which usually occurs 4-5 days after passage. With each passage, cells are dissociated into single cells and transferred to new Matrigel at a dilution of 1:5-1:10. TM (BD Biosciences). For defined, growth factor-free cultures, cell suspensions were added to tissue culture plates pre-coated with 1% gelatin (Mediatech). Cells were maintained and passaged as described above, except that SMC4 medium was essentially free of all cytokines and growth factors, including bFGF.

[0243] For freezing purposes, cells were dissociated into single cells, resuspended in SMC4+ fibronectin with 10% v / v DMSO (Mediatech) added and placed in cryovials (Nalgene). Once the lid was closed, the cryovials were placed in Mr. Frosty (Nalgene) and kept overnight at -80°C. The next day, the cryovials were transferred to liquid nitrogen for long-term storage. To thaw, the cryovials were placed in a 37°C water bath for approximately 1-2 minutes until most of the ice melted. The thawed cell solution was then gently mixed with fresh conventional hESC culture medium and spun down at 300g for 5 minutes. The cell solution was resuspended in SMC4+ fibronectin culture medium and transferred to Matrigel TM (BD Biosciences) coated tissue culture plates. As with all other cell culture incubations, cells were maintained in a humidified incubator set at 37°C with 5% CO2.

[0244] B. Induction of Reprogramming

[0245] To initiate the reprogramming process, ectopic expression of reprogramming factors (variable combinations of human Oct4, Sox2, Klf4, c-Myc, Lin28, and Nanog) was obtained using lentiviral transduction or other methods such as protein treatment alone. In most cases, starting cells were cultured at 10% confluency (i.e., 1x10 5Cells / wells of 6-well plates) were seeded onto a gelatin (Mediatech) coated surface. For viral infection methods, freshly collected lentivirus was added to the starting cells at a dilution of 1:2, 4 μg / mL polybrene (Millipore) was added, and the infection was spun at 650g for 1.5 hours at 32°C. The culture was transferred to 37°C and 5% CO2 for another 7 hours. After incubation, the cells were washed three times with PBS and supplied with fresh culture medium. For cells that are difficult to infect, such as IMR90 fibroblasts in a feeder-free culture system, this process was repeated again 48 hours after the initial infection. For non-genetic methods of inducing reprogramming, such as direct protein application to cells, a protein mixture or a mixture consisting of reprogramming factors was added to the cell solution at 8 μg / mL and kept for 24 hours before the culture medium was changed. Repeat this step an additional 2-4 times. All starting cells were cultured in their own somatic cell culture medium until the starting protein was added after the 4th day, at which point the culture medium was converted to a portion of somatic cell culture medium and a portion of conventional hESC culture medium. According to fusion (usually in 4-6 days), cells are trypsinized, mixed with equal parts of culture medium, spun down at 300g for 5 minutes, resuspended in 1:1 somatic / conventional hESC culture medium and expanded to larger culture plates at 1:4-1:6. For example, cells in two wells of a 6-well plate are usually expanded to a 10 cm dish. The next day after expansion, the culture medium is completely converted to conventional hESC culture medium. Once the expanded cells reach fusion (usually in 8-12 days), they will be enriched (see unique group enrichment). In all cases, the culture medium is routinely changed every other day.

[0246] C. Unique group enrichment

[0247] After inducing the reprogramming of the starting cells with various strategies, including individual lentiviral constructs or polycistronic vectors containing Oct4 and / or Klf4 and / or Sox2 and / or Myc and culturing for about 8-12 days (see above), the cells are 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. In short, the dissociated cells are resuspended in a staining solution containing Hanks' balanced salt solution (Invitrogen), 4% fetal bovine serum (Invitrogen) and 10mM Hepes (Invitrogen) and kept on ice. According to the dilution recommended by the manufacturer, the first antibody bound is added to the cell solution and the solution is incubated on ice for 15 minutes. The cell solution is washed, resuspended in staining buffer and kept on ice. At this point, various enrichment / depletion strategies are used, including fluorescence-activated cell sorting (BD Biosciences, see below) and magnetic bead cell sorting (Miltenyi Biotec, see below).

[0248] Flow cytometry sorting was performed on a FACS Aria (BD, Biosciences). The primary antibodies used included SSEA4 (BD Biosciences), Tra-181 (Biosciences), Tra-161 (BD Biosciences), CD30 (BD Biosciences), and CD50 (BD Biosciences) as indicated. The sorted cells were then spun down and resuspended in SMC4+fibronectin medium and transferred to Matrigel. TM Coated tissue culture plates. When sorting to microwells, i.e. 96-well plates, the plates were spun down at 300 g for 2 minutes. The SMC4+ fibronectin medium was replaced every other day for 3-4 days. After 3-4 days, the SMC4+ fibronectin medium was usually replaced by the SMC4 medium for the remaining time of the culture. Colony formation was usually seen 7-9 days after sorting. Flow cytometry analysis was performed on Guava EasyCyte 8HT (Millipore).

[0249] According to the experimental scheme, MACS microbeads (Miltenyi Biotec) are separated. Briefly, cells are dissociated into single cells (see cell culture of pluripotent stem cells) and the first antibody combined with appropriate FITC-, according to the instructions, including SSEA4 (BD Biosciences), Tra-181 (Biosciences), Tra-161 (BD Biosciences), CD30 (BD Biosciences) and CD50 (BD Biosciences). Then the cells are magnetically labeled by anti-FITC microbeads (Miltenyi Biotec). Then the labeled cell suspension is loaded on the LS MACS column (Miltenyi Biotec). The cells collected from the positive or negative selected parts are spun down at 300g for 5 minutes and resuspended in SMC4+ fibronectin, transferred to Matrigel TM (BD Biosciences) coated tissue culture plates. The next day, fresh culture medium is added to the culture and then replaced every other day. After 3-4 days, SMC4+ fibronectin culture medium is usually replaced by SMC4 culture medium during the remaining time of culture. Colonies usually appear 5-7 days after sorting.

[0250] D. Alkaline phosphatase staining

[0251] Cells were fixed for 30 seconds in 4% v / v paraformaldehyde (Alfa Aesar), washed three times with PBS and stained with an alkaline phosphatase staining kit (Sigma-Aldrich). Briefly, 1 mL sodium nitrite solution was added to 1 mL FRV-alkaline solution, mixed and incubated for 2 minutes at 25 ° C. The solution was then mixed with 45 mL H2O, followed by addition of 1 mL naphthol AS-BI alkaline solution. Alkaline staining mixture was added to fixed cells and incubated for 15 minutes at 25 ° C and subsequently washed with PBS. Cells were then scored in the presence of alkaline phosphatase.

[0252] E. Immunofluorescence staining

[0253] Cells were fixed in 4% v / v paraformaldehyde (Alfa Aesar) for 15 minutes, washed three times with PBS containing 0.2% v / v Tween (PBST) (Fisher Scientific) and permeabilized in PBS for 1 hour at 25°C using 0.15% v / v TritonX-100 (Sigma-Aldrich). After permeabilization, cells were blocked with 1% v / v bovine serum albumin (BSA) (Invitrogen) in PBST (PBSTB) (Fisher Scientific) for 30 minutes at 25°C. After gentle removal of PBSTB, cells were incubated with primary antibodies in PBSTB at 4°C overnight. The 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 antibody (Alexa 488or 555; Invitrogen) at 1:200 dilution in PBSTB for 1 hour at 25°C. Cells were washed three times in PBST and stained with Hoechst dye (Invitrogen). Images of stained cells were captured using a fluorescent microscope or CCD camera.

[0254] F. Differentiation induction and teratoma formation

[0255] Feeder-free iPSCs are differentiated into monolayers and embryoid bodies. For monolayer differentiation, iPSCs are allowed to reach near fusion before changing the differentiation medium, because once differentiated cells usually reduce their proliferation. Briefly, once fused, SMC4 culture medium is changed to a differentiation medium containing DMEM / F12 (Mediatech), 20% fetal bovine serum (Invitrogen), 1% non-essential amino acids (Mediatech), 2mM L-glutamyl (Mediatech) and 100μM β-mercaptoethanol. Once the culture medium is changed, iPSCs are allowed to differentiate for 14 days. Change the culture medium every 2-3 days. For embryoid body ("EB") formation and differentiation, hiPSCs are single cells dissociated with Accutase (Millipore) and resuspended in differentiation medium at a final concentration of 75,000 cells / mL, adding 5μM Thiazovivan. Cells are inoculated in V-bottom 96-well non-tissue culture plates (Nunc) at 100μL / well and separated by centrifuge at 950g for 5 minutes. The next day, the compact "ball-like mass" was transferred to an ultra-low binding 6-well plate (Corning) using a P1000 at approximately 30-40 EBs / well. After 7 days, EBs were transferred to a Matrigel-coated 6-well plate at a 1:1 transfer ratio. After 3 weeks of culture, the cells were fixed and stained.

[0256] Teratoma inhibition and analysis were performed by applied stem cells (Menlo Park, CA). Briefly, 1000,000-2000,000 single-cell dissociated hiPSCs were mixed in 100 μL SMC4 medium supplemented with culture medium and 100 μL Matrigel and introduced into the kidney capsule and testis of light brown SCID mice. Developed teratomas were harvested, sliced ​​and analyzed for various differentiated cell types and structures.

[0257] G. RT-qPCR and qPCR analysis

[0258] RNA was isolated using the PicoPure RNA Isolation Kit (MDS Analytical Technologies) and 0.5 μg RNA was used to generate the first strand of cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). Relative gene expression levels were determined using TaqMan Fast Universal PCR Master Mix (Applied Biosystems) with the FAM-labeled TaqMan probes listed in Table 3 below.

[0259] Table 3. ABI primers and probes

[0260]

[0261]

[0262]

[0263] H. Gene expression analysis

[0264] Total RNA was isolated from cells using Pico Pure RNA Isolation Kit (Molecular Devices, Sunnyvale, CA). In short, the standard protocol of MessageAmp II α RNA Amplification Kit (Applied Biosystems / Ambion, Austin, TX) was used, and an optional second round of amplification was then used to transcribe to biotin-labeled α RNA using the MessageAmp II biotin enhancement kit (Applied Biosystems / Ambion, Austin, TX) standard protocol to prepare biotinylated α RNA. Biotin-labeled α RNA was purified and fragmented according to Affymetrix recommendations. At 45°C, 20 μg of fragmented α RNA was used for hybridization with human genome U133 Plus 2.0 chips (Affymetrix Inc. Santa Clara, CA) for 16 hours. The array was washed and stained in Affymetrix incubation device 450, and scanned using Affymetrix gene chip scanner 3000 7G. Using Affymetrix expression console software, the default analysis settings were used to analyze image data. Arrays were normalized by log-scaled robust multiarray analysis (RMA) and visualized in Spotfire for Genomes 3.1 (Tibco Spotfire, Palo Alto, CA).

[0265] I. Karyotype Analysis and Copy Number Variation Analysis

[0266] Cytogenetic analysis was performed on 20G band-dissociated metaphase cells by Cell Line Genetics in Madison, WI.

[0267] High-resolution comparative genomic hybridization (NimbleGen 12x135k) and subsequent copy number variation analysis were performed by WiCell (Madison, WI).

[0268] Example 2: Cell culture conditions and methods to enable feeder-free culture environment and enzymatic single cell dissociation and passaging of pluripotent stem cells

[0269] This embodiment relates to the culture and dissociation of pluripotent cell groups. Such cell groups include, but are not limited to, embryonic stem cells and induced pluripotent cells, such as those produced by somatic cell nuclear transplantation (SCNT) or by the introduction of pluripotency factors-induced pluripotent stem cells (iPSC). Pluripotent stem cell culture conditions traditionally include the use of feeder cells, which inactivate mitotic dissociation via irradiation or mitomycin C treatment, but provide growth factors and nutrients required to support stem cell culture. The culture of stem cell groups without feeder cells is advantageous for research and industrial applications, where homologous stem cell groups are necessary or where conversion, industrial behavior requires xenogeneic, specific stem cell production culture conditions. In this embodiment, several specific small molecule regulators of cell signaling pathways are tested to establish if individual small molecules or combinations of small molecules ("mixtures") can be used to enhance the culture of pluripotent cells in feeder-free systems.

[0270] When culturing pluripotent cell populations without feeder layers and instead using more defined extracellular matrices such as Matrigel in conventional hESC cell culture media TM Conventional / basal media (such as those described in the first section of Tables 1 and 2) do not support cell viability and pluripotency (Figure 1). However, the use of small molecule inhibitors of Rock kinases improves cell viability under these conditions. Furthermore, the use of small molecule inhibitors of MAP kinase, TGFβ, and Wnt / β-catenin pathways maintains the pluripotent nature of the cells in the absence of feeder layers, although changes in cell viability may be noted. The combination of Rock kinase inhibitors, in combination with MEK, TGFβ, and GSK3 inhibitors all maintain the viability and pluripotency of pluripotent stem cells when cultured in a feeder-free environment.

[0271] Pluripotent cells, such as ESC or iPSC, usually grow as clumps. Traditionally, these cells are expanded and passaged by artificial selection of colonies of morphology identified by researchers skilled in the art. Example 1 herein describes such a procedure (described as passage in clumps). The selected colonies are then broken and the dissociated cells are re-seeded on a plate. The rapid expansion of pluripotent cell populations benefits from the use of enzymatic, single-cell passage. Enzymes such as trypsin and accutase are commonly used for single-cell dissociation of cells during passage.

[0272] In a particular demonstration, iPSC cells showed a significant decrease in viability when enzymatically passaged and plated as single cells in a feeder-free environment, such as by Figure 3BBy using the culture platform of the present invention comprising culture medium components such as those listed in Tables 1 and 2, culturing a pluripotent cell population in a feeder-free culture medium and enzymatically dissociating a single cell for passage, the viability of the cells and the maintenance of pluripotency are greatly improved. More specifically, by using a combination of conventional / basic hESC culture medium formulations and regulators of cell signaling pathways such as the MAP kinase pathway, the TGFβ pathway, the Wnt / β-catenin pathway, and the Rho / Rock pathway, the viability and pluripotency of pluripotent stem cells are maintained during applications requiring single cell dissociation and feeder-free culture.

[0273] As shown in FIG. 2 , iPSCs generated on feeder-free cell culture medium and passaged using pellets are single cells dissociated using an enzyme such as accutase and plated as single cells in a feeder-free system such as Matrigel. TM Use of SMC4 or SMC4+fibronectin culture medium compositions described in Table 1. Only when the dissociated cells are placed in a culture medium containing a combination of small molecules described in these culture media, cell viability is maintained. The use of conventional culture media, such as the basal culture medium formulations in Table 1, results in a large amount of cell loss when pluripotent cells are enzymatically dissociated into single cells and plated in a feeder-free environment. Once the cells are adapted to feeder-free culture media and single cell passage, they are constantly maintained in this manner using SMC4 culture media. All features of hiPSC cells adapted to feeder-free but cell passage conditions are shown in Figure 3. The pluripotent state of these cells is maintained after multiple passages: pluripotency markers are identified by immunofluorescence and gene expression. Moreover, about 99.8% of the cells maintain SSEA4 and Tra1-81 positive staining, as measured by flow cytometry. The gene expression profiles of these cells are similar to those of human ESCs cultured on feeder layers. The transgene silencing in hiPSC is maintained, and the karyotype is normal and the same clones grown in feeder cell culture are the same. Furthermore, hiPSCs passaged and maintained in this manner differentiated into the three germ layers, as demonstrated by in vitro differentiation and teratoma formation ( Fig. 3I and 3J ) as shown.

[0274] Example 3: Cell culture conditions and methods to enable single cell sorting of pluripotent cells while maintaining pluripotency and cell viability

[0275] As described in Example 2, the stem cell culture of ESC or iPSC is routinely cultured on feeder cells and then mechanically dissociated before the plate is re-seeded by artificial selection of cell colonies. Skilled researchers can identify stem cell colonies with multipotent, non-differentiated characteristics based on colony morphology and use this as a method for selecting pluripotent cells. Therefore, it is possible to select and manually enrich pluripotent groups or groups with ideal characteristics from some of these cells with less than ideal characteristics, such as cells showing differentiation signs in culture or agglomerates of dead cells. This process is laborious and depends on skilled researchers to select ideal cell groups. Wherein the use of cell enrichment or sorting techniques selected individually according to ideal characteristics is of great benefit to the art. Using currently available technology, such as magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS), such enrichment steps will require enzymatic passage of pluripotent cell groups to single cell format before enrichment and inoculation back into culture medium. Moreover, the use of feeder-supported cultures is less ideal for these technologies, so it is necessary to use feeder-free culture systems.

[0276] In a specific embodiment, using the media components of the invention as described in Table 1 and the methods described in Examples 1 and 2, the pluripotent cell population is dissociated into single cells, enriched using a cell sorting process such as magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS) and plated on feeder-free culture without loss of pluripotency or cell viability.

[0277] In a specific example, as shown in FIG4 , based on the cell surface marker SSEA4 + / Tra181 + Positive cell selection and sorting The dissociated pluripotent cells were maintained as single cell populations in SMC4 medium (Table 1). These surface antigens are commonly used as pluripotency markers. The sorted double positive populations were then transferred to feeder-free culture (Matrigel TM ECM coating) and allowed to grow in SMC4 medium (Table 1) or SMC4 + fibronectin medium for 2-4 days before switching back to SMC4 medium (Table 1). Cell division was seen within 24 hours of sorting and the cells were nearly confluent on the 5th day after sorting. The sorted cells were single cells cultured for another 5 passages and were shown to not only express markers of the undifferentiated state but also represent a substantially pure population of pluripotent cells, according to SSEA4 + / Tra181 + Cell double staining flow cytometry ( Figure 4B and 4C )judge.

[0278] In the example of enrichment and sorting of pluripotent stem cells during culture, for cells positive for Tra181, the enrichment includes culturing of pluripotent and differentiated cells, and then Tra181 + Continued passaging of the cells maintains a pure population of pluripotent stem cells.

[0279] Study the efficiency of single cell sorting of pluripotent cells. Figure 4D The number of various post-sorting events seen in E, as measured by FACS instrumentation, were plated in a feeder-free culture system using SMC4 medium or SMC4 + fibronectin medium and the number of cells positive for alkaline phosphatase was scored. Alkaline phosphatase is an early inducer of multipotent colony formation. Quantified post-sorting plating efficiency, as shown by the number of alkaline-positive colonies / number of cells plated, was seen in this system at approximately 8-10% ( Figure 4E ). To demonstrate the potential of single cell sorting in the maintenance of an otherwise pluripotent cell population, iPSCs were cultured as dissociated single cells labeled with fluorescently conjugated antibodies specific for the pluripotency markers SSEA4 and Tra181, applied to FACS and selected based on these markers. SSEA4 selected cells were plated directly from the FACS instrument at 1-9 events / well + / Tra181 + Cells were plated in 96-well plates. As few as 1 event / well produced cloned alkaline phosphatase-positive colonies ( Figure 4F and 4G ). Therefore, such single cell sorting systems are used for clonal selection of pluripotent cells, based on features that are preferentially associated with certain cell surface markers.

[0280] Example 4: Methods and conditions to enable efficient cell reprogramming to a pluripotent state in a feeder-free culture system

[0281] The use of iPSCs for industrial and / or clinical applications necessitates the generation, selection and maintenance of cells in well-defined culture conditions, particularly xeno-free conditions. Therefore, cell reprogramming in feeder-free culture conditions is highly desirable. However, although fibroblasts and keratinocytes are the most commonly used cell types for reprogramming due to access via skin biopsies or hair follicles, the efficiency of reprogramming of these cell types is very low, and effective methods for reprogramming these cells in feeder-free culture have yet to be demonstrated.

[0282] As described in Example 2, conventional hESC stem cell culture media enables the growth and maintenance of pluripotent cultures in the absence of feeder cells and the expansion of these cultures using enzymatic single cell passaging in the presence of inhibitors of cell signaling pathways - specifically the MAP kinase pathway, the TGFβ pathway, the Wnt / β-catenin pathway, and the Rho / Rock pathway. 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. As described in Example 1, the cells were plated on Matrigel TM Reprogramming protocols were performed with feeder cells instead of feeder cells.

[0283] Compared with reprogramming by feeder-free cells, conventional hESC stem cell culture medium or conventional hESC culture medium with the addition of specific pathway regulators listed in SMC4 culture medium (Table 1) was used. As can be seen in Figure 5, individual lentivirus was used to express Oct4, Klf4, Sox2 and Myc, and after induction of reprogramming, many iPSC-like colonies were seen in feeder-free culture with the addition of SMC4 culture medium, while colonies were rarely seen or not seen in cultures maintained in conventional hESC culture medium alone. The subsequent characteristics of colonies produced in SMC4 culture medium showed that many of them were true iPSC colonies. The pluripotency of cells was determined by the expression of pluripotency markers Oct4, Nanog, Sox2, KLF4, SSEA4 and TRA 1-81, and immunofluorescence, flow cytometry and gene expression profiles ( Fig. 13C -E). Moreover, the cells were shown to efficiently differentiate into all three germ layers when cultured in differentiation medium. Thus, the present invention demonstrates the efficient generation of iPSCs in a feeder-free culture system. The efficiency of this method, compared with other reprogramming methods, is described in Example 8 and Table 4.

[0284] Example 5. Cell culture composition for production and maintenance of pluripotent cells in the naive state

[0285] Recent studies have shown that by epigenetic reprogramming, terminally differentiated cells have the ability to turn to progenitor cell-like states (Xie, H., Ye, M., Feng, R. and Graf, T. 2004), different differentiated cell states (Szabo, Bhatia 2010) or even return to embryonic states (Takahashi et al., 2006), such as iPSC. Although the generation of iPSC has become more conventional, only a small percentage of somatic cells are reprogrammed to iPSC in a given experiment. Several parameters attributed to this low efficiency include the proliferative state of somatic cells, resulting in additional mutation formation of gene activation or inhibition, the format of gene delivery and environmental cues. It has also been reported that not all cells identified as iPSCs perform similarly to ESCs. For example, gene expression profiles have demonstrated that many iPSCs show significant differences in their ESC counterparts' expression profiles. In addition, studies of Xist activity and X chromosome reactivation analysis showed that while some ESCs are in a naive state (i.e., the ground state of pluripotency), most, if not all, derived iPSCs are in a mature state (i.e., mature to differentiate). Combined, these differences may contribute to reduced pluripotency and low efficiency in iPSC differentiation toward specific cell types, reducing the value of iPSCs in regenerative medicine.

[0286] By targeting key cellular pathways involved in the mechanisms behind the naive and mature states, the inventors have demonstrated the ability to transform pluripotent stem cells, including conventional iPSCs, that exist in a mature state into a naive state. Using the culture medium components listed in Tables 1 and 2, it is possible that such mature pluripotent cells and conventional iPSCs are further reprogrammed to a naive state. More specifically, by reprogramming somatic cells or culturing iPSCs in a culture medium containing regulators of cell signaling pathways, such as MAP kinase pathways, TGFβ pathways, and / or Wnt / β-catenin pathways, the gene expression signals of the generated or cultured iPSCs become more like ESCs than conventional iPSCs (iPSCs generated and / or cultured in conventional culture medium and not in contact with regulators of cell signaling pathways). As shown in the hierarchical clustering, conventional culture medium and SMC4 culture medium for deriving hiPSCs are similar to each other and are different from their parental line 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 naive status ( Fig. 6E Moreover, iPSCs cultured in SMC4 medium significantly reduced Xist activity and enhanced X chromosome gene expression ( Figure 6C and 6DFinally, 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, retaining only the ability of cells in the original state ( Figure 3H ). Therefore, culturing pluripotent stem cells, including iPSCs in a mature state, in SMC4 medium promotes the naive state and enhances differentiation potential.

[0287] To determine the pluripotent state of hiPSC clones generated using the SMC4-supplemented culture medium and cell sorting platform (Figure 7), hiPSCs were derived from the same starting fibroblast line and a 3-factor polycistronic vector expressing Oct4, Klf4, and Myc but maintained in conventional culture, including pellet passaging and feeder cells (FTi99). Affymetrix global gene expression analysis of various clones and mRNA derived from hESCs was performed ( Figure 7B ). It can be seen that all pluripotent cell lines have distinct lineages from the control fibroblast line (FTC1, Pearson score 0.886) and substantially similar expression lineages to each other (Pearson score 0.969). The heat map signatures generated represent the hESC / FTi99 (hESC or hiPSC generated and maintained on feeder cells and conventional medium) and FTi91 / FTi93 (hiPSC generated and maintained on feeder-free SMC4 medium) groups ( Figure 7C ). In-depth analysis of the 1739 differentially expressed transcripts identified an interesting trend: while several pluripotency genes normally associated with the naive state were upregulated in the FTi91 / FTi93 group, more significantly, many differentiation genes associated with the mature state were repressed in this group ( Fig.7D ). Full analysis of hiPSCs generated from SMC4 medium suggests that culture conditions play a more influential role in determining the undifferentiated state than the starting cell line or derivation strategy. The data also suggest that clones derived from or adapted to SMC4 culture conditions exhibit primitive features associated with preferred quality, such as a highly undifferentiated state ( Fig. 7E ).

[0288] Example 6: Identification of an Antibody Cocktail to Identify Bona Fide hiPSCs

[0289] In addition to SSEA4 and Tra181 expression, CD30 and CD50 expression were also identified and considered to represent additional pluripotency surface markers ( Fig.10). Cells reprogrammed using polycistronic lentivirus expressing Oc4, Klf4, and Sox2 representing various potential states were identified with very few cells expressing true pluripotency markers, such as Nanog. To date, there is no reliable method for identifying truly pluripotent hiPSCs based on surface marker expression. For example, as seen in FIG11 , some clonal populations identified as positive for SSEA4, Tra181, and CD9 do not express Nanog and are incorrectly identified as hiPSCs.

[0290] The current invention provides a combination of cell surface markers to identify a population of cells expressing Nanog, a marker of truly pluripotent cells. Specifically, cells positive for CD30, SSEA4, and Tra181 surface markers identify cells expressing Nanog (Figure 11). This study demonstrates that the combination of CD30 / SSEA4 / Tra181 expression represents a unique combination of surface markers associated with Nanog expression and truly pluripotent hiPSCs.

[0291] In other examples of increased enrichment, the cell population undergoing reprogramming is sorted to identify cells that are positive for expression of the CD13 surface marker and these CD13 + Cells are removed from the reprogrammed cell population. CD13 associated somatic cells + population with non-reprogrammed cells, as well as depletion of CD13 + The reprogrammed cell population has enhanced enrichment of SSEA4 / Tra181 positive cells ( Fig.12 ).like Fig.12 As shown in , when somatic cells were reprogrammed for 21 days using a polycistronic vector expressing Oct4, Klf4, and Sox2, cells with various surface marker expression patterns were established, with only a small subset of cells representing SSEA4 and Tra181 positive cells (dashed arrows, Fig.12 However, when based on CD13 expression (solid arrow, Fig.12 ) When the same population was first evaluated, the subset of the population negative for CD13 (i.e., depleted of CD13 cells) represented a population that was significantly enriched for SSEA4 and Tra181 expressing cells (11.04%, Fig.12 In contrast, the subset of cells expressing high levels of CD13 represented a minority of cells expressing SSEA4 and Tra181 (1.14%, Fig.12 ).

[0292] Example 7: Use of Single Cell Sorting in the Generation of Induced Pluripotent Stem Cells from Differentiated Cells

[0293] like Fig. 8AAs seen in Figure 8, early fibroblast cultures in the reprogramming process, where individual lentiviruses expressing Oct4, Klf4, Sox2, and myc were used to induce reprogramming, contained colonies of cells with different morphologies. Some of these cells were positive for pluripotency markers while others were only transformed, fast-growing cells. At this stage of reprogramming, it is not clear that the cell colonies will continue to form iPSCs by individual cell morphologies. Faster growth, transformation, but not pluripotent cells quickly occupy the culture medium. It is therefore advantageous to have an enrichment step to select iPSCs early in the reprogramming process. Moreover, as shown in Figure 8, some colonies expressed several pluripotency markers during the reprogramming process and were true iPSCs, while some colonies were not fully reprogrammed and only expressed some pluripotency markers. Figure 8B Colony 1 is positive for both SSEA4 and Tra181, while colonies 4 and 5 are positive for only one or the other marker. Therefore, it is more efficient and less technically challenging to use a cell surface marker or several markers of pluripotency simultaneously rather than selecting pluripotent cells by colony morphology.

[0294] Also provided herein is the use of the cell culture components described herein in combination with cell enrichment and / or sorting methods, it is possible to derive iPSCs in greater numbers and in a shorter period of time by selecting individual cells that display surface markers of pluripotency during the reprogramming process. More specifically, as Fig. 9 As outlined in FIG, two iPSC generation pathways using single cell sorting and pluripotency marker-based enrichment during reprogramming are performed using the methods of the present invention.

[0295] In approach A, after starting reprogramming, a mixed cell population of various potential states is generated. The mixed cell population includes differentiated cells, partially reprogrammed cells, reprogrammed cells, and cells that are reprogrammed. Cell populations are enriched using cells expressing pluripotency markers such as SSEA4 (see Example 1 method) such as magnetic bead sorting or flow cytometry sorting. Once enriched, cells are maintained in SMC4 culture medium (Table 1) or in a specific embodiment, SMC4+ fibronectin culture medium for about 3 days followed by SMC4 culture medium, and after a culture period of about 6-10 days, iPSC colonies are identified based on live culture markers such as SSEA4 and TRA181, selected and sorted for clonal expansion ( Fig. 9 ).

[0296] In the schematic diagram of pathway B ( Fig. 9), early after initiation of reprogramming, the mixed cell population is 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. Selected cells expressing a combination of pluripotency markers are transferred to a culture system supplemented with feeder cells or a feeder-free culture system, particularly those supplemented with the cell culture medium components described in Tables 1 and 2. In this way, iPSC colonies are generated with significantly reduced timelines and technical barriers compared to previously described methods.

[0297] In a specific demonstration of this technique, IMR90 fibroblasts were infected with lentivirus expressing Oct4, Sox2, Klf4, and c-Myc (OSKM). After a few days of feeder-free culture, the reprogrammed cells were switched from their somatic cell culture medium to feeder-free culture medium supplemented with SMC4 (Table 1). On day 8 after reprogramming was initiated, flow cytometric analysis revealed that the infected cell population contained a moderate subpopulation expressing the pluripotency marker SSEA4 ( Fig.13A Using magnetic activated cell sorting as described in Example 1, the pluripotent cell population was enriched 3-fold for cells expressing SSEA4 ( Fig.13A After enriching for SSEA4-expressing cells, the sorted cells were transferred to a medium containing Matrigel. TM And feeder-free culture supplemented with conventional hESC medium or SMC4 medium or, in a specific embodiment, SMC4+fibronectin medium. Alkaline phosphatase staining of the culture showed that the use of small molecule inhibitors of MEK, GSK3, Rock kinase and TGFβ supports the enrichment of pluripotent cells using single cell sorting, while conventional basal medium cannot ( Fig.13A ).

[0298] Quantification from three independent experiments clearly demonstrated that pluripotent cell selection by single-cell sorting was only possible in the presence of small-molecule inhibitors ( Fig. 13B The cell colonies enriched by this method were further cultured in SMC4 medium and characterized as true iPSCs: in immunofluorescence and flow cytometry, true iPSCs stained positive for the pluripotency markers SSEA4 and Tra181 ( Fig. 13C and D); showed gene expression profiles similar to those of human ESCs ( Fig.13E ); showed significant silencing of exogenous transgenes ( Fig.13F ) and is capable of differentiating into all three germ layers ( Figure 13G). When the same experimental protocol was used but with conventional culture conditions (consisting of conventional culture medium containing feeder cells and lacking SMC4), few pluripotent cell colonies were observed. Therefore, during cell reprogramming, the ability to strongly enrich pluripotent cell populations using single cell sorting based on cell surface markers of pluripotency depends on the use of cell signaling pathway inhibitors and additives listed in Tables 1 and 2. In addition, when added fibronectin was used in the culture system for 3 days, increased post-sorting seeding was observed. This reprogramming process was also completed for other starting cell types including human adipose-derived stem cells.

[0299] In other examples of the technology, FACS is 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 a lentivirus (OSKM) expressing Oct4, Sox2, Klf4 / c, and Myc. A few days after fibroblast infection, the cell culture was switched to feeder-free culture in SMC4 medium. The infected cell population contained a moderate cell population (positive for SSEA4 and Tra181 markers of pluripotency) Fig.14A ). Cells positive for both pluripotency markers were selected and sorted from cells negative for both markers or positive for only one marker. Comparison of subsequent feeder-free culture with addition of culture medium as described in Table 1 or 2 showed that cultures of cells positive for both markers formed colonies that were subsequently characterized as pluripotent iPSCs, whereas cells negative for the pluripotency markers by FACS gating did not produce alkaline phosphatase-positive colonies ( Fig.14A , B). More specifically, inhibitors of signal transduction molecules MEK, GSK, Rock, and TGFβ as additives to the basal medium formulation facilitated the use of FACS in early sorting selection and enrichment of pluripotent cells during the reprogramming process. In addition, improved post-sorting seeding was observed when added fibronectin was used in the culture system for 3 days.

[0300] The advantages of SMC4 medium and culture system are secondarily used to develop methods for high-throughput generation of feeder-free and clonally derived hiPSCs. Protocols are designed to treat induced cells with SMC4 medium for reprogramming and select for rare individual cells that faithfully reprogram as indicated by a combination of pluripotency markers. Furthermore, we connect the reprogramming process to multiplex platforms for selection assays based on dual-label flow cytometry, qRTPCR, and immunofluorescence ( Fig.15 ) effectively selects the top clones.

[0301] In an optimized multiplex protocol, reprogramming was initiated using a 3-factor (OKS) polycistronic virus at day 20 post infection followed by FACS sorting of SSEA4 at day 30 post infection. + / Tra181 + Cells were plated in 96-well plates ( Fig.15 ) Completed SSEA4 + / Tra181 + This strategy allows for the derivation of many clones and results in the expression of pluripotency markers, the display of attenuation of exogenous gene activity and demethylation of the Oct4 promoter ( Figures 16A-16C ) clones FTC1 clones 1 and 2 were generated. Through in vitro and in vivo differentiation into three somatic cell lineages ( Figures 16D-16E ), FTC1 clones 1 and 2 also appeared pluripotent. Selected clones were also evaluated for chromosomal integrity and showed minimal copy number variation compared to their parental cell line with normal karyotype even after 20 consecutive single-cell passages in FF culture, a significant improvement over other reprogramming strategies ( Figures 17A-17B ).

[0302] To determine the reproducibility of the platform, additional fibroblast cell lines, FTC5 and FTC7, were induced to reprogram using polycistronic vectors expressing Oct4, Klf4 and Sox2 and applied to Fig.15 As shown in Figure 18, colonies derived from FTC5 and FTC7 fibroblast lines were shown to maintain their undifferentiated state, retain their pluripotency and genomic stability. Therefore, the combination of 3 pluripotency factors, SMC4 medium and multiple characterization platform significantly enhances the kinetics of feeder-free reprogramming while enabling the identification, selection and expansion of clonally derived and genomic stable hiPSCs in a high-throughput manner.

[0303] Example 8: Methods and culture components for rapid generation of multiple iPSC clones

[0304] The generation of human iPSCs by ectopic expression of pluripotency genes such as Oct4, Sox2, Klf4, c-myc, Lin28 and Nanog is an inefficient and technically demanding process. Strategies involving the integration of lentiviral or retroviral transgenes of pluripotency factor transgenes into the host cell genome, combined with feeder cell support, have traditionally been the most effective methods for iPSC generation. A literature review of historical studies using viral and feeder cell methods for human iPSC generation showed that 0.001%-0.01% of infected cells became iPS cells, with potential pluripotent cells seen during days 21-30 after infection and these were derived by artificial "clump" passage clones between days 30-45 after infection.

[0305] Other methods for introducing pluripotency genes include transduction of episomal vector systems and modified proteins. Such cells are considered to be an important development for the ultimate clinical application of iPSC technology. However, these methods are even less efficient than reprogramming using viral systems. Moreover, the efficiency of reprogramming is also reduced or impossible in some cases when feeder-free cell systems are used in combination with conventional stem cell culture medium preparations, hindering the development of iPSC for industrial and therapeutic purposes. Somatic cell reprogramming has been characterized as a random process; most cells will eventually be reprogrammed over time. However, a powerful, technically simple, efficient and scalable method for generating multiple iPSC clones in single-cell reprogramming must also be described.

[0306] The present invention provides cell culture conditions and methods to derive cloned iPSC colonies in a relatively short time and with lower technical barriers than current methods. Specifically, and as Fig. 13B As seen in Figure 2, small molecule inhibitors of specific signaling pathways used as additives to SMC4 medium allowed for the generation of iPSC colonies in a feeder-free culture environment much more efficiently than when SMC4 medium was not used. Inhibitors of the MEK, GSK, Rock, and TGFβ signaling pathways were used to allow efficient reprogramming in a feeder-free environment.

[0307] As shown in Figure 13 and Table 4, in three independent reprogramming experiments, no or only one colony was seen when conventional hESC culture medium was used in the reprogramming process in a feeder-free environment, while small molecule culture additives (i.e., SMC4 culture medium) enhanced the efficiency of feeder-free reprogramming events to 0.035%, and resulted in colonies 14-21 days after infection and the derivation of iPSC clones 28-35 days later. Therefore, the use of small molecule inhibitors increases the efficiency of reprogramming in terms of reprogramming time and the percentage of reprogrammed cells. Colonies generated using this method are characterized as pluripotent using standard procedures such as immunofluorescence and gene expression profiles.

[0308] In another demonstration of this technology, differentiated cells were infected with viruses expressing individual pluripotency genes Oct4, Klf4, Sox2, and Myc, and cultured in SMC4 medium and a feeder-free environment (Table 1) for 8-12 days. At this time point, and as described in Example 6 and Figures 13 and 14, FACS or MACS single-cell sorting was used to enrich cells to obtain small pluripotent cell populations determined by positive staining for pluripotency markers such as SSEA4 and Tra181. Using this method, the iPSC generation timeline was greatly reduced: an efficiency of 0.22% in terms of the percentage of reprogrammed cells was demonstrated, and colonies appeared immediately after sorting (Days 10-16). The efficiency of reprogramming allows the derivation of iPSCs cloned 21-28 days after infection.

[0309] In other demonstrations of improved reprogramming efficiency, a polycistronic vector system in which 3 (Oct4, Klf4, and Sox2) or 4 (Oct4, Klf4, Sox2, and myc) pluripotency factors are expressed from the same promoter element was used in combination with an 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 seen on days 6-8 after infection. This approach was so efficient that iPSC colonies appeared only on day 4 after infection. These techniques represent a significant improvement over traditional iPSC generation methods.

[0310] Table 4: Kinetics of reprogramming in various strategies. NI, unidentified; * morphological manifestation of iPSC-like colonies; ** calculated as SSEA4+ / Tra181+ number / seeded cells; *** time required for iPSC colonies to expand and maintain as clonal cell lines (based on Tra181 / SSEA4 staining). Gray boxes represent data from literature retrieval sources. Individual factor viruses; infection by combining individual viruses expressing each key transcription factor, 3 represents Oct4 / Klf4 / Sox2 combination and 4 represents Oct4 / Klf4 / Sox2 / cMyc combination.

[0311]

[0312]

[0313] Example 9: Method for Depleting a Pluripotent Cell Population from a Differentiated Cell Population Using Single Cell Sorting and Enrichment

[0314] Some clinical applications of drug screening and stem cell biology require the generation of homogenous cell populations that are differentiated into specific lineages from pluripotent cells such as ESC or iPSC. Contact of differentiated cell populations with pluripotent cells leads to misleading screening results or even tumor / teratoma formation in vivo. Methods for enriching cell populations of differentiated cells or depleting pluripotent cells from cell populations may include the sorting techniques described in Examples 3 and 6 herein. The use of small molecule additives in cell culture media to specifically prevent differentiation or partial differentiation of pluripotent cells during the single cell sorting process, as provided by the present invention, allows pluripotent cells to be negatively selected from fully differentiated cell populations. Conversely, under culture conditions where pluripotent cells remain completely positive for surface markers of pluripotency, positive selection of differentiated cells from a cell population by cell sorting can be more efficient. As Fig. 20 As can be seen in Table 1, when cells are pre-cultured in the culture environment described in Table 1, mixed fully differentiated fibroblasts and pluripotent cell populations can be effectively separated using FACS. Small molecule culture additives can also be used during the sorting procedure to stabilize the single cell suspension. Fig. 20 It was seen that in subsequent culture and alkaline phosphatase staining, no pluripotent cells were observed in the cells negatively selected for pluripotency markers.

[0315] Example 10: Cytokine- and growth factor-free culture of pluripotent stem cells on feeder-free culture

[0316] As discussed in Example 2, conventional human pluripotent culture systems include feeder cells and cytokines, such as bFGF, which are used as exogenous stimulation to maintain pluripotent stem cells in an undifferentiated state. Feeder cells and the process of producing recombinant cytokines are used as sources of xenogeneic contamination. In addition, the key factors secreted by feeder cells and the precise cell pathways stimulated by cytokines have not yet been identified. Therefore, conventional culture of human pluripotent stem cells represents an ill-defined system and hinders conversion to clinical-grade large-scale applications.

[0317] To address this situation, the present invention includes additional embodiments in which the feeder-free and single-cell passaging 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, gelatin is used instead of Matrigel. TM Because Matrigel TM represents an extracellular matrix that is of animal origin and not well characterized. These embodiments of the present invention provide a fully defined cytokine-free culture system that allows for endogenous self-renewal and maintenance of pluripotent stem cells, including iPSCs.

[0318] like Fig.21A and 21BAs shown in , human pluripotent stem cells, such as iPSCs generated with a polycistronic vector system containing Oct4, Klf4 and Sox2 and maintained in a feeder-free environment, are easily single-cell passaged to gelatin-coated SMC4 medium culture surfaces lacking additional cytokines or growth factors such as bFGF. After several passages in this fully defined system, iPSCs maintain their pluripotent state as shown by co-expression of Tra181 and SSEA4 ( Fig. 21C ). Moreover, we have demonstrated the generation of iPSCs in this fully defined cytokine-free system. Therefore, in one embodiment, the present invention provides a fully defined culture system lacking growth factors and cytokines, comprising a combination of SMC4 medium and gelatin.

[0319] Example 11. Generation and maintenance of genomic stability in pluripotent stem cells

[0320] Studies suggest that the reprogramming process and subsequent pluripotent stem cell culture can result in a higher propensity for genomic abnormalities. In addition, feeder-free culture has been shown to induce clonal growth of cells with abnormal karyotypes. Fig.17A , Fig. 17B and 18C As shown in, the present invention provides methods of reprogramming to obtain cells with genomic stability and methods of maintaining reprogrammed cells with genomic stability. In the methods of the present invention, genomic stability is maintained during the reprogramming process and during long-term feeder-free culture when cells are reprogrammed using a 3-factor multicistronic construct comprising Oct4, Klf4 and Sox2 and cultured in SMC4 medium. Fig.17A As seen in , high-resolution comparative genomic hybridization demonstrated that minimal copy number variation was detected when hiPSCs were generated using SMC4 medium and maintained in long-term feeder-free culture. Fig. 17B and 18C Genomic stability is maintained during routine long-term feeder-free and single-cell culture as shown in .

[0321] Example 12: Maintaining a population of pluripotent cells using cell surface markers during culture

[0322] It is often necessary or desirable to remove differentiated cells from pluripotent cell cultures to maintain the pluripotency of pluripotent stem cells such as hESC and hiPSC. To date, this process has required manually picking out differentiated cells from cell cultures or collecting undifferentiated cells from extremely differentiated populations. Both processes are labor-intensive, require skill training, and rely on morphology-based cell selection, which is not always an indicator of the true cell state in culture (Figure 8).

[0323] In an improved method, the present invention provides the ability to efficiently and accurately select undifferentiated cells during routine culture. Fig.19A As shown in , hiPSC populations maintained in SMC4 medium and FF culture are easily enriched or sorted to maintain cell cultures of undifferentiated pluripotent cells. In other examples, such as Fig.19B As shown, the majority differentiated cell population, which is usually discarded, can be removed from the cell culture to obtain the majority of undifferentiated pluripotent cells, as described by Tral81 expression. Sequence Listing <110> Fit Therapeutics <120> Cell culture platform for single cell sorting and enhanced iPSC reprogramming <130> D21C14137CN <150> US 61 / 496,991 <151> 2011-06-14 <150> US 61 / 426,369 <151> 2010-12-22 <160> 17 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Transgenic Oct4 forward primer <400> 1 ctggttggag ggaaggtaat ctag 24 <210> 2 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Transgenic Oct4, Klf4, Myc, Lin28, Sox2 and Nanog reverse primers <400> 2 ttttgtaatc cagaggttga ttgttc 26 <210> 3 <211> 13 <212> DNA <213> Artificial sequence <220> <223> Transgenic Oct4, Klf4, Myc, Lin28, Sox2 and Nanog probes <400> 3 ccccgacgcg tct 13 <210> 4 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Transgenic Klf4 forward primer <400> 4 gccttacaca tgaagaggca ttt 23 <210> 5 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Transgenic Myc forward primer <400> 5 tcttgtgcgt aactcgagtc tagag 25 <210> 6 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Transgenic Lin28 forward primer <400> 6 ccggaggcac agaattgac 19 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Transgenic Sox2 forward primer <400> 7 cactgcccct ctcacacatg 20 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Transgenic Nanog forward primer <400> 8 catgcaacct gaagacgtgt aa 22 <210> 9 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Endogenous Oct4 forward primer <400> 9 gggtttttgg gattaagttc ttca 24 <210> 10 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Endogenous Oct4 reverse primer <400> 10 gcccccaccc tttgtgtt 18 <210> 11 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Endogenous Oct4 probe <400> 11 tcactaagga aggaattg 18 <210> 12 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Endogenous Klf4 forward primer <400> 12 agcctaaatg atggtgcttg gt 22 <210> 13 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Endogenous Klf4 reverse primer <400> 13 ttgaaaactt tggcttcctt gtt 23 <210> 14 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Endogenous Klf4 probe <400> 14 agtcttggtt ctaaaggtac c 21 <210> 15 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Endogenous Nanog forward primer <400> 15 tgatgcccat ccagtcaatc t 21 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Endogenous Nanog reverse primer <400> 16 cctcgctgat taggctccaa 20 <210> 17 <211> 0 <212> DNA <213> Artificial sequence <220> <223> Endogenous nanog probe <400> 17 000

Claims

1. A method for increasing the efficiency of reprogramming of a non-pluripotent human cell population, comprising: (a) culturing the non-pluripotent human cells in a culture medium, wherein (i) the culturing comprises inhibiting each of ROCK, GSK3, TGFβ receptor, and MEK; and (ii) the culture medium does not contain non-human animal feeder cells; as well as (b) inducing the non-pluripotent human cells to reprogram into pluripotent cells in the culture medium, wherein (i) the efficiency of reprogramming is increased compared to the efficiency of reprogramming in the absence of inhibition of each of ROCK, GSK3, TGFβ receptor and MEK; (ii) the pluripotent cells express one or more markers of pluripotency and have basal pluripotency; (iii) the one or more markers of pluripotency include SSEA4 or Tra181; and (iv) the basal pluripotency is characterized by displaying reactivated expression of one or more of GATA6, CDX2 or CGB.

2. The method of claim 1, wherein the method further comprises contacting the non-pluripotent human cell with at least one pluripotency factor, wherein the at least one pluripotency factor comprises an Oct4 polypeptide.

3. The method of claim 2, wherein the at least one pluripotency factor further comprises: a Sox polypeptide; a Klf polypeptide; or both a Sox polypeptide and a Klf polypeptide.

4. The method of claim 1, further comprising: The pluripotent cells are treated with one or more dissociation enzymes to obtain single cell dissociated pluripotent cells.

5. The method of claim 4, further comprising: The single cell dissociated pluripotent cells are suspended.

6. The method of claim 4, further comprising: The single cell dissociated pluripotent cells are sorted to enrich for pluripotent cells, wherein the enriched pluripotent cells express one or more pluripotency markers.

7. The method according to any one of claims 4 to 6, further comprising: The pluripotent cells are cultured in the medium, wherein the pluripotent cells maintain pluripotency for at least 5, at least 10, at least 15, or at least 20 passages.

8. The method of claim 1, wherein the ground state pluripotency is further characterized by displaying reactivated expression of genes associated with extraembryonic cells.

9. The method of claim 1, wherein the pluripotent cells have reduced Xist activity and / or enhanced expression of X chromosome genes compared to pluripotent cells obtained without inhibiting each of ROCK, GSK3, TGFβ receptor and MEK during the culturing period.

10. The method of claim 1, wherein the culture medium is free of cytokines and growth factors from any source.

11. The method of claim 1, wherein the pluripotent cells are obtained with higher efficiency compared to conventionally cultured pluripotent cells with or without feeder layer supply.

12. The method of claim 11, wherein the efficiency is increased by at least 10%, at least 50%, at least 100%, at least 300%, or at least 500%.

13. The method of claim 1, wherein: (a) the culture medium contains a ROCK inhibitor, which is thiazovivin or Y27632; (b) the culture medium contains a TGFβ receptor inhibitor, which is A-83-01 or SB431542; (c) the culture medium contains a GSK3 inhibitor, which is CHIR99021 or BIO; (d) the culture medium contains a MEK inhibitor, which is PD98059 or PD0325901; or (e) The culture medium comprises a TGFβ receptor inhibitor, a GSK3 inhibitor, a MEK inhibitor and a Rock inhibitor, wherein the TGFβ receptor inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

14. The method of claim 1, wherein the basal state pluripotency is characterized by displaying reactivated expression of all of GATA6, CDX2, and CGB.

15. A composition comprising human non-pluripotent cells, human pluripotent cells and a preparation mixture, wherein the preparation mixture comprises a ROCK inhibitor, a TGFβ inhibitor, a GSK3 inhibitor and a MEK inhibitor; wherein (i) the human pluripotent cells express one or more markers of pluripotency and have basal pluripotency; (ii) the one or more pluripotency markers include SSEA4 or Tra181; (iii) the basal state pluripotency is characterized by displaying reactivated expression of one or more of GATA6, CDX2 or CGB; and (iv) the human pluripotent cells are induced pluripotent stem cells (iPSCs) that do not require the expression of exogenous reprogramming factors to maintain pluripotency; (v) the composition does not contain non-human animal feeder cells; (vi) the ROCK inhibitor is thiazovivin or Y27632; (vii) the TGFβ receptor inhibitor is A-83-01 or SB431542; (viii) the GSK3 inhibitor is CHIR99021 or BIO; and (ix) The MEK inhibitor is PD98059 or PD0325901.

16. The composition of claim 15, wherein the human non-pluripotent cells comprise reprogrammed cells or somatic cells.

17. The composition of claim 15, wherein the human non-pluripotent cells comprise an exogenous polynucleotide encoding an Oct4 polypeptide.

18. The composition of claim 17, wherein the human non-pluripotent cells further comprise one or more exogenous polynucleotides encoding a Sox polypeptide, a Klf polypeptide, or both a Sox polypeptide and a Klf polypeptide.

19. The composition of claim 15, wherein the TGFβ receptor inhibitor is SB431542, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and the Rock inhibitor is thiazovivin.

20. Use of the composition according to any one of claims 15 to 19 in the preparation of a medicament.

Images