Compositions and methods for cell reprogramming

Abstract

Disclosed herein are compositions and methods for reprogramming induced pluripotent stem cells, for example, from a primed state to a naive state. Disclosed herein are further methods for producing the reprogrammed induced pluripotent stem cells by contacting the stem cells with another population of stem cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 855,548, filed May 31, 2019, which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listing This application is filed with an electronic Sequence Listing. The Sequence Listing is provided in a file named CHMC63_021WOSeqListing.TXT, which was created and last modified on May 28, 2020, and is 3,318 bytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Aspects of the present disclosure generally relate to reprogrammed induced pluripotent stem cell compositions and methods for making same. [Background technology]

[0004] Remarkable progress has been made in cell fate reprogramming, including methods mediated by transcription factor overexpression, nuclear transfer, and cell fusion. However, the epigenetic and phenotypic states of reprogrammed cells are highly variable, limiting their usefulness for biomedical applications. For example, clonogenic variability (or differentiation bias) limits the efficiency of some reprogramming protocols. In addition, some iPSC cells are resistant to differentiation. More robust and reproducible strategies for stabilizing variable reprogrammed states are now needed for applications in precision medicine, drug screening, and cell therapy. The prior art documents relevant to the invention of this application are as follows (including documents cited in the international phase after the international filing date and documents cited when the application entered the national phase in other countries). (Prior art document) (Patent document) (Patent Document 1) U.S. Patent Application Publication No. 2015 / 0153326 (Patent Document 2) International Publication No. 2017 / 175876 (Patent Document 3) U.S. Patent Application Publication No. 2018 / 0112187 Summary of the Invention

[0005] Cell fate reprogramming is an important goal in molecular biology, offering the potential to enable disease modeling, drug discovery, and regenerative medicine. Reprogramming requires significant alterations in gene expression signatures specific to the desired cell type. This has previously been achieved through different experimental approaches, such as nuclear transfer, cell fusion, transcription factor transfection, and small molecules. As disclosed herein, using an experimental co-culture model, cells, such as human cells in a primed pluripotent state, can be reprogrammed to a naive-like state in the presence of a naive pluripotent stem cell population. Importantly, this unique cell-to-cell mRNA exchange phenomenon, which accounts for a measurable portion of the recipient transcriptome, does not require the manual introduction of conventional reprogramming factors. This process is primarily driven by direct cell contact, likely via nanotubes connected to neighboring cells, rather than other indirect mechanisms.

[0006] Some aspects of the present disclosure relate to methods of reprogramming cells. In some embodiments, the method includes contacting an acceptor cell with a donor cell in vitro. In some embodiments, the contacting causes transfer of one or more intracellular components from the donor cell to the acceptor cell. In some embodiments, the acceptor cell is a pluripotent stem cell (PSC). In some embodiments, the acceptor cell is a primed PSC. In some embodiments, the acceptor cell is an induced pluripotent stem cell (iPSC). In some embodiments, the acceptor cell is a primed induced pluripotent stem cell. In some embodiments, the acceptor cell is a mammalian cell. In some embodiments, the acceptor cell is a mouse cell. In some embodiments, the acceptor cell is a human cell. In some embodiments, the acceptor cell is a human induced pluripotent stem cell (hiPSC). In some embodiments, the acceptor cell is a primed hiPSC. In some embodiments, the acceptor cell is an epiblast-derived stem cell (EpiSC). In some embodiments, the acceptor cells are primed cells. In some embodiments, the acceptor cells express primed transcription factors and / or primed cell surface markers. In some embodiments, but without being limited by any mechanism of action, the donor cells comprise tunneling nanotubes (TNTs) or cytonemes on their surfaces. In some embodiments, the donor cells are PSCs. In some embodiments, the donor cells are iPSCs. In some embodiments, the donor cells are mammalian cells. In some embodiments, the donor cells are mouse cells. In some embodiments, the donor cells are human cells. In some embodiments, the donor cells are hiPSCs. In some embodiments, the donor cells are naive PSCs. In some embodiments, the donor cells are naive iPSCs. In some embodiments, the donor cells are naive hiPSCs. In some embodiments, the donor cells are embryonic stem cells.In some embodiments, the donor cells are mouse embryonic stem cells (mESCs). In some embodiments, the donor cells are naive mESCs. In some embodiments, the donor cells are mouse EpiSCs. In some embodiments, the donor cells are naive cells. In some embodiments, the donor cells express naive transcription factors and / or naive cell surface markers. In some embodiments, the donor cells and acceptor cells are contacted in culture medium. In some embodiments, the donor cells and acceptor cells are cultured in naive maintenance medium. In some embodiments, the culture medium is RSet medium, naive human stem cell (NHSM), 5i medium, 4i medium, 3i medium, feeder-independent naive embryo (FINE) medium, mTeSR medium, mTeSR medium with Matrigel, PXGL medium, N2B27 medium, N2 medium, T2iLGo, tt2iLGo medium, 2i medium, or 2i medium with gelatin. In some embodiments, the culture medium comprises one or more of a GSK3 inhibitor, a MAPK inhibitor, LIF, a JNK inhibitor, a p38 inhibitor, bFGF, or TGF-β (e.g., at least 1, 3, 5). In some embodiments, the donor and acceptor cells are not cultured with feeder cells. In some embodiments, the donor and acceptor cells are cultured with feeder cells. In some embodiments, the donor and acceptor cells are not contacted with or cultured with an HDAC inhibitor.In some embodiments, the donor and acceptor cells are at least about 1%:99%, 5%:95%, 10%:90%, 15%:85%, 20%:80%, 25%:75%, 30%:70%, 35%:65%, 40%:60%, 45%:55%, 50%:50%, 55%:45%, 60%:40%, 65%:35%, 70%:30%, 75%:25%, 80%:20%, 85%:15%, 90%:10%, 95%:5%, or 99%:1%, or at least about 1%:99%, 5%:95%, 10%:90%, 15%:85%, 20%:80%, 25%:75%, 30%:70%, 35%:65%, 40%:60%, 45%:55%, 50%:50%, 55%:45%, 60%:40%, 65%:35%, 70%:30%, 75%:25%, 80%:20%, 85%:15%, 90%:10%, 95%:5%, or 99%:1%. The cells are cultured at a ratio of the above percentages, at least about the above percentages, less than or equal to the above percentages, or less than or equal to the above percentages, or at any ratio within a range defined by any two of the above ratios, for example, 1%:99%-99%:1%, 10%:90%-90%:10%, 20%:80%-80%:20%, 30%:70%-70%:30%, 40%:60%-60%:40%, 45%:55%-55%:45%, 1%:99%-50%:50%, or 50%:50%-99%:1%. In some embodiments, donor and acceptor cells are cultured at a ratio of 20%:80%, 40%:60%, 45%:55%, 50%:50%, 55%:45%, 60%:40%, or 80%:20%, about, at least, at least about, no more than, or no more than about. In some embodiments, donor and acceptor cells are cultured at a ratio of 50%:50% (1:1), about 50%:50% (1:1), at least 50%:50% (1:1), at least about 50%:50% (1:1), no more than 50%:50% (1:1), or no more than about 50%:50% (1:1). In some embodiments, the transferred intracellular component comprises RNA, DNA, a nucleic acid, a protein, a polypeptide, a peptide, or an organelle, or any combination thereof. In some embodiments, the intracellular component to be transferred is selected from one or more (e.g., at least 1, 3, 5) of RNA, DNA, nucleic acid, protein, polypeptide, peptide, and organelle. In some embodiments, the intracellular component is RNA.In some embodiments, the intracellular component is mRNA, ncRNA, lncRNA, miRNA, piRNA, siRNA, or shRNA, or any combination thereof. In some embodiments, but without being limited by any mechanism of action, the donor cell transfers the intracellular component via tunneling nanotubes or cytonemes. In some embodiments, after contacting, the acceptor cell contains exogenous mRNA derived from the xenogeneic donor cell. In some embodiments, after contacting, the acceptor cell contains exogenous mRNA derived from the allogeneic or autologous donor cell. In some embodiments, after contacting, the acceptor cells contain exogenous mRNA encoding genes that are, about, at least, at least about, up to, or equal to 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 6382, 7000, 8000, 9000 genes, or a range defined by any two of the foregoing values ​​(e.g., 100-9000, 2000-8000, 4000-8000, 100-500, or 300-2000 genes). In some embodiments, the genes contained in the acceptor cells are naive transcription factors. In some embodiments, the acceptor cells are not modified by transfection, electroporation, or viral transduction, or any combination thereof, prior to contacting. In some embodiments, the donor and acceptor cells are contacted under hypoxic conditions. In some embodiments, the hypoxic conditions comprise, consist essentially of, or consist of a percentage of O that is 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% O, about, at least, at least about, equal to or less than, or equal to or less than about, or any concentration of O within a range defined by any two of the foregoing concentrations, e.g., 0%-20%, 3%-10%, 4%-6%, 0%-5%, or 5%-20%.In some embodiments, the hypoxic conditions comprise, consist essentially of, or consist of a percentage of O that is 3%, 4%, 5%, 6%, or 7% O. In some embodiments, the hypoxic conditions comprise, consist essentially of, or consist of a percentage of O that is about 5% O, at least 5% O, at least about 5% O, or at most 5% O. In some embodiments, the donor and acceptor cells are contacted under stressor conditions. In some embodiments, the stressor conditions comprise, consist essentially of, or consist of contact with a cytotoxic compound, hypoxia, non-physiological temperature, non-physiological pH, electroporation, or any combination thereof. In some embodiments, the donor and acceptor cells are incubated at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C In some embodiments, the donor and acceptor cells are contacted or grown at a temperature that is about 37°C, at least 37°C, at least about 37°C, or equal to or less than 37°C, or equal to or less than about 37°C, or any temperature within a range defined by any two of the above temperatures, e.g., 15°C to 50°C, 20°C to 45°C, 25°C to 40°C, 32°C to 42°C, or 35°C to 39°C. In some embodiments, the donor and acceptor cells are contacted or grown at a temperature that is about 37°C, at least 37°C, at least about 37°C, equal to or less than 37°C, or equal to or less than about 37°C. In some embodiments, the donor and acceptor cells are grown in direct contact of the acceptor and donor cells. In some embodiments, the donor and acceptor cells are brought into direct contact. In some embodiments, the donor and acceptor cells are cultured in direct contact.In some embodiments, the donor cells and the acceptor cells are not separated by a transwell. In some embodiments, the donor cells and the acceptor cells are not contacted in the presence of donor cell-conditioned medium. In some embodiments, the contacting step is carried out until the acceptor cells express or upregulate one or more naive stem cell markers (e.g., CD130, CD77, CD7, CD75, or F11R). In some embodiments, the contacting step is carried out until the acceptor cells show downregulation of one or more primed stem cell markers (e.g., CD90, HLA-ABC, CD24, CD57, or SSEA4). In some embodiments, the contacting step results in increased expression of naive pluripotency markers or transcription factors (e.g., KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, or TBS3) in the acceptor cells and primed pluripotency markers or transcription factors. In some embodiments, the contacting step is performed until dome-shaped naive acceptor colonies are observed. In some embodiments, after contacting, the acceptor cells undergo a change in chromatin accessibility. In some embodiments, the change in chromatin accessibility comprises increased accessibility to binding motifs for SOX2 or TFAP2C, or both. In some embodiments, one or both of the donor or acceptor cells are treated with a compound such as resveratrol, epigallocatechin gallate (EGCG), curcumin, genistein, activin-A, Wnt-3a, sodium butyrate, basic fibroblast growth factor (bFGF), oncostatin M (OSM), dexamethasone (DEX), hepatocyte growth factor (HGF), CHIR-99021, forskolin, or erythrocyte stimulating factor (EFG). , Y-27632 (ROCK inhibitor), (s)-(-)-blebbistatin, IWP2, A83-01, LY294002, SB-431542, NVP-BHG, cyclopamine-KAAD, PD-0325901, FGF4, LDN-193189, insulin-like growth factor (IGF), bone morphogenetic protein 2 (BMP2), transforming growth factor β2 (TGF-β2), BMP4, F The subject is contacted with at least one agent selected from the group consisting of GF-7, platelet-derived growth factor (PDGF) beta 3, epidermal growth factor (EGF), exendin-4, human neuregulin (hHRG) beta 3, retinoic acid (RA), L-ascorbic acid 2-phosphate (AA2P), ascorbic acid, insulin-transferrin-selenoethanolamine solution (ITS-X), insulin, rifampicin, penicillin, streptomycin, 2-mercaptoethanol, 3-mercaptopropane-1,2-diol (thioglycerol), L-proline, L-glutamine, non-essential amino acid mixture (NEAA), sodium pyruvate, trypsin-EDTA, phosphatidylinositol (PI), interleukin, prostaglandin, tumor necrosis factor, or any combination thereof.In some embodiments, one or both of the donor or acceptor cells are contacted with at least one agent selected from the group consisting of a GSK3 inhibitor (e.g., CHIR99021), a MAPK inhibitor (e.g., PD0325901), LIF, a JNK inhibitor (e.g., SP600125), a p38 inhibitor (e.g., SB203580), a ROCK inhibitor (e.g., Y-27632), a PKC inhibitor (e.g., Go6983), a BMP inhibitor (e.g., dorsomorphin), bFGF, activin A, ascorbic acid, a cAMP activator (e.g., forskolin), or a TGF-β inhibitor (e.g., A83-01), or any combination thereof. In some embodiments, the donor and acceptor cells are contacted or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, for about the number of days, for at least the number of days, for at least about the number of days, for the number of days, or for about the number of days. In some embodiments, the donor and acceptor cells are contacted or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, for about the number of days stated above, for at least the number of days stated above, for at least about the number of days stated above, for the number of days stated above, or for about the number of days stated above.

[0007] Some aspects of the present disclosure relate to cell compositions. In some embodiments, the cell composition comprises acceptor cells and donor cells. In some embodiments, the acceptor cells are PSCs. In some embodiments, the acceptor cells are primed PSCs. In some embodiments, the acceptor cells are iPSCs. In some embodiments, the acceptor cells are primed induced pluripotent stem cells. In some embodiments, the acceptor cells are mammalian cells. In some embodiments, the acceptor cells are mouse cells. In some embodiments, the acceptor cells are human cells. In some embodiments, the acceptor cells are hiPSCs. In some embodiments, the acceptor cells are primed hiPSCs. In some embodiments, the acceptor cells are EpiSCs. In some embodiments, the acceptor cells are naive pluripotent stem cells. In some embodiments, the acceptor cells are human naive pluripotent stem cells. In some embodiments, the acceptor cells are human naive iPSCs. In some embodiments, but without being limited by any mechanism of action, the donor cell is any cell having tunneling nanotubes. In some embodiments, the donor cell is a PSC. In some embodiments, the donor cell is an iPSC. In some embodiments, the donor cell is a mammalian cell. In some embodiments, the donor cell is a mouse cell. In some embodiments, the donor cell is a human cell. In some embodiments, the donor cell is a hiPSC. In some embodiments, the donor cell is a naive PSC. In some embodiments, the donor cell is a naive iPSC. In some embodiments, the donor cell is a naive hiPSC. In some embodiments, the donor cell is an embryonic stem cell. In some embodiments, the donor cell is an mESC. In some embodiments, the donor cell is a naive mESC. In some embodiments, the donor cell is a mouse EpiSC.In some embodiments, the acceptor cells are pluripotent stem cells and the donor cells are naive pluripotent stem cells. In some embodiments, the cell composition further comprises a naive maintenance medium. In some embodiments, the naive maintenance medium is mTeSR medium, mTeSR medium containing Matrigel, PXGL medium, N2B27 medium, N2 medium, tt2iLGo medium, 2i medium, or 2i medium containing gelatin. In some embodiments, the cell composition does not comprise feeder cells. In some embodiments, the cell composition comprises feeder cells. In some embodiments, the donor and acceptor cells are 95%, 10%:90%, 15%:85%, 20%:80%, 25%:75%, 30%:70%, 35%:65%, 40%:60%, 45%:55%, 50%:50%, 55%:45%, 60%:40%, 65%:35%, 70%:30%, 75%:25%, 80%:20%, 85%:15%, 90%:10%, 95%:5%, or 99%:1%, or at least about 95% of the above. a ratio that is at least about the above percentages, is no more than the above percentages, or is no more than about the above percentages, or any ratio within a range defined by any two of the foregoing ratios, such as 1%:99%-99%:1%, 10%:90%-90%:10%, 20%:80%-80%:20%, 30%:70%-70%:30%, 40%:60%-60%:40%, 45%:55%-55%:45%, 1%:99%-50%:50%, or 50%:50%-99%:1%. In some embodiments, the donor and acceptor cells are at a ratio of 20%:80%, 40:60%, 45%:55%, 50%:50%, 55%:45%, 60%:40%, or 80%:20%, about, at least, at least about, no more than, or no more than about 50%. In some embodiments, the donor and acceptor cells are at a ratio of about 50%:50% (1:1), at least 50%:50% (1:1), at least about 50%:50% (1:1), no more than 50%:50% (1:1), or no more than about 50%:50% (1:1).In some embodiments, acceptor cells comprise exogenous mRNA derived from xenogeneic donor cells. In some embodiments, acceptor cells comprise exogenous mRNA derived from allogeneic or autologous donor cells. In some embodiments, acceptor cells comprise exogenous mRNA encoding 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 6382, 7000, 8000, 9000 genes, about, at least, at least about, up to, or up to, or a range defined by any two of the foregoing values ​​(e.g., 100-9000, 2000-8000, 4000-8000, 100-500, or 300-2000 genes). In some embodiments, the gene contained in the acceptor cells is a naive transcription factor. In some embodiments, the acceptor cells are not modified by transfection, electroporation, or viral transduction, or any combination thereof. In some embodiments, the acceptor cells express or upregulate the expression of one or more naive stem cell markers (e.g., CD130, CD77, CD7, CD75, or F11R). In some embodiments, the acceptor cells show downregulation of one or more primed stem cell markers (e.g., CD90, HLA-ABC, CD24, CD57, or SSEA4). In some embodiments, the acceptor cells express or upregulate the expression of one or more naive pluripotency markers or transcription factors (e.g., KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, or TBS3). In some embodiments, the acceptor cells downregulate expression of one or more primed pluripotency markers or transcription factors (e.g., ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, or XIST).In some embodiments, the cell composition is selected from the group consisting of resveratrol, epigallocatechin gallate (EGCG), curcumin, genistein, activin-A, Wnt-3a, sodium butyrate, basic fibroblast growth factor (bFGF), oncostatin M (OSM), dexamethasone (DEX), hepatocyte growth factor (HGF), CHIR-99021, forskolin, Y-27632 (ROCK inhibitor), (s)-(-)-blebbistatin, IWP2, A83-01, LY294002, SB-431542, NVP-BHG, cyclopamine-KAAD, PD-0325901, FGF4, LDN-193189, insulin-like growth factor (IGF), bone morphogenetic protein 2 (BMP2), transforming growth factor beta 2 (TGF-β2), BMP4, FGF-7, platelet-derived The composition further comprises at least one agent selected from the group consisting of: plant growth factor (PDGF) β3, epidermal growth factor (EGF), exendin-4, human neuregulin (hHRG) β3, retinoic acid (RA), L-ascorbic acid 2-phosphate (AA2P), ascorbic acid, insulin-transferrin-selenoethanolamine solution (ITS-X), insulin, rifampicin, penicillin, streptomycin, 2-mercaptoethanol, 3-mercaptopropane-1,2-diol (thioglycerol), L-proline, L-glutamine, non-essential amino acid mixture (NEAA), sodium pyruvate, trypsin-EDTA, phosphatidylinositol (PI), interleukin, prostaglandin, tumor necrosis factor, or any combination thereof. In some embodiments, the cell composition further comprises at least one agent selected from the group consisting of a GSK3 inhibitor (e.g., CHIR99021), a MAPK inhibitor (e.g., PD0325901), LIF, a JNK inhibitor (e.g., SP600125), a p38 inhibitor (e.g., SB203580), a ROCK inhibitor (e.g., Y-27632), a PKC inhibitor (e.g., Go6983), a BMP inhibitor (e.g., dorsomorphin), bFGF, activin A, ascorbic acid, a cAMP activator (e.g., forskolin), a TGF-β inhibitor (e.g., A83-01), or any combination thereof.

[0008] The embodiments of the invention provided herein are described by the following numbered alternatives: 1. A method comprising contacting an acceptor cell with a donor cell, said contacting causing transfer of an intracellular component from said donor cell to said acceptor cell. 2. The method of alternative 1, wherein said acceptor cells are pluripotent stem cells (PSCs). 3. The method of alternative 1 or 2, wherein said acceptor cells are primed human induced pluripotent stem cells ("primed hiPSCs"). 4. The method of any of the preceding alternatives, wherein the donor cells are any cells that have nanotubes. 5. The method of any of the preceding alternatives, wherein said donor cells are cells in a naive state that express naive transcription factors. 6. The method of any of the preceding alternatives, wherein said donor cells are naive mouse embryonic stem cells (naive mESCs). 7. The method of any of the preceding alternatives, wherein said donor cells and said acceptor cells are contacted in culture medium. 8. The method of any of the preceding alternatives, wherein said donor cells and said acceptor cells are cultured in a 1:1 ratio. 9. The method of any of the preceding alternatives, wherein the intracellular component is selected from one or more of RNA, protein, and organelle. 10. The method of any of the preceding alternatives, wherein the intracellular component is RNA. 11. The method of any of the preceding alternatives, wherein said donor cells transfer intracellular components via tunneling nanotubes or cytonemes. 12. The method of the preceding alternative, wherein said donor and acceptor cells are contacted under hypoxic conditions. 13. The method of alternative 12, wherein the hypoxic condition is about 5% O2. 14. The method of any of alternatives 1-11, wherein the donor cells and the acceptor cells are contacted under stressor conditions, the stressor conditions being selected from contact with a cytotoxic compound, hypoxia, a non-physiological temperature, a non-physiological pH, electroporation, or any combination thereof. 15. The method of any of alternatives 1-11, wherein the cells are contacted or grown at about 37°C. 16. The method of any of the preceding alternatives, wherein the acceptor cells and the donor cells are expanded by direct contact of the acceptor cells and the donor cells. 17. The method of any of the preceding alternatives, wherein said contacting step is carried out until said acceptor cells express naive stem cell markers (CD130, CD77). 18. The method of any of the preceding alternatives, wherein said contacting step is performed until said acceptor cells exhibit downregulation of primed stem cell markers (CD90, HLA-ABC). 19. The method of the preceding alternative, wherein said contacting step results in increased expression of naive pluripotency markers (DPPA3, TFCP2L1, DNMT3L, KLF4 and KLF17) and downregulation of primed pluripotency markers (DUSP6, THY1) in said acceptor cells. 20. The method of any of the preceding alternatives, wherein said contacting step results in expression of naive markers KLF17 and TFAP2C in said acceptor cells. 21. The method of any of the preceding alternatives, wherein said contacting step is performed until dome-shaped naive acceptor colonies are observed. 22. The donor cells or acceptor cells, or both, are treated with resveratrol, epigallocatechin gallate (EGCG), curcumin, genistein, activin-A, Wnt-3a, sodium butyrate, basic fibroblast growth factor (bFGF), oncostatin M (OSM), dexamethasone (DEX), hepatocyte growth factor (HGF), CHIR-99021, forskolin, Y-27632 ( ROCK inhibitor), (s)-(-)-blebbistatin, IWP2, A83-01, LY294002, SB-431542, NVP-BHG, cyclopamine-KAAD, PD-0325901, FGF4, LDN-193189, insulin-like growth factor (IGF), bone morphogenetic protein 2 (BMP2), transforming growth factor β2 (TGF-β2), BMP4, FGF-7, platelet-derived growth factor

[0023] The method of any of the preceding alternatives, comprising contacting the patient with at least one agent selected from the group consisting of: peptide (PDGF) beta 3, epidermal growth factor (EGF), exendin-4, human neuregulin (hHRG) beta 3, retinoic acid (RA), L-ascorbic acid 2-phosphate (AA2P), ascorbic acid, insulin-transferrin-selenoethanolamine solution (ITS-X), insulin, rifampicin, penicillin, streptomycin, 2-mercaptoethanol, 3-mercaptopropane-1,2-diol (thioglycerol), L-proline, L-glutamine, non-essential amino acid mixture (NEAA), sodium pyruvate, trypsin-EDTA, phosphatidylinositol (PI), interleukin, prostaglandin, tumor necrosis factor, or any combination thereof. [Brief explanation of the drawings]

[0009] In addition to the features described above, additional features and modifications will be readily apparent from the following drawings and description of exemplary embodiments, which should be understood to illustrate embodiments and are not intended to limit the scope.

[0010] [Figure 1A]Figure 1 illustrates an outline of an embodiment of the experimental design of human iPSCs (hiPSCs) expressing EGFP co-culture with mouse feeder (mFeeder) SNL cells. Human and mouse cell fractions were sorted by flow cytometry for downstream analysis. Bar, 200 µm. [Figure 1B] 1 illustrates an embodiment of RT-PCR analysis of human / mouse-specific ACTB / Actb and NEAT1 / Neat1 expression levels in sorted hiPSCs and SNL 76 / 7 mouse feeder (mFeeder) fractions after 5 days of co-culture. NEAT1 / Neat1, which is present in the cell nucleus and therefore cannot be transferred between cells, is shown as a negative control. [Figure 1C] Figure 1 illustrates an embodiment of RT-PCR analysis of human / mouse-specific ACTB / Actb expression levels in sorted hiPSCs cocultured with SNL 76 / 7 mouse feeder cells versus hiPSCs or SNL 76 / 7 mouse feeder cells cultured alone, as well as sorted mouse SNL cells cocultured with hiPSCs versus hiPSCs or SNL cells cultured alone for 5 days using the cell lines TkDA3-4-GFP, 1383D6-GFP, 317D6-GFP, and FF-I01-GFP. After coculture, hiPSCs expressed mouse Actb mRNA, and mouse SNL cells expressed human ACTB mRNA. [Figure 1D] Illustrated are embodiments of hiPSCs cultured either alone (top panel) or with feeder cells (bottom panel) as observed by SEM. hiPSCs cultured with SNL cells exhibit protruding nanotube structures extending between the two cell types. [Figure 1E] Illustrated are embodiments of mESCs cultured with m-feeder cells with visible nanotube structures (upper panel) and impaired nanotube formation between mESCs and m-feeder cells by LPS treatment (lower panel) as observed by SEM. The dotted line indicates the cell-cell interface. The double-headed arrow indicates the gap between the cells. [Figure 1F]Illustrates an embodiment of nanotube structures between m-feeders and hiPSCs observed by SEM (top row) that were compromised by LPS treatment (bottom row). The dotted line indicates the cell-cell interface. [Figure 1G] An embodiment of quantitative RT-PCR analysis of endogenous and translocated human / mouse-specific ACTB / Actb is illustrated in FIG. 1F. [Figure 2A] Illustrates an overview of an embodiment of the experimental design for hiPSC co-culture with mouse embryonic stem cells (mESCs) followed by serial sorting of hiPSCs. [Figure 2B] Figure 2B illustrates an embodiment of quantitative RT-PCR analysis of mouse-specific Actb and Nanog expression levels in sorted hiPSCs after 5 days of co-culture with mESCs (top graph). Values ​​indicate levels detected in hiPSCs compared to endogenous expression in mESCs. Figure 2B also illustrates quantitative RT-PCR analysis of human-specific ACTB and NANOG expression levels in sorted mESCs after co-culture with hiPSCs (bottom graph). Values ​​indicate levels detected in mESCs compared to endogenous expression in hiPSCs. [Figure 2C] Illustrated are embodiments of immunofluorescence micrographs of morphological changes in three hiPSC clones (317-12-EGFP, 317D6-EGFP, TKDA-mCherry) co-cultured with mESCs or mESC-OCT4-EGFP on day 5. [Figure 2D] Illustrates an embodiment of a heat map of differentially expressed mouse genes in hiPSCs and the gene ontology (GO) categories of the genes. The top 75 genes with the highest variance in gene expression between samples are plotted in the heat map. Increases in detected mouse genes are seen in all four tested cell lines. [Figure 2E] Illustrates an embodiment of selected naive and primed pluripotency-associated mRNAs in hiPSCs after co-culture with mESCs by RNA-seq. [Figure 3A]Illustrates an embodiment of proliferation and morphological changes in mESCs, hiPSCs, and hiPSCs mixed with mESCs from day 1 to day 5. The light gray signal in the right panel corresponds to 317D6-EGFP hiPSC cells. [Figure 3B] Figure 1 illustrates an embodiment of quantitative RT-PCR analysis of primed state-related (DUSP6) and naive state-related (DPPA3, DNMT3L, TFCP2L1, KLF4, KLF17) gene expression in hiPSCs after co-culture with mESCs. The graph shows the fold change of sorted hiPSCs and hiPSCs experiencing co-culture with mESCs only from three independent experiments. [Figure 3C] Figure 3C illustrates an embodiment of quantitative RT-PCR analysis of naive-state-related (DPPA3, DNMT3L, TFCP2L1, KLF4, KLF17) gene expression in hiPSCs after culture in mESC-conditioned medium or with mESCs in a transwell coculture assay (top graph). The graph shows the fold change of these hiPSCs compared to sorted hiPSCs cultured with mESCs. Figure 3C also illustrates quantitative RT-PCR analysis of mouse Actb in hiPSCs after coculture with mESCs, in a transwell coculture assay with mESCs, or after culture in mESC-conditioned medium. [Figure 3D] Illustrates an embodiment of flow cytometry analysis of primed specific markers (CD90 and HLAABC) and naive specific markers (CD130 and CD77) in hiPSC cultures either alone or with mESCs. [Figure 3E] Illustrated are embodiments of immunostaining for human naive markers KLF4, TFCP2L1, or human nuclear antigen (HuNu) in parental hiPSCs, sorted and expanded hiPSCs after co-culture with mESCs, or hiPSCs after chemical resetting. [Figure 3F]This figure illustrates an embodiment of principal component analysis (PCA) based on genes differentially expressed between naive and conventional PSCs for chemically reset cells (cR), co-cultured cells with mESCs (mixed), their parent iPSCs (primed), deposition dataset, Shef6-primed ESCs (Shef6-primed) from the RNA-seq dataset, and the chemically reset cells (Shef6-cR). PC1 explains 57% of the analyzed gene set, and PC2 explains 18%. Each set has three dots corresponding to three replicates. [Figure 3G] 1 illustrates an embodiment of hierarchical clustering of a dataset obtained by RNA-seq. [Figure 3H] Illustrated are embodiments of averaged normalized RNA-seq counts of naive and primed markers in cells from Figures 3E-G. [Figure 3I] Illustrated is an embodiment of quantitative RT-PCR analysis of human primed (DUSP6) and naive (DPPA3, DNMT3L, TFCP2L1, KLF4, KLF17) gene expression in different hiPSC cell lines (317-12, 317D6, TKDA) cultured with mESCs. [Figure 3J] Illustrated is an embodiment of quantitative RT-PCR analysis of human primed (DUSP6) and naive (DPPA3, DNMT3L, TFCP2L1, KLF4, KLF17) gene expression in hiPSC cell lines cultured with mESCs at different ratios (2:8, 5:5, 8:2, 10:0). hiPSCs showed that the higher the relative number of mESCs, the more naive marker genes were expressed. [Figure 3K] 1 illustrates SEM analysis of an embodiment of hiPSCs co-cultured with mESCs showing the loss of nanotubes and the transfer of mouse Actb and Nanog in hiPSCs in the presence of LPS. LPS low: 100 ng / mL; LPS high: 500 ng / mL. [Figure 3L] Illustrates one embodiment of the morphology of hiPSCs (expressing mCherry) co-cultured with mESCs (expressing GFP) under LPS treatment. [Figure 3M] Illustrated is an embodiment of quantitative RT-PCR analysis of human primed (DUSP6) and naive (DPPA3, DNMT3L, TFCP2L1, KLF4, KLF17) gene expression in hiPSCs undergoing co-culture with mESCs in the presence or absence of LPS treatment. LPS low: 100 ng / mL; LPS high: 500 ng / mL. [Figure 4A] Illustrates one embodiment of ATAC-seq analysis of hiPSCs before and after co-culture with mESCs. One culture using 317-12 and two replicate cultures using 317-D6 were analyzed. [Figure 4B] Figure 1 illustrates an example of a global view of chromatin accessibility changes in hiPSCs before and after co-culture with mESCs. ATAC-seq was performed in three separate experiments. ATAC-seq peaks were identified and categorized into peaks shared between the two conditions (top), peaks that closed during co-culture (middle), and peaks that opened during co-culture (bottom). [Figure 4C] Illustrates an embodiment of transcription factor (TF) binding site motif enrichment analysis in hiPSC 317-D6 line ATAC-seq peaks (replicate 1) with and without mESC co-culture. Each point represents a TF binding motif. The X-axis shows the enrichment of motifs in the "co-culture loss" peak. The Y-axis shows the enrichment of motifs in the "co-culture gain" peak. [Figure 4D] Illustrates an embodiment of a similar TF binding site motif enrichment analysis shown in Figure 4C for 317-D6 (replication 2) and 317-12 hiPSC lines. [Figure 4E] 1 illustrates an embodiment of a screenshot from the UCSC genome browser depicting the promoter region of the human TFAP2C gene. ATAC-seq signals of hiPSCs in co-culture or with co-culture with mESC cells are shown. [Figure 4F]Illustrates an embodiment of a schematic of mRNA transfer-induced naive-like conversion in human pluripotent stem cells co-cultured with mESCs. a': Introduction of mRNA encoding a TF occurs between adjacent primed hiPSCs and naive mESCs during co-culture. b': Chromatin reorganization and epigenetic modifications at the corresponding TF-bound loci are induced during conversion to a naive-like state. c': Primed hiPSCs are reprogrammed into early naive-like cells. [Figure 5A] Illustrates embodiments of shRNAs used herein that target mouse Klf4, Tfcp2l1, and Tfap2c. Shows the sequence comparison of each shRNA's target sequence with its human orthologue. Shows the number of mismatches between the mouse target sequence and its human orthologue. [Figure 5B] Illustrates one embodiment of validation of shRNA knockdown efficiency in mouse ESCs. Quantitative RT-PCR analysis of Klf4, Tfcl2l1, Tfap2c, Pou5f1, and Nanog from mouse ESCs infected with luciferase- (shLuc) or mouse transcription factor-targeting shRNAs. Data are expressed as folds of the shLuc value. Values ​​are shown as mean ± SEM (n = 3). Differences were analyzed by ANOVA with Tukey's post-hoc test. **P<0.01, ***P<0.001, and ****P<0.0001 vs. shLuc. [Figure 5C] 1 shows an embodiment of quantitative RT-PCR analysis of KLF4, TFCP2L1, TFAP2C, POU5F1, and NANOG derived from human iPSCs infected with luciferase (shLuc) or mouse transcription factor-targeting shRNA. Data are expressed as multiples of the shLuc value. Values ​​are shown as mean ± SEM (n=3). [Figure 5D] Illustrates an embodiment of an experimental design overview of human iPSCs expressing mouse transcription factors targeting shRNA (puromycin-resistant) co-culture with mouse ESCs (puromycin-sensitive), followed by puromycin selection and growth of human iPSCs in PGXL medium. [Figure 5E]Illustrates an embodiment of the formation of growing human iPSC colonies expressing shRNA-targeted mouse transcription factors after puromycin selection. Bar, 100 μm. [Figure 5F] One embodiment of quantification of the number of dome-shaped colonies is shown in Figure 5E. Colonies were counted from at least 15 different observation fields. [Figure 5G] Illustrated are embodiments of brightfield (left) and immunostained (right) images of hiPSCs before and after mouse ESC co-culture followed by puromycin selection. Bar, 100 μm (brightfield) or 10 μm (immunostained). [Figure 6A] 1 shows an embodiment of immunostaining of pan-Oct4 (clone: ​​C30A3) and mouse-specific Oct4 (clone: ​​D6C8T) antibodies for human iPSCs and mouse ESCs. [Figure 6B] 1 illustrates one embodiment of immunostaining of human-specific Nanog (clone: ​​D73G4) and mouse-specific Nanog (clone: ​​D2A3) antibodies on human iPSCs and mouse ESCs. [Figure 6C] This figure illustrates an embodiment of immunostaining for mouse-specific Oct4 protein in human iPSC (GFP-positive) co-cultures with mouse ESCs (tdTomato-positive) or human iPSCs alone. A representative area at high magnification, enclosed by a dashed rectangle, is also shown in the inset. The arrow indicates that human iPSCs are weakly positive for mouse Oct4 protein. [Figure 6D] This figure shows an example of immunostaining for mouse-specific Nanog protein in human iPSC (GFP-positive) co-cultures with mouse ESCs (tdTomato-positive) or human iPSCs alone. A representative area at high magnification, enclosed by a dashed rectangle, is also shown in the inset. The arrow indicates that human iPSCs are weakly positive for mouse Nanog protein. DETAILED DESCRIPTION OF THE INVENTION

[0011] Described herein are cell compositions comprising acceptor cells and donor cells, wherein the acceptor cells and donor cells are pluripotent stem cells. In some embodiments, the acceptor cells are primed pluripotent stem cells and the donor cells are naive pluripotent stem cells. In some embodiments, direct cell-to-cell contact between the acceptor cells and the donor cells reprograms the acceptor cells from a primed state to a naive state. In some embodiments, but without being limited by any mechanism of action, this reprogramming can occur by tunneling nanotubes. In some embodiments, the acceptor cells and donor cells are of the same species (allogeneic), such as human. In other embodiments, the acceptor cells and donor cells are of different species (xenogeneic). For example, the acceptor cells are human cells and the donor cells are mouse cells. Also described herein are methods of preparing reprogrammed naive acceptor cells by contacting the acceptor cells with naive donor cells.

[0012] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols generally identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0013] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood when read in light of this disclosure by one of ordinary skill in the art to which this disclosure belongs. For purposes of this disclosure, the following terms are described below.

[0014] As used herein, the articles "a" and "an" are used to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0015] "About" means a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 10% from the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0016] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" are understood to mean the inclusion of the recited step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" means that the recited elements are required or essential, and that no other elements may be present. "Consisting essentially of" means including the elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they significantly affect the activity or action of the recited elements.

[0017] As used herein, the terms "individual," "subject," or "patient" have their plain and ordinary meanings as understood in light of the present specification, and refer to human or non-human mammals, such as dogs, cats, mice, rats, cows, sheep, pigs, goats, non-human primates, or birds, such as chickens, as well as other vertebrates or invertebrates. The term "mammal" is used in its ordinary biological sense. Thus, it specifically includes, but is not limited to, primates, including monkeys (chimpanzees, apes, monkeys) and humans, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, etc.

[0018] As used herein, the terms "effective amount" or "effective dose" have their plain and ordinary meaning as understood in light of the specification and refer to that amount of the recited composition or compound that produces an observable effect. The actual dosage level of the active ingredients in the active compositions of the presently disclosed subject matter can be varied to administer an amount of the active composition or compound effective to achieve the desired response for a particular subject and / or application. The selected dosage level will depend on various factors, including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimum dose is administered, and in the absence of dose-limiting toxicity, the dose is increased to the minimum effective amount. Determination and adjustment of the effective dose, as well as evaluation of when and how to make such adjustments, are contemplated herein.

[0019] As used herein, the terms "function" and "functional" have their plain and ordinary meaning as understood in light of this specification and refer to biological, enzymatic, or therapeutic function.

[0020] The term "inhibit," as used herein, has its plain and ordinary meaning as understood in light of the present specification and can refer to a reduction or prevention of biological activity. The reduction can be a percentage of O that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, about, at least, at least about, less than, or less than about, or a percentage of O that is within a range defined by any two of the foregoing values. The term "delay," as used herein, has its plain and ordinary meaning as understood in light of the specification and refers to a delay, postponement, or postponement of a biological event to a time later than would otherwise be expected. The delay can be a percentage of O that is 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, about a percent of O, at least a percent of O, at least about a percent of O, or less than a percent of O, or less than a percent of O, or a percentage of O within a range defined by any two of the foregoing values. The terms suppression and delay do not necessarily indicate 100% suppression or delay. Partial suppression or delay may be achieved.

[0021] As used herein, the term "isolated" has its plain and ordinary meaning as understood in light of the specification and refers to (1) a substance and / or entity that is separated from at least some of the components with which it is associated when it is originally produced (in nature and / or in an experimental setting), and / or (2) when manufactured, prepared, and / or created by the hand of man. Isolated substances and / or entities can be separated from equal to, about, at least about, no more than, or no more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, substantially 100%, or 100% of other components with which they are originally associated (or a range including and / or spanning the stated values). In some embodiments, an isolated agent is about, at least, at least about, no more than, or no more than about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, substantially 100%, or 100% pure (or a range including and / or spanning the above values). As used herein, an "isolated" material can be "pure" (e.g., substantially free from other components). As used herein, the term "isolated cell" can refer to a cell that is not contained in a multicellular organism or tissue.

[0022] As used herein, "in vivo" is given its plain and ordinary meaning in light of the present specification and refers to the performance of methods within living organisms, usually animals, mammals, including humans, and plants, as opposed to tissue extracts or dead organisms.

[0023] As used herein, "ex vivo" is given its plain and ordinary meaning in light of this specification and refers to the performance of methods outside of a living body that do not significantly alter natural conditions.

[0024] As used herein, "in vitro" is given its plain and ordinary meaning in light of the specification and refers to the performance of a method outside biological conditions, for example, in a petri dish or test tube.

[0025] As used herein, the terms "nucleic acid" or "nucleic acid molecule" have their plain and ordinary meaning as understood in light of the present specification and refer to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), that naturally occur in cells, oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have alterations in the sugar moiety and / or pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Additionally, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of base moiety modifications include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Phosphodiester bond analogs include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, or phosphoramidates. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. Nucleic acids can be either single-stranded or double-stranded. "Oligonucleotide" can be used interchangeably with nucleic acid and can refer to either double-stranded or single-stranded DNA or RNA.The nucleic acid(s) can be contained in a nucleic acid vector or construct (e.g., a plasmid, virus, retrovirus, lentivirus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC), which can be used to amplify and / or express the nucleic acid(s) in various biological systems. Typically, the vector or construct will also contain elements including, but not limited to, a promoter, enhancer, terminator, inducer, ribosome binding site, translation initiation site, start codon, stop codon, polyadenylation signal, origin of replication, cloning site, multiple cloning site, restriction enzyme site, epitope, reporter gene, selection marker, antibiotic selection marker, target sequence, peptide purification tag, or accessory gene, or any combination thereof.

[0026] RNA is a nucleic acid polymer molecule that performs a wide range of functions in biological systems. Messenger RNA is responsible for protein expression derived from sequence information stored in genomic DNA. During transcription, pre-mRNA transcripts are processed (e.g., intron splicing, 5' capping, polyadenylation) and exported from the nucleus as mature mRNA, which moves freely through the cytoplasm until binding to ribosomes for translation. Other RNA molecules, including but not limited to noncoding RNA (ncRNA), antisense RNA (asRNA), long noncoding RNA (lncRNA), microRNA (miRNA), Piwi-interacting RNA (piRNA), small interfering RNA (siRNA), or short hairpin RNA (shRNA), or combinations thereof, play a major role in gene regulation, usually by binding to and subsequent degradation or inactivation of complementary mRNA and pathways such as the RNA-induced silencing complex (RISC). This knowledge has led to a rich toolset for engineering cells to transiently express proteins (mRNA) or downregulate their expression by transfecting synthetic or isolated RNA molecules. Similarly, the delivery of RNA molecules during cell-to-cell transfer (eg, by TNTs or microvesicles) has considerable effects on recipient cells.

[0027] A nucleic acid or nucleic acid molecule can contain one or more sequences encoding different peptides, polypeptides, or proteins, which can be adjacent within the same nucleic acid or nucleic acid molecule or can be joined with, for example, linkers, repeats, or restriction enzyme sites, or extra nucleic acid between any other sequences that are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, about, at least, at least about, up to, or about, up to, or any length within the range defined by any two of the foregoing base lengths. As used herein, the term "downstream" of a nucleic acid refers to the sequence on the strand containing the coding sequence (sense strand) that is after the 3' end of the preceding sequence when the nucleic acid is double-stranded. As used herein, the term "upstream" of a nucleic acid refers to the sequence on the strand containing the coding sequence (sense strand) that is before the 5' end of the following sequence when the nucleic acid is double-stranded. As used herein, the term "grouped" in reference to nucleic acids refers to two or more sequences occurring in close proximity, either directly or with extra nucleic acid between them, but usually without intervening sequences encoding functional or catalytic polypeptides, proteins, or protein domains, for example, linkers, repeats, or restriction enzyme sites, or sequences that are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, about, at least, at least about, up to, or up to, or any base length within the range defined by any two of the foregoing base lengths.

[0028] The nucleic acid described herein comprises nucleobases.The primary, standard, natural or unmodified bases are adenine, cytosine, guanine, thymine and uracil.Other nucleobases include, but are not limited to, purine, pyrimidine, modified nucleobase, 5-methylcytosine, pseudouridine, dihydrouridine, inosine, 7-methylguanosine, hypoxanthine, xanthine, 5,6-dihydrouracil, 5-hydroxymethylcytosine, 5-bromouracil, isoguanine, isocytosine, aminoallyl base, dye-labeled base, fluorescent base or biotin-labeled base.

[0029] As used herein, the terms "peptide," "polypeptide," and "protein" have their plain and ordinary meaning as understood in light of the present specification and refer to polymers composed of amino acids linked by peptide bonds. The many functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymatic, structural, transport, defensive, hormonal, or signal transduction functions. Peptides, polypeptides, and proteins are often, but not always, produced biologically by ribosomal complexes using nucleic acid templates, although chemical synthesis is also available. By manipulating nucleic acid templates, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed. These fusions of more than one peptide, polypeptide, or protein can be joined adjacently within the same molecule, or with extra amino acids between them, for example, linkers, repeats, epitopes, or tags, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases in length, about the above-mentioned bases in length, at least the above-mentioned bases in length, at least about the above-mentioned bases in length, or up to the above-mentioned bases in length, or up to the above-mentioned bases in length, or any other sequence within the range defined by any two of the foregoing base lengths. As used herein, the term "downstream" on a polypeptide refers to the sequence after the C-terminus of the preceding sequence. As used herein, the term "upstream" on a polypeptide refers to the sequence that precedes the N-terminus of the subsequent sequence.

[0030] The term "purity" of any given substance, compound, or material, as used herein, has its plain and ordinary meaning as understood in light of the present specification and refers to the actual abundance of the substance, compound, or material relative to the expected abundance. For example, the substance, compound, or material is about, at least, at least about, no more than, or no more than about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure (including all decimal points therebetween). Purity may be affected by unwanted impurities or contaminants, including, but not limited to, nucleic acids, DNA, RNA, nucleotides, proteins, polypeptides, peptides, amino acids, lipids, cell membranes, cell debris, small molecules, degradation products, solvents, carriers, vehicles, or any combination thereof. In some embodiments, the substance, compound, or material is substantially free of host cell proteins, host cell nucleic acids, plasmid DNA, contaminating viruses, proteasomes, host cell culture components, process-related components, mycoplasma, pyrogens, bacterial endotoxins, and adventitious infectious agents. Purity can be measured using techniques including, but not limited to, electrophoresis, SDS-PAGE, capillary electrophoresis, PCR, rtPCR, qPCR, chromatography, liquid chromatography, gas chromatography, thin-layer chromatography, enzyme-linked immunosorbent assay (ELISA), spectroscopy, UV-visible spectroscopy, infrared spectroscopy, mass spectrometry, nuclear magnetic resonance, gravimetry, or titration, or any combination thereof.

[0031] The term "yield" of any given substance, compound, or material, as used herein, has its plain and ordinary meaning as understood in light of the specification and refers to the actual total amount of the substance, compound, or material relative to the expected total amount. For example, the yield of a substance, compound, or material may be about, at least, at least about, no more than, or no more than about 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (including all decimal points therebetween) of the expected total amount. Yield may be affected by the efficiency of the reaction or process, undesired side reactions, decomposition, the quality of the input substances, compounds, or materials, or loss of the desired substance, compound, or material at any stage of production.

[0032] The term "% w / w" or "% weight / weight" as used herein has its plain and ordinary meaning as understood in the context of the present specification and refers to a percentage expressed in terms of the weight of a component or agent relative to the total weight of the composition, multiplied by 100. The term "% v / v" or "% volume / volume" as used herein has its plain and ordinary meaning as understood in the context of the present specification and refers to a percentage expressed in terms of the liquid volume of a compound, substance, component, or composition relative to the total liquid volume of the composition, multiplied by 100.

[0033] stem cells As used herein, the term "totipotent stem cells" (also known as omnipotent stem cells) has its plain and ordinary meaning as understood in light of this specification and refers to stem cells that can differentiate into embryonic and extraembryonic cell types. Such cells are capable of building complete, viable organisms. These cells are produced from the fusion of egg and sperm cells. Cells produced by the first few divisions of a fertilized egg are also totipotent.

[0034] As used herein, the term "embryonic stem cells (ESCs), commonly abbreviated as ES cells, has its plain and ordinary meaning as understood in light of the present specification and refers to cells that are pluripotent and derived from the inner cell mass of an early embryo, the blastocyst. For purposes of the present invention, the term "ESCs" is sometimes used broadly to encompass embryonic germ cells as well.

[0035] As used herein, the term "pluripotent stem cells (PSCs)" has its plain and ordinary meaning as understood in light of the present specification and encompasses any cell that can differentiate into almost any cell type in the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (stomach lining, digestive tract, lungs), mesoderm (muscle, bone, blood, urogenital tract), and ectoderm (epidermal tissue and nervous system). PSCs may be the descendants of inner cell mass cells of a preimplantation blastocyst or may be obtained by the induction of non-pluripotent stem cells, e.g., adult somatic cells, by forcing the expression of specific genes. Pluripotent stem cells may be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.

[0036] As used herein, the term "induced pluripotent stem cells (iPSCs)," commonly abbreviated as iPS cells, has its plain and ordinary meaning as understood in light of the present specification and refers to a type of pluripotent stem cell artificially derived, typically from a non-pluripotent stem cell, e.g., an adult somatic cell, by inducing the "forced" expression of specific genes. hiPSCs refer to human iPSCs. In several art-known methods, iPSCs can be derived by transfecting specific stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection can be achieved by viral transduction using viruses such as retroviruses or lentiviruses. Transfected genes can include the master transcriptional regulators Oct-3 / 4 (POU5F1) and Sox2, although other genes may increase the efficiency of induction. After 3-4 weeks, a small number of transfected cells begin to resemble pluripotent stem cells morphologically and biochemically and are typically isolated by morphological selection, doubling time, or reporter gene and antibiotic selection. As used herein, iPSCs include first-generation iPSCs, second-generation iPSCs in mice, and human induced pluripotent stem cells.In some methods, retrovirus systems are used to transform human fibroblasts into pluripotent stem cells using four essential genes: Oct3 / 4, Sox2, Klf4, and c-Myc.In other methods, retrovirus systems are used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28.Genes whose expression is induced in iPSCs include, but are not limited to, Oct-3 / 4 (POU5F1), certain members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15), certain members of the Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, LIN28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or any combination thereof.

[0037] The term "progenitor cell," as used herein, has its plain and ordinary meaning as understood in light of the present specification and encompasses any cell that can be used in the methods described herein, whereby one or more progenitor cells acquire the ability to regenerate themselves or differentiate into one or more specialized cell types. In some embodiments, progenitor cells are pluripotent or have the ability to become pluripotent. In some embodiments, progenitor cells are subjected to treatment with external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, progenitor cells can be totipotent (or omnipotent) stem cells, pluripotent stem cells (induced or non-induced), multipotent stem cells, oligopotent stem cells, and unipotent stem cells. In some embodiments, progenitor cells can be derived from an embryo, an infant, a child, or an adult. In some embodiments, progenitor cells can be somatic cells that have been subjected to treatment to confer pluripotency via genetic manipulation or protein / peptide treatment. Progenitor cells include embryonic stem cells (ESCs), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSCs).

[0038] In some embodiments, one step is to obtain stem cells that are pluripotent or can be induced to become pluripotent. In some embodiments, the pluripotent stem cells are derived from embryonic stem cells, which are derived from totipotent cells of early mammalian embryos and are capable of unlimited undifferentiated proliferation in vitro. Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of an early-stage embryo, the blastocyst. Methods for deriving embryonic stem cells from blastocysts are well known in the art. Human embryonic stem cells H9 (H9-hESC) are used in the exemplary embodiments described in this application, but it will be understood by those skilled in the art that the methods and systems described herein can be applied to any stem cells.

[0039] Additional stem cells that can be used in embodiments of the present invention include, but are not limited to, those provided by or described in databases hosted by the National Stem Cell Bank (NSCB), the Human Embryonic Stem Cell Research Center at the University of California, San Francisco (UCSF), the WISC cell Bank at the Wi Cell Research Institute, the University of Wisconsin Stem Cell and Regenerative Medicine Center (UW-SCRMC), Novocell, Inc. (San Diego, Calif.), Cellartis AB (Göteborg, Sweden), ES Cell International Pte Ltd (Singapore), the Technion at the Israel Institute of Technology (Haifa, Israel), and the Stem Cell Database hosted by Princeton University and the University of Pennsylvania. Exemplary embryonic stem cells that can be used in embodiments according to the present invention include, but are not limited to, SA01 (SA001), SA02 (SA002), ES01 (HES-1), ES02 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6), BG01 (BGN-01), BG02 (BGN-02), BG03 (BGN-03), TE03 (13), TE04 (14), TE06 (16), UCOI (HSF1), UC06 (HSF6), WA01 (HI), WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14). Exemplary human pluripotent cell lines include, but are not limited to, TkDA3-4, 1231A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34-1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF, or 317-12 cells.

[0040] In developmental biology, cell differentiation is the process by which less specialized cells become more specialized cell types. As used herein, the term "directed differentiation" describes the process by which less specialized cells become specific specialized target cell types. The specificity of the specialized target cell type can be determined by any applicable method that can be used to define or change the fate of the original cell. Exemplary methods include, but are not limited to, genetic manipulation, chemical treatment, protein treatment, and nucleic acid treatment.

[0041] In some embodiments, adenoviruses can be used to deliver the necessary pluripotency factors into cells, resulting in iPSCs that are virtually identical to embryonic stem cells. Because adenoviruses do not combine their own genes with those of the target host, the risk of tumor formation is eliminated. In some embodiments, non-viral techniques are used to generate iPSCs. In some embodiments, reprogramming can be achieved via plasmids without the use of any viral transfection system at all, albeit with very low efficiency. In other embodiments, iPSCs are generated using direct protein delivery, thereby eliminating the need for viral or genetic modification. In some embodiments, mouse iPSCs can be generated using similar methodologies. Repeated treatment of cells with specific proteins delivered to the cells via polyarginine anchors was sufficient to induce pluripotency. In some embodiments, the expression of pluripotency-inducing genes can also be increased by treating somatic cells with FGF2 under hypoxic conditions.

[0042] As used herein, the term "naive" has its plain and ordinary meaning as understood in light of the present specification and refers to the state of pluripotent stem cells in early stages of development, such as those comprising the inner cell mass or epiblast during embryonic development (e.g., around days 4-9 after fertilization in humans). These cells can be easily cultured or genetically manipulated, proliferate rapidly, exhibit high single-cell clonogenicity, and can be induced to differentiate efficiently into tissues of all three germ layers. Consequently, these cells can be used to form chimeras. In culture, these cells typically grow as dome-shaped colonies and depend on LIF for maintenance and growth. They exhibit global DNA hypomethylation and have not undergone X-chromosome inactivation. Typical examples of naive stem cells are mouse ESCs, such as those from the inner cell mass of preimplantation embryos, and mouse iPSCs. Naive transcription factors or other related proteins include, but are not limited to, KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, or TBS3, or any combination thereof.Naive cell surface markers include, but are not limited to, CD7, CD75, CD77, CD130, or F11R, or any combination thereof. In some embodiments, a "naive" cell is a cell that expresses at least one, or at least three, or each of the following transcription factors or other related proteins: KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, and TBS3, or a "naive" cell is a cell that has higher expression of at least one, or at least three, or each of KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, and TBS3 compared to a "primed" cell.In some embodiments, a "naive" cell is a cell that expresses at least one, or at least three, or each of the following transcription factors or other related proteins: DPPA, TFCP2L1, DNMT3L, KLF4, or KLF17, or a "naive" cell is a cell that has higher expression of at least one, or at least three, or each of the following proteins: DPPA, TFCP2L1, DNMT3L, KLF4, and KLF17 compared to a "primed" cell. In some embodiments, a "naive" cell is a cell that expresses at least one, or at least three, or each of the following cell surface markers: CD7, CD75, CD77, CD130, and F11R, or a "naive" cell is a cell that has higher expression of at least one, or at least three, or each of the following proteins: CD7, CD75, CD77, CD130, and F11R compared to a "primed" cell. In some embodiments, "naive" cells are cells that express at least one or each of the following cell surface markers: CD130 and CD77, or "naive" cells are cells that have higher expression of at least one or each of the proteins CD130 and CD77 compared to "primed" cells.In some embodiments, a "naive" cell is a cell that expresses at least one, or at least three, or each of the following transcription factors, cell surface markers, or other related proteins: KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, TBS3, CD7, CD75, CD77, CD130, and F11R, or a "naive" cell is a cell that has higher expression of at least one, or at least three, or each of the following proteins: KLF4, KLF5, KLF17, TFCP2L1 DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, TBS3, CD7, CD75, CD77, CD130, and F11R compared to a "primed" cell. In some embodiments, a "naive" cell is a cell that expresses at least one, or at least three, or each of the following transcription factors, cell surface markers, or other related proteins: DPPA3, TFCP2L1, DNMT3L, KLF4, KLF17, CD130, and CD77, or a "naive" cell is a cell that has higher expression of at least one, or at least three, or each of the following proteins: DPPA3, TFCP2L1, DNMT3L, KLF4, KLF17, CD130, and CD77, compared to a "primed" cell. In some embodiments, "naive" cells do not express one or more (e.g., at least 1, 3, 5) of the following "primed" transcription factors or other related proteins: ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, or XIST, or "naive" cells have reduced expression of one or more (e.g., at least 1, 3, 5) of ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, or XIST compared to "primed" cells.In some embodiments, "naive" cells do not express one or more (e.g., at least one) of DUSP6 or THY1, or "naive" cells have reduced expression of one or more (e.g., at least one) of DUSP6 or THY1 compared to "primed" cells. In some embodiments, "naive" cells do not express one or more (e.g., at least one, three, five) of the following "primed" cell surface markers: CD24, CD57, CD90, SSEA4, or HLAABC, or "naive" cells have reduced expression of one or more (e.g., at least one, three, five) of CD24, CD57, CD90, SSEA4, or HLAABC compared to "primed" cells. In some embodiments, "naive" cells do not express one or more (e.g., at least one) of CD90 or HLAABC, or "naive" cells have reduced expression of one or more (e.g., at least one) of CD90 or HLAABC compared to "primed" cells. In some embodiments, "naive" cells do not express one or more (e.g., at least 1, 3, 5, 10) of the following proteins: ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, XIST, CD24, CD57, CD90, SSEA4, or HLAABC, or "naive" cells have reduced expression of one or more (e.g., at least 1, 3, 5, 10) of the following proteins: ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, XIST, CD24, CD57, CD90, SSEA4, or HLAABC compared to "primed" cells. In some embodiments, a "naive" cell refers to a "primed" cell that has been reprogrammed to a naive state according to one of the methods described herein, and the relative expression of one or more (e.g., at least 1, 3, 5, 10) of the proteins listed herein for the "naive" cell is compared to when the "naive" cell was a "primed" cell (i.e., in a "primed" state) prior to the reprogramming step according to one of the methods described herein.

[0043] The term "primed" as used herein has its plain and ordinary meaning as understood in light of the present specification and refers to the state of pluripotent stem cells that represent a later stage of development, e.g., pluripotent stem cells that comprise the three primary germ layers (ectoderm, mesoderm, and endoderm) after differentiation of the epiblast during embryonic development (e.g., after day 9 post-fertilization in humans). These cells are less suitable for chimera formation because they are more difficult to genetically manipulate, have poor single-cell clonogenicity, and tend to differentiate into cell lineages belonging to any one specific germ layer. In culture, these cells typically grow as flat monolayers and depend on activin or FGF2 for maintenance and growth. They exhibit global DNA methylation and have undergone or are in the process of undergoing X-chromosome inactivation. Primed stem cells typically include isolated human ESCs, human iPSCs, and mouse epiblast stem cells (EpiSCs). Primed transcription factors or other related proteins include, but are not limited to, ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, or XIST, or any combination thereof. Primed cell surface markers include, but are not limited to, CD24, CD57, CD90, SSEA4, or HLAABC, or any combination thereof. In some embodiments, a "primed" cell is a cell that expresses at least one, or at least three, or each of the following "primed" transcription factors or other related proteins: ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, or XIST, or a "primed" cell is a cell that has higher expression of one or more of ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, or XIST compared to "naive" cells (e.g., at least 1, 3, 5). In some embodiments, a "primed" cell is a cell that expresses one or more DUSP6 or THY1, or has higher expression of one or more DUSP6 or THY1 compared to a "naive" cell (e.g., at least one).In some embodiments, "primed" cells express one or more of the following "primed" cell surface markers: CD24, CD57, CD90, SSEA4, or HLAABC, or "primed" cells have higher expression of one or more (at least 1, 3, 5) of CD24, CD57, CD90, SSEA4, or HLAABC compared to "naive" cells. In some embodiments, "primed" cells express one or more of CD90 or HLAABC, or have higher expression of one or more (e.g., at least 1) of CD90 or HLAABC compared to "naive" cells. In some embodiments, "primed" cells express one or more of the following proteins: ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, XIST, CD24, CD57, CD90, SSEA4, or HLAABC, or "primed" cells have higher expression of one or more (at least 1, 3, 5) of ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, XIST, CD24, CD57, CD90, SSEA4, or HLAABC compared to "naive" cells. In some embodiments, a "primed" cell is a cell that does not express one or more of the following "naive" transcription factors or other related proteins: KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, and TBS3, or a "primed" cell is a cell that has reduced expression of one or more (e.g., at least 1, 3, 5, 10) of the following proteins compared to a "naive" cell: KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, and TBS3.In some embodiments, a "primed" cell is a cell that does not express one or more of the following "naive" transcription factors or other related proteins: DPPA, TFCP2L1, DNMT3L, KLF4, or KLF17, or a "primed" cell is a cell that has reduced expression of one or more (e.g., at least 1, 3, 5) of the following proteins: DPPA, TFCP2L1, DNMT3L, KLF4, or KLF17 compared to a "naive" cell. In some embodiments, a "primed" cell is a cell that does not express one or more of the following cell surface markers: CD7, CD75, CD77, CD130, or F11R, or a "primed" cell is a cell that has reduced expression of one or more (e.g., at least 1, 3, 5) of the following proteins: CD7, CD75, CD77, CD130, or F11R compared to a "naive" cell. In some embodiments, "primed" cells are cells that do not express one or more of the proteins CD130 or CD77, or that have reduced expression of one or more (e.g., 1) of the proteins CD130 or CD77 compared to "naive" cells. In some embodiments, a "primed" cell is a cell that does not express one or more of the following transcription factors, cell surface markers, or other related proteins: KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, TBS3, CD7, CD75, CD77, CD130, or F11R, or a "primed" cell is a cell that has reduced expression of one or more (at least 1, 3, 5, 10) of the following proteins compared to a "naive" cell: KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, TBS3, CD7, CD75, CD77, CD130, or F11R.In some embodiments, a "primed" cell is a cell that does not express one or more of the following transcription factors, cell surface markers, or other related proteins: DPPA3, TFCP2L1, DNMT3L, KFL4, KLF17, CD130, or CD77, or a "primed" cell is a cell that has reduced expression of one or more (e.g., at least 1, 3, 5) of the following proteins compared to a "naive" cell: DPPA3, TFCP2L1, DNMT3L, KLF4, KLF17, CD130, or CD77. In some embodiments, a "primed" cell refers to a cell that has not yet been reprogrammed to a "naive" state according to one of the methods described herein, and the relative expression of one or more (e.g., at least 1, 3, 5, 10) of the proteins listed herein for a "primed" cell is compared to the expression levels found in a cell after reprogramming to a "naive" state according to one of the methods described herein.

[0044] To effectively utilize pluripotent stem cells for purposes such as stem cell research, disease modeling, drug screening, and cell-based therapy, the use of naive stem cells has been shown to be more preferable, and therefore, significant efforts have been made to transform primed stem cells into a naive state. Previously developed techniques for transitioning iPSCs to a naive state generally involve genetic manipulation to express naive factors (e.g., KLF4, KLF2) or the use of the following small molecule compounds, but are not limited to: glycogen synthase kinase 3β (GSK3) inhibitors (e.g., CHIR99021), mitogen-activated protein kinase (MAPK) (also known as extracellular signal-regulated kinase [ERK1 / 2]) inhibitors (e.g., PD0325901), leukemia inhibitory factor (LIF), c-Jun N-terminal kinase (JNK) inhibitors (e.g., SP600125), p38 inhibitors (e.g., SB203580), Rho kinase (ROCK) inhibitors (e.g., Y-27632), and protein kinase C (PKC) inhibitors (e.g., Go). 6983), bone morphogenetic protein (BMP) inhibitors (e.g., dorsomorphin), basic fibroblast growth factor (bFGF, FGF-2), activin A, ascorbic acid, cAMP activators (e.g., forskolin), TGF-β, or TGF-β inhibitors (e.g., A83-01), or any combination thereof. In some embodiments, the cells are grown in 2i medium, which may include a GSK3 inhibitor and a MAPK inhibitor. In some embodiments, the cells are grown in mTeSR, RSet, naive human stem cell (NHSM), T2iLGo, tt2iLGo, 5i, 4i, 3i, or feeder-independent naive embryo (FINE) medium, which may include LIF, a MAPK inhibitor, a GSK3 inhibitor, a JNK inhibitor, a p38 inhibitor, bFGF, or TGF-β, or any combination thereof. In some embodiments, the stem cells are not grown with a feeder cell substrate. In some embodiments, the stem cells are grown with a feeder cell substrate.Additional information regarding transforming stem cells to a naive state can be found in Collier et al (2018) and Kumari (2016), each of which is expressly incorporated by reference in its entirety.

[0045] The term "naive maintenance medium" as used herein has its plain and ordinary meaning as understood in light of the present specification and refers to a growth medium that supports naive pluripotent stem cells and maintains them in a naive state. The naive maintenance medium can be any medium disclosed herein, such as PXGL medium, N2B27 medium, N2 medium, tt2iLGo medium, 2i medium, or 2i medium containing gelatin. The naive maintenance medium can also be supplemented with any of the small molecule compounds, inhibitors, activators, or growth factors described herein, such as GSK3 inhibitors, MAPK inhibitors, LIF, JNK inhibitors, p38 inhibitors, ROCK inhibitors, PKC inhibitors, BMP inhibitors, bFGF, activin A, ascorbic acid, cAMP activators, TGF-β, or TGF-β inhibitors. In some embodiments, the naive maintenance medium allows naive pluripotent stem cells to survive or proliferate and maintain a naive state for a culture period of, about, at least, at least about, up to, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, for example. In some embodiments, the percentage of naive pluripotent stem cells that maintain their naive state after a certain period of culture is about, at least, at least about, no more than, or no more than about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or any percentage within the range defined by any two of the foregoing, e.g., 40%-99%, 50%-90%, 60%-80%, 40%-70%, or 60%-99%. In some embodiments, naive maintenance medium can also be used to support primed pluripotent stem cells.In some embodiments, the naive maintenance medium is maintained for about, at least, at least about, up to, or about the following number of days of culture after a culture period: e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, The primed pluripotent stem cells are reprogrammed to a naive state after a culture period of 1, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, at about, at least, at least about, less than, or equal to the number of days in culture. In other embodiments, the naive maintenance medium does not support primed pluripotent stem cells, where the number, viability, or clonogenicity of primed pluripotent stem cells declines over time. In some embodiments, the percentage of primed pluripotent stem cells that lose viability or clonogenicity after a certain period of culture is about, at least, at least about, or no more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or any percentage within a range defined by any two of the foregoing percentages, e.g., 10%-99%, 30%-80%, 40%-70%, 10%-60%, or 40%-99%.

[0046] The term "chemical reset (cR) cells" as used herein has its plain and ordinary meaning as understood in light of the present specification, and refers to primed pluripotent stem cells that are reprogrammed to naive state using histone deacetylase (HDAC) inhibitors.Exemplary HDAC inhibitors used for chemical reset include but are not limited to valproic acid, sodium butyrate, vorinostat, panobinostat, belinostat, gibinostat, dacinostat, PCI-24781, CHR-3996, JNJ-26481585, SB939, AR-42, ACY-1215, romidepsin, α-ketomide, HKI 46F08, phenylbutyrate, pivanex, entinostat, mocetinostat, tasedinaline, or CUDC-101, or any combination thereof. Additional information regarding cR cells can be found in Guo, G et al (2017), Epigenetic resetting of human pluripotency. Development. 144, 2748-2763, the entire contents of which are expressly incorporated herein by reference.

[0047] The term "feeder cells," as used herein, has its plain and ordinary meaning as understood in light of the present specification and refers to cells that support the growth of pluripotent stem cells, such as by secreting growth factors into the medium or displaying them on the cell surface. Feeder cells are generally adherent cells and can be growth-arrested. For example, feeder cells can be growth-arrested by irradiation (e.g., gamma rays), mitomycin C treatment, electric pulses, or mild chemical fixation (e.g., formaldehyde or glutaraldehyde). However, feeder cells do not necessarily need to be growth-arrested. Feeder cells can serve purposes such as secreting growth factors, displaying growth factors on the cell surface, detoxifying the medium, or synthesizing extracellular matrix proteins. In some embodiments, feeder cells are allogeneic or xenogeneic to the supported target stem cells, which can affect downstream applications. In some embodiments, feeder cells are mouse cells. In some embodiments, feeder cells are human cells. In some embodiments, the feeder cells are mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse 3T3 cells, mouse SNL 76 / 7 cells, human fibroblasts, human forehead fibroblasts, human skin fibroblasts, human adipose mesenchymal cells, human bone marrow mesenchymal cells, human amniotic mesenchymal cells, human amniotic epithelial cells, human umbilical cord mesenchymal cells, human fetal muscle cells, human fetal fibroblasts, or human adult fallopian tube epithelial cells. In some embodiments, conditioned medium prepared from feeder cells is used instead of or in combination with feeder cell co-culture. In some embodiments, feeder cells are used during the growth of target stem cells. In some embodiments, feeder cells are not used during the growth of target stem cells.

[0048] As used herein, the term "intercellular transfer" has its plain and ordinary meaning as understood in light of this specification and refers to the transport of biological materials from one cell to another. While the plasma membrane normally functions as a barrier, mechanisms exist by which cells exchange materials, including, but not limited to, fluids, salts, nutrients, sugars, small molecule compounds, organelles, mitochondria, endosomes, vesicles, proteins, polypeptides, peptides, nucleic acids, DNA, or RNA, or any combination thereof. RNA may include mRNA, miRNA, siRNA, shRNA, or other types of RNA disclosed herein or known in the art, which may result in the expression of exogenous proteins or the downregulation of gene expression via endogenous silencing pathways. Intercellular transfer, without being limited by any mechanism, may occur via tunneling nanotubes (TNTs) or cytonemes, which are long, actin-containing membrane protrusions that connect two or more cells and facilitate transport. As disclosed herein, these TNTs can be selective for specific categories of biological materials (e.g., size, polarity, charge, stability, etc.) and even for specific species within a category (e.g., some RNAs are transferred more efficiently than others). Proinflammatory stimuli, such as lipopolysaccharide (LPS) or IFN-γ, reduce TNT formation. Another method of cell-to-cell transfer is via microvesicles and exosomes. These small membrane-bound vesicles have been shown to be capable of carrying RNAs, such as mRNA and miRNA. As shown herein, these two types of cell-to-cell transfer can be distinguished by examining conditioned media or by transwell assays, which allow the transfer of floating microvesicles and exosomes but do not allow the direct cell-cell contact required for TNTs.

[0049] Some embodiments described herein relate to pharmaceutical compositions consisting essentially of, or consisting of, an effective amount of a cell composition described herein and a pharmaceutically acceptable carrier, excipient, or combination thereof. The pharmaceutical compositions described herein are suitable for human and / or veterinary use.

[0050] As used herein, "pharmaceutically acceptable" has its plain and ordinary meaning as understood in light of the specification and refers to a carrier, excipient, and / or stabilizer that is nontoxic or has an acceptable level of toxicity to cells or mammals exposed thereto at the dosages and concentrations employed. As used herein, "pharmaceutically acceptable," "diluent," "excipient," and / or "carrier" have their plain and ordinary meaning as understood in light of the specification and contemplate any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with administration to a human, feline, canine, or other vertebrate host. Typically, pharmaceutically acceptable diluents, excipients, and / or carriers are those diluents, excipients, and / or carriers approved by a regulatory agency of the federal government, state government, or other regulatory body for use in animals, including humans, and non-human mammals such as cats and dogs, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia. The terms diluent, excipient, and / or "carrier" have their plain and ordinary meaning as understood in light of this specification and may refer to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutical diluents, excipients, and / or carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water, saline, and aqueous solutions of dextrose and glycerol can be used as liquid diluents, excipients, and / or carriers, particularly for injectable solutions. Suitable pharmaceutical diluents and / or excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. A non-limiting example of a physiologically acceptable carrier is a pH-buffered aqueous solution.Physiologically acceptable carriers can also contain one or more (e.g., at least 1, 3, 5, 10) of the following: antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids, carbohydrates such as glucose, mannose, or dextrin, chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, salt-forming counterions such as sodium, and non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®. The compositions may contain minor amounts of wetting agents, bulking agents, emulsifying agents, or pH buffering agents, as desired. These compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations, etc. The formulation should be compatible with the mode of administration.

[0051] Cryoprotectants are additives to cell compositions that improve the efficiency and yield of cryopreservation by preventing the formation of large ice crystals. Cryoprotectants include, but are not limited to, DMSO, ethylene glycol, glycerol, propylene glycol, trehalose, formamide, methylformamide, dimethylformamide, glycerol 3-phosphate, proline, sorbitol, diethyl glycol, sucrose, triethylene glycol, polyvinyl alcohol, polyethylene glycol, or hydroxyethyl starch. Cryopreservatives can be used as part of a cryopreservation medium, which contains other components such as nutrients (e.g., albumin, serum, bovine serum, fetal calf serum [FCS]) to enhance the survival rate of cells after thawing. In these cryopreservation media, at least one cryoprotectant may be found at a concentration that is, about, at least, at least about, less than, or equal to about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or any percentage within a range defined by any two of the foregoing values.

[0052] Additional excipients with desirable properties include, but are not limited to, preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizers, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate, sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. Some excipients may be residual amounts or contaminants from the manufacturing process, including, but not limited to, serum, albumin, ovalbumin, antibiotics, inactivating agents, formaldehyde, glutaraldehyde, β-propiolactone, gelatin, cell debris, nucleic acids, peptides, amino acids, or growth media components, or any combination thereof. The amount of excipient may be found in the composition at 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w, about the above %; at least the above %; at least about the above %; or less than or equal to the above %; or any weight percentage within a range defined by any two of the foregoing values.

[0053] The term "pharmaceutically acceptable salts," as understood in light of this specification, has its plain and ordinary meaning and includes relatively non-toxic inorganic and organic acid or base addition salts of compositions or excipients, including, but not limited to, analgesics, therapeutic agents, other materials, and the like. Examples of pharmaceutically acceptable salts include those derived from mineral acids such as hydrochloric acid and sulfuric acid, and those derived from organic acids such as ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like. Examples of inorganic bases suitable for forming salts include hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, zinc, and the like. Salts can also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For example, classes of such organic bases may include, but are not limited to, mono-, di-, and trialkylamines, including methylamine, dimethylamine, and triethylamine, mono-, di-, or trihydroxyalkylamines, including mono-, di-, and triethanolamine; amino acids such as glycine, arginine, lysine, and the like; guanidine; N-methylglucosamine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; and trihydroxymethylaminoethane.

[0054] Appropriate formulation depends on the selected route of administration.The techniques for formulating and administering the compounds described herein are known to those skilled in the art.Multiple techniques for administering compounds exist in the art, including but not limited to enteral, oral, rectal, topical, sublingual, buccal, intraaural, epidural, intradermal, aerosol, parenteral delivery (including intramuscular, subcutaneous, intraarterial, intravenous, intraportal, intraarticular, intradermal, peritoneal, intramedullary injection, intrathecal, direct intraventricular, intraperitoneal, intranasal or intraocular injection).Pharmaceutical compositions will generally be adjusted to suit the specific intended route of administration.

[0055] As used herein, "carrier" has its plain and ordinary meaning as understood in light of the present specification and refers to a compound, particle, solid, semi-solid, liquid, or diluent that facilitates the passage, delivery, and / or uptake of a compound into cells, tissues, and / or bodily organs.

[0056] As used herein, the term "diluent" has its plain and ordinary meaning as understood in light of the specification and refers to an ingredient in a pharmaceutical composition that lacks pharmacological activity but may be pharmaceutically necessary or desirable. For example, a diluent may be used to increase the bulk of a potent drug whose mass is too small to manufacture and / or administer. It may also be a liquid for dissolving a drug to be administered by injection, ingestion, or inhalation. A common form of diluent in the art is a buffered aqueous solution, such as, but not limited to, phosphate-buffered saline, which mimics the composition of human blood.

[0057] The present invention is generally disclosed herein using affirmative language to describe numerous embodiments. The present invention also includes embodiments in which subject matter, such as substances or materials, method steps and conditions, protocols, or procedures, is completely or partially excluded.

[0058] Methods and compositions for producing reprogrammed naive stem cells Compositions containing reprogrammed naive stem cells and methods for their preparation are described herein.These reprogrammed naive stem cells exhibit characteristics found in naive stem cells involved in embryogenesis (i.e., cells that constitute the inner cell mass and epiblast of developing embryos before the epiblast differentiates into three germ layers), or in naive cells reprogrammed from stem cells primed by other methods known in the art, such as transgenic expression of naive transcription factors or the use of small molecule compounds, inhibitors, or activators that transition primed stem cells to a naive state.These characteristics include, but are not limited to, the expression of naive transcription factors, the expression of naive cell surface markers, dome-like cell colony morphology, the ability to persist and grow in cell culture media known to be incompatible with primed stem cells, and modifications in the chromatin state that allow access to genes encoding transcription factors, other proteins, and non-coding RNAs associated with the naive state, while downregulating the expression of transcription factors, other proteins, and non-coding RNAs associated with the primed state or somatic state. These reprogrammed naive stem cells are not necessarily totipotent stem cells, as they lack the capacity to form extraembryonic cells and tissues.

[0059] Described herein are methods for reprogramming cells from a primed state to a naive state. The methods involve contacting an acceptor cell with a donor cell in vitro. In some embodiments, the contact results in the transfer of intracellular components from the donor cell to the acceptor cell. As shown herein, in some embodiments, this reprogramming phenomenon is induced by direct cell-cell contact between one cell (the "donor cell") and a second cell (the "acceptor cell"). Without being limited by any mechanism of action, it is believed that the donor and acceptor cells form a cytoplasmic bridge via long protrusions called tunneling nanotubes (TNTs) or cytonemes. These TNTs allow the transfer of cellular materials, including RNA (including mRNA, ncRNA, lncRNA, miRNA, piRNA, siRNA, or shRNA). Among these mRNAs, at least 491 donor-derived naive-state-associated transcription factors have been shown to transfer to the acceptor cell. The presence of these mRNAs encoding naive transcription factors in acceptor cells is strong enough to induce reprogramming from a primed state to a naive state. The use of inflammatory stimulatory molecules, such as LPS, prevents the formation of TNTs and thus inhibits this naive reprogramming. Neither donor cell-conditioned medium nor transwells separating the two cell populations can induce naive reprogramming in acceptor cells, indicating that other known cell-to-cell transfer mechanisms (e.g., microvesicles, exosomes) are sufficient for reprogramming; direct cell-cell contact and the formation of TNTs are required. In some embodiments, this process of naive reprogramming is not achieved by viruses. In some embodiments, this process of naive reprogramming is not achieved by adeno-associated viruses or lentiviruses. In some embodiments, this process of naive reprogramming is not achieved by transfection, transduction, or electroporation.

[0060] Acceptor cells herein are cells that receive genetic and other cellular material from donor cells by co-culture with the donor cells, where the donor and acceptor cells are in direct contact, possibly, but not limited by any mechanism, via TNT. In some embodiments, the acceptor cell is a pluripotent stem cell. In some embodiments, the acceptor cell is an iPSC. In some embodiments, the acceptor cell is a primed pluripotent stem cell. In some embodiments, the acceptor cell is a primed iPSC. In some embodiments, the acceptor cell is a mammalian cell. In some embodiments, the acceptor cell is a mouse cell. In some embodiments, the acceptor cell is a human cell. In some embodiments, the acceptor cell is a human PSC. In some embodiments, the acceptor cell is an hiPSC. In some embodiments, the acceptor cell is a primed hiPSC. In some embodiments, the acceptor cell is a cell in a primed state. In some embodiments, acceptor cells express primed transcription factors and / or primed cell surface markers.In some embodiments, acceptor cells are TkDA3-4, 1231A3, 317-D6, 317-A4, CDH1, 5-T-3, 3-34-1, NAFLD27, NAFLD77, NAFLD150, WD90, WD91, WD92, L20012, C213, 1383D6, FF, or 317-12 cell lines.After directly contacting acceptor cells with donor cells, acceptor cells receive materials such as mRNA, ncRNA, protein, and organelles from donor cells.In some embodiments, contacting occurs in cell culture.In some embodiments, the contacting occurs for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, for about the number of days, for at least the number of days, for at least about the number of days, for up to, or for up to about the number of days, or for a range.

[0061] In some embodiments, the number of genes expressed in the acceptor cells after contact with the donor cells that were not expressed prior to contact is about, at least, at least about, no more than, or no more than about 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 6000, 6382, 7000, 8000, 9000 additional genes, or within a range defined by any two of the foregoing values, e.g., 100-9000, 2000-8000, 4000-8000, 100-500, or 300-2000 additional genes. In some embodiments, the additional genes expressed in the acceptor cells are expressed from exogenous mRNA received from the donor cells. In some embodiments, the exogenous mRNA from the donor cells encodes a naive transcription factor. In some embodiments, the exogenous mRNA from the donor cell encodes a native cell surface marker. In some embodiments, the additional genes expressed include genes involved in membrane protein targeting, symbiotic processes, translation initiation, and RNA processing and localization. In some embodiments, the acceptor cell and the donor cell are of different species, and the exogenous mRNA is heterologous. In some embodiments, the acceptor cell and the donor cell are of the same species, and the exogenous mRNA is allogeneic or autologous. In some embodiments, the acceptor cell and the donor cell have different genomic DNA, and the exogenous mRNA differs in gene sequence from the genomic DNA of the acceptor cell. In some embodiments, some mRNAs, ncRNAs, or proteins are transferred from the donor cell to the acceptor cell in a more favorable manner than other mRNAs, ncRNAs, or proteins. For example, Wnt1, Wnt5a, Hoxa11, Fgf13 are genes that are highly expressed in donor cells (i.e., multiple copies of mRNA are present) but are not transferred with high efficiency into acceptor cells.

[0062] In some embodiments, after contacting with donor cells, acceptor cells transition from a primed state to a naive state. In some embodiments, after contacting with donor cells, acceptor cells express or up-regulate the expression of one or more naive pluripotency markers or transcription factors. In some embodiments, after contacting with donor cells, acceptor cells express or up-regulate the expression of one or more (for example, at least 1, 3, 5 or 10) naive pluripotency markers or transcription factors KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C or TBS3. In some embodiments, after contact with donor cells, acceptor cells express or upregulate the expression of one or more (e.g., at least 1, 3, or 5) of the naive pluripotency markers or transcription factors DPPA3, TFCP2L1, DNMT3L, KLF4, or KLF17. In some embodiments, after contact with donor cells, acceptor cells express or upregulate the expression of one or more naive cell surface markers. In some embodiments, after contact with donor cells, acceptor cells express or upregulate the expression of one or more, or all (e.g., at least 1, 3, 5) of the naive cell surface markers CD130, CD77, CD7, CD75, or F11R. In some embodiments, after contact with donor cells, acceptor cells express or upregulate the expression of one or more (e.g., at least 1) of the naive cell surface markers CD130 or CD77. In some embodiments, after contact with the donor cells, the acceptor cells express or upregulate the expression of one or more (e.g., at least 1, 3, 5, 10) of the following naive pluripotency markers, transcription factors, or cell surface markers: KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, TBS3, CD130, CD77, CD7, CD75, or F11R.In some embodiments, after contact with the donor cells, the acceptor cells express exogenous genes from the donor cells. In some embodiments, after contact with the donor cells, the acceptor cells express exogenous actin from the donor cells. In some embodiments, after contact with the donor cells, the acceptor cells express exogenous NANOG from the donor cells.

[0063] In some embodiments, after contact with donor cells, acceptor cells do not express or down-regulate one or more primed pluripotency markers or transcription factors. In some embodiments, after contact with donor cells, acceptor cells do not express or down-regulate one or more (e.g., at least 1, 3, 5) of primed pluripotency markers or transcription factors ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, or XIST. In some embodiments, after contact with donor cells, acceptor cells do not express or down-regulate the primed pluripotency markers or transcription factors DUSP6 or THY1. In some embodiments, after contact with donor cells, acceptor cells do not express or down-regulate one or more primed cell surface markers. In some embodiments, after contact with donor cells, acceptor cells do not express or down-regulate expression of one or more (e.g., at least 1, 3, 5) of the primed cell surface markers CD90, HLA-ABC, CD24, CD57, or SSEA4. In some embodiments, after contact with donor cells, acceptor cells do not express or down-regulate expression of one or more (e.g., at least 1) of the primed cell surface markers CD90 or HLAABC. In some embodiments, after contact with donor cells, acceptor cells do not express or down-regulate expression of one or more (e.g., at least 1, 3, 5, 10) of the primed pluripotency markers, transcription factors, or cell surface markers ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, XIST, CD90, HLA-ABC, CD24, CD57, or SSEA4.

[0064] In some embodiments, after contact with the donor cells, the acceptor cells undergo a change in chromatin accessibility. In some embodiments, the change in chromatin accessibility allows for the expression of naive pluripotency markers, transcription factors, or cell surface markers. In some embodiments, after contact with the donor cells, the acceptor cells undergo a change in more than half of the total open chromatin regions of their genome. In some embodiments, after contact with the donor cells, the acceptor cells undergo a change in chromatic accessibility, wherein the binding motifs for SOX2 and / or TFAP2C are increased in accessibility.

[0065] As used herein, a donor cell is a cell that provides genetic and other cellular material to an acceptor cell through direct cell-cell contact. While not limited to a particular mechanism, it is believed that transfer occurs via TNT. In some embodiments, the donor cell is a pluripotent stem cell. In some embodiments, the donor cell is an iPSC. In some embodiments, the donor cell is a naive PSC. In some embodiments, the donor cell is a mammalian cell. In some embodiments, the donor cell is a mouse cell. In some embodiments, the donor cell is a human cell. In some embodiments, the donor cell is a human PSC. In some embodiments, the donor cell is a mouse PSC. In some embodiments, the donor cell is a mouse iPSC. In some embodiments, the donor cell is a mouse ESC. In some embodiments, the donor cell is a naive mouse ESC. After direct contact between the donor cell and the acceptor cell, the donor cell provides material, such as mRNA, ncRNA, proteins, and organelles, to the acceptor cell. In some embodiments, the contacting occurs in cell culture. In some embodiments, the contacting occurs for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 days, for about said number of days, for at least said number of days, for at least about said number of days, for no more than said number of days, or for no more than about said number of days.

[0066] In some embodiments, the donor cell and the acceptor cell are of different species, and the donor cell provides exogenous mRNA that is xenogeneic to the acceptor cell. In some embodiments, the donor cell and the acceptor cell are of the same species, and the donor cell provides exogenous mRNA that is allogeneic or to the acceptor cell. In some embodiments, the donor cell and the acceptor cell are from the same individual, and the donor cell provides exogenous mRNA that is autologous to the acceptor cell. In some embodiments, the donor cell and the acceptor cell have different genomic DNA, and the donor cell provides exogenous mRNA to the acceptor cell that differs in gene sequence from the acceptor cell. In some embodiments, after contacting, the acceptor cell expresses a protein from the exogenous mRNA from the donor cell. In some embodiments, after contacting, the acceptor cell expresses a protein from the xenogeneic exogenous mRNA. In some embodiments, after contacting, the acceptor cell expresses a protein from the allogeneic exogenous mRNA. In some embodiments, the acceptor cell expresses a protein from an exogenous mRNA that is autologous.

[0067] In some embodiments, after contact with acceptor cells, donor cells remain in a naive state. In some embodiments, after contact with acceptor cells, donor cells remain in a naive state when cultured in a naive maintenance medium or a naive stem cell expansion medium. In some embodiments, after contact with acceptor cells, donor cells express or highly express one or more (e.g., at least 1, 3, 5, 10) of naive pluripotency markers, transcription factors, or cell surface markers: KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, TBS3, CD130, CD77, CD7, CD75, or F11R. In some embodiments, after contact with the acceptor cells, the donor cells do not express or express low levels of one or more (e.g., at least 1, 3, 5, 10) of the following primed pluripotency markers, transcription factors, or cell surface markers: ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, XIST, CD90, HLA-ABC, CD24, CD57, or SSEA4. In some embodiments, after contact with the acceptor cells, the donor cells transition from a naive state to a primed state. In some embodiments, after contact with acceptor cells, donor cells do not express or down-regulate the expression of one or more (e.g., at least 1, 3, 5, or 10) of the following naive pluripotency markers, transcription factors, or cell surface markers: KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, TBS3, CD130, CD77, CD7, CD75, or F11R. In some embodiments, after contact with acceptor cells, donor cells express or up-regulate the expression of one or more (e.g., at least 1, 3, 5, or 10) of the following primed pluripotency markers, transcription factors, or cell surface markers: ZIC2, ZIC3, OTX2, DUSP6, THY1, FOXA2, XIST, CD90, HLA-ABC, CD24, CD57, or SSEA4.In some embodiments, after contact with the acceptor cells, the donor cells comprise exogenous mRNA derived from the acceptor cells. In some embodiments, after contact with the acceptor cells, the donor cells comprise exogenous mRNA derived from xenogeneic acceptor cells. In some embodiments, after contact with the acceptor cells, the donor cells comprise exogenous mRNA derived from allogeneic or autologous acceptor cells. In some embodiments, the donor and acceptor cells have different genomic DNA, and the donor cells comprise exogenous mRNA derived from the acceptor cells whose gene sequence differs from the genomic DNA of the donor cells. In some embodiments, the donor cells comprise exogenous actin or NANOG mRNA derived from the acceptor cells.

[0068] In some embodiments, the acceptor cells and donor cells are 1%:99%, 5%:95%, 10%:90%, 15%:85%, 20%:80%, 25%:75%, 30%:70%, 35%:65%, 40%:60%, 45%:55%, 50%:50%, 55%:45%, 60%:40%, 65%:35%, 70%:30%, 75%:25%, 80%:20%, 85%:15%, 90%:10%, 95%:5%, or 99%:1%, or at least about the above percentages. In some embodiments, the acceptor cells and donor cells are contacted or cultured at a ratio of about 50%:50%, at least about 50%:50%, at least about 50%:50%, or at about 50%:50% or less, or any ratio within a range defined by any two of the foregoing ratios, for example, 1%:99%-99%:1%, 10%:90%-90%:10%, 20%:80%-80%:20%, 30%:70%-70%:30%, 40%:60%-60%:40%, 45%:55%-55%:45%, 1%:99%-50%:50%, or 50%:50%-99%:1%. In some embodiments, the acceptor cells and donor cells are contacted or cultured at a ratio of about 50%:50%, at least 50%:50%, at least about 50%:50%, or at about 50%:50% or less. In some embodiments, the acceptor cells and donor cells are contacted or cultured at a ratio of about 20%:80%, at least 20%:80%, at least about 20%:80%, no more than 20%:80%, or no more than about 20%:80%. In some embodiments, the acceptor cells and donor cells are contacted or cultured at a ratio of about 80%:20%, at least 20%:80%, at least about 80%:20%, no more than 80%:20%, or no more than about 80%:20%. In some embodiments, the donor cells are in dome-shaped colonies before contacting with the acceptor cells. In some embodiments, the acceptor cells are in a flat monolayer before contacting with the donor cells. In some embodiments, the acceptor cells are in dome-shaped colonies after contacting with the donor cells. In some embodiments, the acceptor cells and donor cells are in direct contact with each other.In some embodiments, the acceptor cells and donor cells are not separated by a transwell.

[0069] In some embodiments, the acceptor cells and donor cells are contacted or cultured under hypoxic conditions. In some embodiments, the hypoxic conditions comprise, consist essentially of, or consist of an O concentration of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, about, at least, at least about, or equal to or less than about, or equal to or less than about, or any concentration within a range defined by any two of the foregoing concentrations, for example, 0%-20%, 3%-10%, 4%-6%, 0%-5%, or 5%-20% O. In some embodiments, the hypoxic conditions comprise, consist essentially of, or consist of a concentration of O2 that is about, at least, at least about, no more than, or no more than about: 4%, 5%, or 6% O2. In some embodiments, the hypoxic conditions comprise, consist essentially of, or consist of a concentration of O2 that is about, at least, at least about, no more than, or no more than about 5% O2, at least 5% O2, no more than 5% O2, or no more than about 5% O2.

[0070] In some embodiments, the acceptor cells and donor cells are incubated at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97° The acceptor cells and donor cells are contacted or cultured at a temperature that is 0° C., about 0° C., at least 0° C., at least about 0° C., or equal to or less than 0° C., or any temperature within a range defined by any two of the above temperatures, e.g., 15° C. to 50° C., 20° C. to 45° C., 25° C. to 40° C., 32° C. to 42° C., or 35° C. to 39° C. In some embodiments, the acceptor cells and donor cells are contacted or cultured at a temperature that is about 37° C., at least 37° C., at least about 37° C., equal to or less than 37° C., or equal to or less than about 37° C.

[0071] In some embodiments, the acceptor cells may not be maintained or grown in naive stem cell growth medium or maintenance medium before contacting with the donor cells. In some embodiments, the donor cells may be maintained or grown in naive stem cell growth medium or maintenance medium before contacting with the acceptor cells. In some embodiments, after contacting with the donor cells, the acceptor cells may be reprogrammed and maintained or grown in naive stem cell growth medium or maintenance medium. In some embodiments, the naive stem cell growth medium or maintenance medium is RSet medium, naive human stem cell (NHSM), 5i medium, 4i medium, 3i medium, feeder-independent naive embryo (FINE) medium, mTeSR medium, mTeSR medium, Matrigel, PXGL medium, N2B27 medium, N2 medium, T2iLGo, tt2iLGo medium, 2i medium, or 2i medium containing gelatin. In some embodiments, the naive stem cell growth medium or naive maintenance medium comprises one or more (e.g., at least 1, 3, 5) of a GSK3 inhibitor, a MAPK inhibitor, LIF, a JNK inhibitor, a p38 inhibitor, bFGF, or TGF-β. In some embodiments, the acceptor cells or donor cells, or both, are grown on a feeder cell substrate. In some embodiments, the acceptor cells or donor cells, or both, are not grown on a feeder cell substrate. In some embodiments, after contact with the donor cells, the acceptor cells are reprogrammed and can be grown without a feeder cell substrate (although a feeder cell substrate would otherwise be required). In some embodiments, the donor cells are not chemically reset cells. In some embodiments, the acceptor cells are not chemically reset cells. In some embodiments, the donor and acceptor cells are not contacted or cultured with an HDAC inhibitor.In some embodiments, the donor and acceptor cells are not contacted or cultured with one or more (e.g., at least 1, 3, 5) of valproic acid, sodium butyrate, vorinostat, panobinostat, belinostat, gibinostat, dacinostat, PCI-24781, CHR-3996, JNJ-26481585, SB939, AR-42, ACY-1215, romidepsin, alpha-ketomide, HKI 46F08, phenylbutyrate, pivanex, entinostat, mocetinostat, tacedinaline, or CUDC-101, or any combination thereof. In some embodiments, the donor cells are not modified by transfection, electroporation, or viral transduction, or any combination thereof, prior to contact. In some embodiments, the acceptor cells are not modified by transfection, electroporation, or viral transduction, or any combination thereof, prior to contacting. In some embodiments, the acceptor cells are not modified to exogenously express transcription factors prior to contacting. In some embodiments, the acceptor cells are not modified to exogenously express naive transcription factors prior to contacting. In some embodiments, the acceptor cells are not modified to exogenously express one or more (e.g., at least 1, 3, 5) of Oct-3 / 4, Soxl, Sox2, Sox3, Sox15, Klfl, Klf2, Klf4, Klf5, C-myc, L-myc, N-myc, Nanog, LIN28, Tert, Fbx15, ERas, ECAT15-1, ECAT15-2, Tcl1, β-catenin, ECAT1, Esg1, Dnmt3L, ECAT8, Gdf3, Fth117, Sal14, Rex1, UTF1, Stella, Stat3, Grb2, Prdm14, Nr5a1, Nr5a2, or E-cadherin, or any combination thereof, prior to contact.

[0072] In some embodiments, the donor and acceptor cells are contacted or cultured under stressor conditions, wherein the stressor conditions are selected from contact with a cytotoxic compound, hypoxia, non-physiological temperature, non-physiological pH, electroporation, or any combination thereof. In some embodiments, the donor or acceptor cells, or both, are contacted or cultured under stressor conditions, wherein the stressor conditions are selected from contact with a cytotoxic compound, hypoxia, non-physiological temperature, non-physiological pH, electroporation, or any combination thereof. In some embodiments, the donor or acceptor cells are contacted or cultured under stressor conditions, wherein ... Phosphorus, Y-27632 (ROCK inhibitor), (s)-(-)-blebbistatin, IWP2, A83-01, LY294002, SB-431542, NVP-BHG, cyclopamine-KAAD, PD-0325901, FGF4, LDN-193189, insulin-like growth factor (IGF), bone morphogenetic protein 2 (BMP2), transforming growth factor β2 (TGF-β2), BMP4, F The subject is contacted with at least one agent selected from the group consisting of GF-7, platelet-derived growth factor (PDGF) beta 3, epidermal growth factor (EGF), exendin-4, human neuregulin (hHRG) beta 3, retinoic acid (RA), L-ascorbic acid 2-phosphate (AA2P), ascorbic acid, insulin-transferrin-selenoethanolamine solution (ITS-X), insulin, rifampicin, penicillin, streptomycin, 2-mercaptoethanol, 3-mercaptopropane-1,2-diol (thioglycerol), L-proline, L-glutamine, non-essential amino acid mixture (NEAA), sodium pyruvate, trypsin-EDTA, phosphatidylinositol (PI), interleukin, prostaglandin, tumor necrosis factor, or any combination thereof.

[0073] In the embodiments described herein, the acceptor cells receive genetic and other cellular material, and the donor cells provide the genetic and other cellular material. In some embodiments, the genetic and other cellular material includes mRNA. In some embodiments, the acceptor cells receive genetic and other cellular material by direct contact with the donor cells. In some embodiments, the donor cells provide genetic and other cellular material by direct contact with the acceptor cells. In some embodiments, the acceptor cells are primed stem cells. In some embodiments, the donor cells are naive stem cells. In some embodiments, the acceptor cells are stem cells and the donor cells are stem cells. In some embodiments, the acceptor cells are primed stem cells and the donor cells are naive stem cells. In some embodiments, the acceptor cells are primed iPSCs and the donor cells are naive iPSCs. In some embodiments, the acceptor cells are human stem cells and the donor cells are human stem cells. In some embodiments, the acceptor cells are human iPSCs and the donor cells are human iPSCs. In some embodiments, the acceptor cells are primed human iPSCs and the donor cells are naive human iPSCs. In some embodiments, the acceptor cells are human stem cells and the donor cells are mouse stem cells. In some embodiments, the acceptor cells are human iPSCs and the donor cells are mouse iPSCs. In some embodiments, the acceptor cells are human iPSCs and the donor cells are mouse ESCs. In some embodiments, the acceptor cells are primed hiPSCs and the donor cells are naive mouse iPSCs. In some embodiments, the acceptor cells are human iPSCs and the donor cells are naive mouse ESCs. In some embodiments, the acceptor cells are naive human iPSCs and the donor cells are naive human iPSCs. In some embodiments, the acceptor cells are naive human iPSCs and the donor cells are naive mouse iPSCs.In some embodiments, the acceptor cells are naive human iPSCs and the donor cells are naive mouse ESCs.

[0074] Reprogrammed naive stem cells, such as human reprogrammed naive stem cells, can be prepared by the methods provided herein (i.e., by co-culturing in direct contact with a naive stem cell population), as included in the Examples. Reprogrammed naive stem cells prepared according to these methods exhibit superior properties compared to alternative primed-to-naive reprogramming protocols. These alternative protocols generally involve the transgenic expression of pluripotency transcription factors, or the use of small molecule compounds or growth factors, such as leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF, FGF-2), transforming growth factor β (TGF-β), c-Jun N-terminal kinase (JNK), Rho kinase (ROCK), bone morphogenetic protein (BMP), activin A, or combinations thereof, or inhibitors or activators, or combinations of inhibitors or activators. These alternative protocols may also require the use of feeder cells, such as mouse fibroblasts, mouse embryonic fibroblasts, mouse STO cells, mouse SNL 76 / 7 cells, human fibroblasts, human forehead fibroblasts, human dermal fibroblasts, human amniotic mesenchymal cells, or human umbilical cord mesenchymal cells. In some embodiments of the methods and compositions of the invention, the culture does not contain feeder cells in addition to the acceptor and donor cells.

[0075] The method provided herein for preparing reprogrammed naive stem cells can be faster than previous protocols.For example, naive transcription factors or markers can be observed in reprogrammed acceptor cells after contact with donor cells for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 days, for about these days, for at least these days, for at least about these days, for less than these days, or for less than these days. The methods provided herein can be performed without the need for one or more small molecule compounds or growth factors, or without the use of feeder cells.

[0076] Reprogrammed naive stem cells prepared according to the methods provided herein have faster doubling times and can differentiate into a broader range of lineages both in vitro and in vivo compared to the parental primed stem cells. This allows for rapid expansion of stem cells, which can ultimately be used to generate desirable cell types and assemblies, such as cell cultures, tissues, and organoids. Improved differentiation capacity can lead to tissues or organoids that more closely resemble animal tissues and organs.

[0077] In some embodiments, the doubling time of the reprogrammed naive stem cells is about, at least, at least about, not more than, or not more than about: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or any time within a range defined by any two of the foregoing times, e.g., 5-24 hours, 10-20 hours, 14-18 hours, 5 hours to 20 hours, or 15 hours to 24 hours. In some embodiments, the doubling time of the reprogrammed naive stem cells is about, at least, at least about, no more than, or no more than about 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the doubling time of the parental primed stem cells, or any percentage within a range defined by any two of the foregoing doubling time percentages, e.g., 20%-99%, 40%-70%, 50%-60%, 20%-60%, or 40%-99%.

[0078] In some embodiments, the single-cell clonogenicity of the reprogrammed naive stem cells is about, at least, at least about, no more than, or no more than about 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, of the single-cell clonogenicity of the parental primed stem cells. 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, or 500%, or any percentage within a range defined by any two of the aforementioned single cell clonogenic percentages, e.g., 100%-500%, 120%-300%, 150%-250%, 100%-200%, or 150%-500%.

[0079] In some embodiments, reprogrammed naive stem cells can be reprimed to a primed pluripotent stem cell state. In some embodiments, reprogrammed naive stem cells can be differentiated into mesodermal cells, mesodermal lineage cells, ectodermal cells, ectodermal lineage cells, endodermal cells, or endodermal lineage cells, or any combination thereof. In some embodiments, reprogrammed naive stem cells can be differentiated into somatic cells, hematopoietic cells, endothelial cells, muscle cells, stromal cells, bone cells, epidermal cells, epithelial cells, hepatocytes, gastrointestinal cells, gastric cells, parietal cells, alveolar cells, pancreatic cells, neural cells, neurons, neural crest cells, melanocytes, or keratinocytes, or any combination thereof. In some embodiments, reprogrammed naive stem cells can be differentiated into a broader range of somatic cells compared to parentally primed stem cells.

[0080] Embodiments of the present disclosure include cell compositions comprising, consisting essentially of, or consisting of two populations of cells. In some embodiments, the cell compositions comprise, consist essentially of, or consist of a population of acceptor cells and a population of donor cells. In some embodiments, the cell compositions comprise, consist essentially of, or consist of acceptor cells and donor cells. In some embodiments, the acceptor cells and donor cells are induced pluripotent stem cells. In some embodiments, the acceptor cells are human cells and the donor cells are mouse cells. In some embodiments, the acceptor cells are primed stem cells and the donor are naive stem cells. In some embodiments, the acceptor cells are transitioned from a primed state to a naive state (reprogrammed acceptor cells or reprogrammed naive stem cells) by direct contact with the donor cells. In some embodiments, the cell compositions comprise, consist essentially of, or consist of reprogrammed acceptor cells and donor cells. In some embodiments, both the reprogrammed acceptor cells and donor cells are naive stem cells or exhibit characteristics of naive stem cells. In some embodiments, both the reprogrammed acceptor cells and donor cells are not primed stem cells or do not exhibit characteristics of naive stem cells. Embodiments herein also include cell cultures comprising, consisting essentially of, or consisting of acceptor cells and donor cells. In some embodiments, the cell composition or cell culture further comprises, consists essentially of, or consists of a growth medium. In some embodiments, the growth medium is a medium that supports naive stem cells but not primed stem cells. In some embodiments, the growth medium comprises one or more small molecules, activators, or inhibitors described herein. In some embodiments, the growth medium further comprises a cryoprotectant. Embodiments herein also include methods of preparing the cell compositions or cell cultures described herein.In some embodiments, the method comprises, consists essentially of, or consists of contacting or culturing a population of acceptor cells with a population of donor cells. In some embodiments, the method comprises, consists essentially of, or consists of contacting or culturing acceptor cells with donor cells. Embodiments herein also include a population of reprogrammed naive stem cells or reprogrammed naive stem cells prepared according to the methods provided herein. In some embodiments, the population of reprogrammed naive stem cells or reprogrammed naive stem cells differs from reprogrammed naive stem cells generated by other methods. In some embodiments, the population of reprogrammed naive stem cells or reprogrammed naive stem cells is not genetically engineered. In some embodiments, the population of reprogrammed naive stem cells or reprogrammed naive stem cells is not contacted with one or more small molecules, activators, or inhibitors described herein (e.g., molecules used in other methods to reprogram stem cells from a primed state to a naive state). The embodiments herein also include cell populations, cultures, tissues, organoids, or organs produced by differentiating reprogrammed naive stem cells or a population of reprogrammed naive stem cells.In some embodiments, the cell populations, cultures, tissues, organoids, or organs comprise, consist essentially of, or consist of reprogrammed naive stem cells or a population of reprogrammed naive stem cells that are differentiated back to a primed state.In some embodiments, the cell populations, cultures, tissues, organoids, or organs comprise, consist essentially of, or consist of reprogrammed naive stem cells or a population of reprogrammed naive stem cells that have differentiated into somatic cells.In some embodiments, only a percentage of the reprogrammed naive stem cells or population of reprogrammed naive stem cells, e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, about, at least, at least about, less than, or less than about, or 99%, or any percentage of cells within a range defined by any two of the foregoing percentages (e.g., 5%-99%, 20%)-80%, 40%-60%, 5%-60%, or 40%-99%), are differentiated. [Example]

[0081] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Those skilled in the art will appreciate that many other embodiments are also within the scope of the present invention, as described above and in the claims herein.

[0082] Example 1 Nanotube-dependent mRNA transfer between mouse and human cells Human induced pluripotent stem cells (hiPSCs) and the mouse feeder cell line SNL 76 / 7 were co-cultured (Figure 1A). After co-culture, mouse mRNA was reproducibly detected in purified human cells after direct co-culture, and vice versa. RT-PCR of sorted cell fractions using human / mouse-specific primers revealed that mouse-specific β-actin mRNA (Actb) was detected in sorted hiPSCs, whereas human β-actin mRNA (ACTB) was detected in sorted mouse feeder cells (Figure 1B). These patterns were confirmed in all four tested hiPSC clones transgenically engineered to express EGFP (TkDA3-4-EGFP [RRID:CVCL_RJ54], 1383D6-EGFP [RRID:CVCL_UP39], 317D6-EGFP [RRID:CVCL_K092], and FF-I01-EGFP) (Figure 1C). RRID stands for Research Resource Identifiers and is a unique identifier for referencing scientific resources, accessible, for example, through SciCrunch (available on the World Wide Web at scicrunch.org / resources). Importantly, not all transcripts were identified in this manner. The long non-coding human RNA NEAT1 was not detected in mouse feeder cells (Figure 1B), consistent with studies showing cell-to-cell movement of β-actin mRNA, but not nuclear long non-coding RNAs (Haimovich et al. 2017).

[0083] Tunneling nanotubes (TNTs) enable intercellular transport of mRNA, microRNA, proteins, and organelles. Therefore, intercellular connections between mouse and human cells were investigated using scanning electron microscopy (SEM). Compared to hiPSCs, which exhibit short protrusions on their surface when cultured alone (Figure 1D), a significant number of nanotubes protruding from the surface of SNL cells from hiPSCs were observed. Similar protruding structures were also observed at the interface between mouse embryonic stem cells (mESCs) and SNL cells (Figure 1E). Inflammatory stimulation treatments, such as lipopolysaccharide (LPS), which is known to inhibit cell-cell junctions in vitro, effectively prevented nanotube connections between co-cultured cells (Figure 1F, 1E). Quantitative analysis revealed that a fraction of mouse Actb mRNA, corresponding to approximately 0.1% of its expression in SNL cells, was detected in hiPSCs under normal conditions, whereas only a negligible amount was detected in LPS-treated cells (Figure 1G). These data indicate that a subset of mRNAs can potentially be transferred between hiPSCs and mouse feeder cells via intercellular nanotubes.

[0084] Example 2 Transcriptomic analysis of mRNA via cell-to-cell transfer from mouse to human To test whether this transition occurs in other pluripotent stem cell types, hiPSCs were co-cultured with mESCs. mESCs (naive) and hiPSCs (primed) (Figure 2A) are in unique pluripotent states that require different media. hiPSCs are maintained in mTeSR / Matrigel conditions in a two-dimensional flat shape, whereas mESCs are maintained in 2i / gelatin culture, which has a three-dimensional colony shape. When hiPSCs are cultured under 2i / gelatin conditions, the cells are unable to sustain proliferation and are quickly eliminated. However, we found that hiPSCs can adapt to 2i / gelatin culture and proliferate well when co-cultured with mESCs (Figure 2A). To investigate whether mRNA transfer contributes to this unique adaptation, we assessed the mRNA expression levels of hiPSCs after two rounds of flow cytometry isolation from mESCs based on the use of two different fluorescent reporter lines to minimize mouse cell contamination and / or fusion (Figure 2A). Mouse-specific Actb mRNA was again identified in hiPSC-derived transcripts after coculture, but not in monoculture (Figure 2B). Conversely, mESCs contained human-specific ACTB mRNA (Figure 2B). The amount of mouse Actb and Nanog mRNA detected in hiPSCs cocultured with mESCs was similar to that observed in cocultures of hiPSCs and SNL (Figure 2B). All hiPSC clones (317-12, 317D6, TkDA3-4) underwent similar cell morphology transformation in the presence of mESCs (Figure 2C) and mouse Actb and Nanog mRNA transfer (Figure 2B), confirming the consistency of the mRNA transfer mechanism across multiple hiPSC lines.

[0085] The identity of the mobile mRNAs was then determined by RNA-seq and subsequent isolation of human and mouse sequences in the resulting reads. Co-culture with mESCs increased the amount of mouse RNA detected in hiPSCs, increasing the percentage of mouse reads relative to total reads found in hiPSC clones 317-12 and 317D6 from 0.05% (6,817 mouse reads) and 0.07% (8,186 mouse reads) to 0.3% (44,176 mouse reads) and 2.8% (311,859 mouse reads), respectively. The average number of unique mouse genes expressed per sample was nearly three-fold higher in the co-cultured samples: 9,953 genes compared to 3,571 genes in monocultured cells. Of the top 75 genes with the highest variance in gene expression, all had higher expression in the co-cultured cell lines (Figure 2D). Gene Ontology (GO) enrichment analysis revealed that many of these genes are involved in membrane protein targeting, symbiotic processes, translation initiation, and RNA processing and localization. Among the mouse genes detected in the coculture samples, 491 mouse-derived naive-state-associated transcription factor (TF) mRNAs were found in hiPSCs sorted after coculture with mESCs (Figure 2E). In contrast, several mouse-derived RNAs known to be highly expressed in mESCs, including Wnt1, Wnt5a, and Hoxa11, were not detected, while Fgf13 had only one read across all samples, indicating differential migration abilities among different mRNAs.

[0086] Example 3. Mouse ESC-derived mRNA transfer enables naive conversion of human iPSCs. The presence of numerous naive mouse TF mRNAs raised the possibility that adaptation to growth in 2i / gelatin conditions triggered reprogramming of hiPSCs to a naive pluripotent state by mouse-derived TFs. Following morphological conversion of hiPSCs around days 3 to 5 (Figure 3A), quantitative analysis of sorted hiPSCs revealed strong expression of core naive pluripotency markers (DPPA3, TFCP2L1, DNMT3L, KLF4, and KLF17) and downregulation of a primed marker (DUSP6) (Figure 3B). Conditioned medium from mESC and transwell coculture assays failed to induce dome-shaped hiPSCs and produced negligible induction of naive pluripotency markers without appreciable mouse-specific Actb mRNA (Figure 3C), suggesting that naive-associated mRNA induction in hiPSCs requires cell-cell contact.

[0087] Flow cytometry analysis using multiplexed naive-specific and primed-specific antibodies revealed that hiPSCs alone highly expressed the primed-specific markers CD90 and HLAABC, but did not express the naive-specific markers CD130 and CD77. Conversely, after 10 days of coculture with mESCs and subsequent sorting, the putative naive hiPSC population expressed the naive-specific markers CD130 and CD77, along with downregulation of the primed-specific markers (Figure 3D). Purified human naive marker-expressing cells could be repeatedly expanded in human naive maintenance medium (PXGL). KLF17 and TFAP2C are established human / primate-specific naive pluripotency regulators. To confirm complete phenotypic conversion to the naive state after passaging, immunofluorescence analysis was performed on expanded putative naive hiPSCs. Similar to the chemically reset cells described in Guo et al. (2017), hiPSCs undergoing coculture with mESCs expressed both KLF17 and TFAP2C, along with human-specific nuclear antigens, whereas parental unmixed hiPSCs did not (Figure 3E). RNA-seq was also performed on cells transformed via mixed culture (mixed), chemically reset cells (cR), and their parental iPSCs, and compared them with the initially reported naive human PSCs (Figure 3F, 3G). Principal component analysis (PCA) and hierarchical clustering were performed based on differentially expressed genes between naive and conventional PSCs. In PCA, mixed / cR cells were separated from parental primed PSCs in PC1 (explaining 57% of the variance), but there were still differences between the samples and the sedimented dataset in PC2 (explaining 18% of the variance) (Figure 3F). In our dataset, the naive markers KLF17, DNMT3L, TFCP2L1, DPPA3, and DPPA5 were exclusively or highly expressed in mixed / cR cells. The primed markers DUSP6 and THY1 were exclusively expressed in parentally primed PSCs. Meanwhile, expression of the commonly expressed gene NANOG was unchanged compared to these markers (Figure 3H).Importantly, the gene expression profiles of in-house cR cells and mixed cells were indistinguishable, indicating that co-cultured primed stem cells could be reprogrammed to a naive state without the need for chemical compounds required for cR cells. The co-culture method was also applied to three different hiPSC lines (TkDA3-4, 317-12, and 317D6), revealing that all lines exhibited strong induction of naive-like characteristics (Figure 3I). Interestingly, a positive correlation was found between the conversion efficiency and the hiPSC / mESC ratio. The higher the relative number of mESCs, the higher the expression of naive marker genes in hiPSCs (Figure 3J). These results indicate that naive reprogramming is reproducible and can be promoted by direct co-culture with mESCs.

[0088] Given that LPS disrupts nanotube connectivity in the hiPSC / mESC and feeder cell coculture model (Example 1), we investigated the effect of LPS on naive-like conversion of hiPSCs. Similar to the previous example, LPS treatment eliminated nanotube formation between hiPSCs and mESCs and transferred mouse-specific Actb and Nanog mRNAs to hiPSCs (Figure 3K). Consistent with these results, cocultured hiPSCs under LPS treatment resulted in the emergence of a greater number of flat-shaped hiPSCs (Figure 3L), followed by the downregulation of naive-related genes (Figure 3M). Collectively, these data indicate that reprogramming via mRNA transfer potentially requires direct cell-cell contact via nanotubes.

[0089] Example 4. Mouse ESC co-culture induces dynamic changes in accessible chromatin in hiPSCs To clarify whether mESC coculture had a global effect on the chromatin state of hiPSCs, we performed ATAC-seq experiments on hIPSCs before and after coculture using two biological replicates of 317-12 and 317-D6 cells. Across all three experiments, we observed strong agreement between the resulting ATAC-seq peaks (Figure 4A). Next, we identified regions of open chromatin that were altered in the presence of cocultured mouse cells. More than half of all open chromatin regions showed significantly altered accessibility after coculture. For example, of the total 59,360 ATAC-seq peaks identified in experimental replicate 1 for 317-D6 cells, 29,079 (49%) did not change significantly between conditions ("invariant"), 24,307 (41%) had significantly less ATAC-seq signal in the presence of mouse cells ("coculture loss"), and 5,974 (10%) had significantly more signal ("coculture gain"); similar percentages were observed in the other two experiments (Figure 4B).

[0090] To identify specific TFs that may respond to these large-scale chromatin accessibility changes, we performed TF binding site motif enrichment analysis of the ATAC-seq peak sets corresponding to each of these three categories. Surprisingly, we observed highly significant motif enrichment for many of the same TFs whose mRNAs were transcribed from mESCs. For example, in the "co-culture gain" peak from 317-D6 (replicate 1), binding motifs for SOX2 and TFAP2C were highly enriched (Figure 4C), and high mouse-specific gene expression was detected in hiPSCs when co-cultured with mESCs (Figure 2). These results are highly consistent across three different cell types and highly consistent with previously reported enriched motifs for the same set of TFs identified in the 317-D6 (replicate 2) and 317-12 experiments (Figure 4D). Notably, many of the identified regions are located proximal to biologically important genes. For example, a highly reproducible "co-culture gain" peak is located immediately upstream of the TFAP2C promoter (Figure 4E). Taken together, these data indicate that a fraction of TF mRNAs is transferred from naive mESCs ("influencers") to adjacent primed hiPSCs ("recipients" or "acceptors"), and then induces chromatin reorganization at the corresponding TF-bound loci to reprogram hiPSCs to a naive-like pluripotent state ( Figure 4F ).

[0091] MicroRNAs and incomplete mRNAs are transferred via extracellular vesicles (e.g., exosomes). In contrast to this diffusion-based transfer mechanism, reports using immortalized cells or fibroblasts suggest that full-length mRNAs can also undergo direct cell-cell transfer via cytoplasmic extensions characteristic of membrane nanotubes connecting influencer and acceptor cells. Without being limited by any mechanism of action, described herein is evidence that mRNAs, including those encoding pioneer transcription factors, can be transferred between contacting cells, resulting in changes to the acceptor cell transcriptome and epigenome.

[0092] Example 5 Silencing of mouse Tfcp2l1 and Tfap2c in human iPSCs inhibits naive conversion induced after co-culture with mouse ESCs. To further confirm that the mRNA transferred from mouse donor ESCs reprogrammed the recipient human-primed iPSCs to a naive state, we designed shRNAs specific for mouse Klf4, Tfcp2L1, and Tfap2c. The shRNA sequences were designed within the region of divergence between the mouse and human genomic sequences to avoid off-target effects due to native human KLF4, TFCP2L1, and TFAP2C (Figure 5A). The efficacy of the designed shRNAs was first demonstrated in mouse ESCs. Viral shRNA vectors were prepared and used to infect mouse ESCs, and the expression of Klf4, Tfcp2L1, Tfap2c, Pou5f1, and Nanog was assessed by qRT-PCR. Significant downregulation of the expression of the corresponding genes was observed with shRNAs targeting the three transcription factors, while the expression of Pou5f1 and Nanog was unaffected (Figure 5B). Conversely, human iPSCs infected with shRNA vectors showed no gene downregulation, indicating that mouse-specific shRNAs had no effect on the human orthologs (Figure 5C). Figure 5D shows the experimental design for evaluating the effect of shRNA silencing on naive reprogramming. Briefly, puromycin-resistant hiPSCs expressing mouse-specific shRNAs and puromycin-sensitive mESCs were co-cultured to induce hiPSC reprogramming to a naive state. After 5 days of co-culture, puromycin was added to the medium to select hiPSCs. Among the shRNAs tested (Klf4#1, Klf4#3, Tfcp2l1#1, Tfcp2l1#3, and Tfap2c#3), the number of dome-shaped colonies, indicating naive hiPSCs, was significantly reduced in the Klf4#3, Tfcp2l1#1, Tfcp2l1#3, and Tfap2c#3 shRNA conditions (Figure 5E-F). In control hiPSCs (expressing luciferase-specific shRNA [shLuc]), expression of TFCP2L1 is observed in coculture with mESCs and after puromycin selection (Figure 5G), indicating that silencing of transferred mouse naive transcription factors reduces the efficiency of reprogramming primed hiPSCs to a naive state.

[0093] Example 6 Expression of mouse-derived transcription factor proteins in human iPSCs induced after co-culture with mouse ESCs We examined the expression of mouse transcription factor proteins after coculture. hiPSCs and mESCs were stained with pan- and mouse-specific Oct4 (Figure 6A) and human- and mouse-specific Nanog (Figure 6B) antibodies. While the pan-Oct4 and human-specific Nanog antibodies strongly labeled hiPSCs, these cells did not show cross-reactivity with the mouse-specific Oct4 and Nanog antibodies. After coculture of GFP-expressing hiPSCs and tdTomato-expressing mESCs, the hiPSCs showed weaker reactivity with the mouse-specific Oct4 and Nanog antibodies compared with adjacent mESCs, suggesting that cell-to-cell transfer of mRNA from mESCs results in low levels of mouse-specific protein expression in the hiPSCs (Figure 6C-D).

[0094] Example 7 Materials and Methods Cell culture: SNL 76 / 7 feeder cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 0.1 mM MEM non-essential amino acids, and 2 mM L-glutamine.

[0095] Primed human iPSCs (hiPSCs) were routinely cultured in mTeSR1 on Matrigel growth factor-reduced (BD Biosciences)-coated plates, or in StemFit (AK03N, Ajinomoto) on laminin-511 E8 (Nippi)-coated dishes for TKDA3-4, 317D6, and 317-12 iPSC clones. For passaging, 80-90% confluent hiPSCs were dissociated using Accutase (Millipore) and then cultured in 1:30 diluted Matrigel growth factor-reduced or 0.5 μg / cm in 10 μM ROCK inhibitor Y-27632 for 1 day and fresh medium without Y-27632 for subsequent days. 21 x 10 cells per well on a Laminin-511 E8-coated 6-well tissue culture plate (Corning) 5 The cells were replated.

[0096] hiPSC lines (TKDA3-4, 1383D6) stably expressing GFP were maintained on SNL feeder cells in StemFit (AK02N, Ajinomoto) to analyze intercellular RNA transfer. After 3–5 days, cells were sorted using a FACSAria II cell sorter (BD Biosciences) for GFP-positive and GFP-negative cells, followed by total RNA extraction.

[0097] Naive mouse ESCs (mESCs) were cultured in a 1:1 mixture of DMEM / F12 and Neurobasal medium, 1x N2 supplement, 1x B27 supplement, 2mM glutamine, 50U / ml and 50µg / ml penicillin-streptomycin (all from ThermoFisher Scientific), at a concentration of 1.5x10 -4 1 × 10 cells per 6-well plate were cultured in the following conditions: M monothioglycerol (Sigma M6145), 50 μg / ml bovine serum albumin (Sigma), 10 ng / ml recombinant mouse LIF (Millipore), 3 μM CHIR99021 (Tocris), and 1 μM PD0325901 (Sigma). 5 Cells were cultured on a MEF layer seeded at a density of 1000 x g / ml. Cells were passaged by incubation with Accutase (ThermoFisher Scientific) for 5 min.

[0098] For conversion of primed hiPSCs to the naive state, naive mESCs and primed hiPSCs were dissociated into single cells using Accutase, with 5 x 10 cells per 12-well plate. 5 cells (2.5×10 5 hiPSCs + 2.5 x 10 5 A total of 0.5 x 10 mESCs were cultured per 12-well plate in naive mESC medium with 10 µM Y-27632. 6The cells were seeded onto MEFs seeded at a density of 1000 x 1000 cells / well. The next day, the medium was changed to naive mESC medium or PXGL medium without Y-27632. Dome-shaped naive colonies were visible as early as 5 days after seeding. On day 5, the cells were treated with Accutase to dissociate into single cells, and then FACS was used to selectively sort hiPSCs. The sorted cells were maintained in PXGL medium at 37°C under 5% O2, and naive markers could be detected 7–10 days after coculture. During the 5-day coculture, LPS was added to the mixed cells at a low concentration of 100 ng / ml and a high concentration of 500 ng / ml.

[0099] PXGL medium is prepared by supplementing N2B27 medium with 1 μM PD0325901 (MEK inhibitor), 2 μM XAV939 (Wnt inhibitor), 2 μM Go6983 (PKC inhibitor), and 10 ng / mL human LIF. N2B27 medium is prepared by mixing 487 mL of DMEM / F12 and 487 mL of Neurobasal medium and supplementing with 10 mL of B27 supplement, 5 mL of N2 supplement, 10 mL of 200 mM L-glutamine, and 1 mL of 0.1 M β-mercaptoethanol. N2 medium. Non-commercially available N2 supplement is prepared by supplementing DMEM / F12 basal medium with 0.4 mg / mL insulin, 10 mg / mL apotransferrin, 3 μM sodium selenite, 1.6 mg / mL putrescine, and 2 μg / mL progesterone.

[0100] Mitotic inactivation of feeder cells: To prepare feeder cells, MEF and SNL cells that had reached 90% confluency were treated with 10 μg / mL mitomycin C (Fujifilm Wako) for 3 h. After extensive washing with PBS and trypsinization, the mitomycin C-treated cells were plated onto gelatin-coated culture dishes at a density of 7.5 × 10 4 cells / cm 2 The feeder cell dishes were used within one week of seeding. The original medium was replaced with stem cell expansion medium immediately before adding the hiPSCs.

[0101] Flow cytometry: Primed naive hiPSCs were dissociated into single cells with Accutase, washed, and passed through a 30-40 μm cell strainer. Conjugated antibodies were mixed with 50 μL of PBS (BD Biosciences) and applied to 50-100 μL of cells (2-5 x 10 per reaction). 5 Cells were incubated in the dark at 4°C for 30 minutes, washed twice with buffer (2% FBS in PBS), and centrifuged at 300 x g for 5 minutes. Cells were resuspended in buffer and analyzed on a BD LSRFortessa cell analyzer (BD Biosciences) or BD FACSAria Fusion for cell sorting. Single-stained cells or OneComp eBeads (eBioscience) were used for compensation calculations. Data were analyzed using FlowJo V10.1 software (FlowJo, LLC).

[0102] Immunofluorescence microscopy: hiPSCs were seeded onto Matrigel and MEF feeder-coated Lab-Tek II chamber slides (Nunc) and cultured at 37°C under 5% O for 2 days. Cells were fixed with 4% paraformaldehyde (Wako) for 10 minutes at room temperature (RT) and permeabilized with 0.3% Triton X-100 (Sigma) in PBS for 10 minutes at room temperature. Cells were blocked with MAXblock blocking medium (Active Motif) for 1 hour. Primary and secondary antibodies were diluted in MAXblock blocking medium and applied for 1 hour and 20 minutes, respectively. DNA was counterstained with 1 μg / mL DAPI (Thermo Fisher) for 15 minutes. Samples were washed twice with PBS between each step. Images were captured using an FV3000 confocal microscope (Olympus).

[0103] qPCR: Total RNA was extracted using the RNeasy Mini Kit (QIAGEN). 1 μg of RNA was reverse transcribed using SuperScript III (Thermo Fisher) followed by quantitative PCR using Taqman universal master mix and Taqman assays (Thermo Fisher) using the StepOnePlus Real-Time PCR System (Thermo Fisher). RNA samples from three or four biological replicates were used for each condition.

[0104] RT-PCR: Total RNA from sorted cells was extracted with TRI Reagent (Molecular Research Center, Inc.). First-strand cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara). RT-PCR was performed using Tks Gflex DNA polymerase (Takara) and specific primers described below. Quantitative PCR was performed using Thunderbird SYBR qPCR Mix (Toyobo) on an ABI StepOnePlus Real Time PCR System (Applied Biosystems). To normalize relative expression, a standard curve was generated for each gene for relative quantification, and the expression level of each gene was normalized to the ribosomal 28S RNA gene.

[0105] Scanning electron microscopy: Cells were cultured on gelatin-coated cell-tight C-1 cell disks LF (MS-0113K; Sumitomo Bakelite). After the indicated treatments, they were fixed for 2 h with 2.5% glutaraldehyde in 0.1 M phosphate buffer. They were washed overnight at 4°C in the same buffer and postfixed for 2 h with 1% OsO4 buffered in 0.1 M phosphate buffer. Samples were dehydrated in a graded series of ethanol and dried in a critical point dryer (JCPD-5; JEOL) using liquid CO2. They were sputter-coated with platinum and examined by scanning electron microscopy (S-4500; Hitachi, Tokyo, Japan).

[0106] RNA sequencing (RNA-seq): Total RNA was purified using the RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. RNA quality and quantity were checked using Bioanalyzer (Agilent) and Qubit (Life Technologies) instruments, respectively. The initial amplification step was performed using the NuGEN Ovation RNA-Seq System v2, which facilitates the assay of amplifying RNA samples to create double-stranded cDNA. Libraries were then generated using the Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the Illumina HiSeq 2500 system. RNA-seq data analysis was performed using the BioWardrobe Experiment Management System (accessible on the World Wide Web at github.com / Barski-lab / biowardrobe). Briefly, reads were mapped to the mm10 genome using TopHat (version 2.0.9) and assigned to RefSeq genes (with one annotation per gene) using the BioWardrobe algorithm. PCA was performed using the top 1000 most variable genes across experimental conditions. The first and second principal components were plotted. Differential gene expression analysis was performed via DESeq2 in the BioWardrobe environment. Gene ontology analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID).

[0107] To separate mouse and human sequences, we used the bbsplit function in the bbtools suite (BBTools) to bin each read from the RNA-seq fastq into separate fastq files based on whether the read was specific to the human (hg19) or mouse (mm10) genome. To create the most stringent conditions, we set complete mode to true and discarded all ambiguous reads. Mouse-specific reads in human cells were aligned to the mm10 genome using STAR (v2.5.1b). The resulting aligned sam files were sorted using the sort function in samtools and converted to bam files. Reads for each gene were counted using the R function summarizeOverlaps in the GenomicAlignments library using the Union counting mode. DESeq2 was used to quantify differential gene expression between samples. To compare the gene expression profiles of established human naive cell lines with the first reported naive PSC line, we deposited sequence data for Shef6 prime (accession numbers: ERR1924246, ERR1924247, ERR1924248) and Shef6-cR (accession numbers: ERR1924234, ERR1924235, ERR1924236) from the European Nucleotide Archive. The sequence data were uploaded to the Galaxy Web platform and analyzed using the public server at usegalaxy.org. Adapter sequences were removed using Trimmomatic Galaxy version 0.36.5 on the Galaxy server. Transcript abundance was quantified using Salmon Galaxy version 0.11.2. Transcript abundances were converted to count data using the Bioconductor package tximport 1.12.0 (Soneson, Love, and Robinson F1000Res 2015) and summarized to the gene level after normalization with the Bioconductor package DESeq2 1.24.0.Gene annotations for Homo sapiens (GRCh38) were obtained from Ensemble. Principal component analysis (PCA) was performed on differentially expressed genes in naive and primed PSCs using the prcomp function based on log2-transformed normalized counts calculated with the scale function in R 3.6.0. Euclidean distances were estimated based on the log2-transformed normalized counts, and cluster analysis was performed on the same genes using the heatmap.2 function in gplots 3.0.1.1.

[0108] ATAC-seq Library Preparation and Sequencing: An ATAC-seq library was prepared. Approximately 1,000,000 primed human iPSCs and naive human PSCs were used. Briefly, samples were lysed in 50 μL of lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.1% NP-40). Immediately after lysis, nuclei were spun at 500 × g for 5 min, and the supernatant was removed. Next, nuclei were incubated with Tn5 transposase and tagging buffer (Illumina) at 37 °C for 30 min. After tagging, the transposed DNA was purified with a MinElute kit (Qiagen). Polymerase chain reaction (PCR) was performed to amplify the library using the following conditions: The thermal cycle consisted of 72°C for 5 minutes; 98°C for 30 seconds; and a thermal cycle of 98°C for 10 seconds, 63°C for 30 seconds, and 72°C for 1 minute, with a final extension of 72°C for 5 minutes. qPCR was used to estimate the number of additional cycles required to generate product at 25% saturation. Typically, 2–5 additional PCR cycles were added to the initial set of 5 cycles. Libraries were purified using AMPure XP beads (Beckman). Size selection of the library pool was achieved by agarose gel electrophoresis, excising gel slices ranging from 250 to 500 bp. The purified pools from the gel slices were analyzed on an Agilent Bioanalyzer, and 75-bp single-read sequencing was performed using an Illumina HiSeq2500 platform according to standard operating procedures.

[0109] ATAC-seq Data Analysis: FASTQ files from ATAC-seq experiments were analyzed using the MARIO next-generation sequencing pipeline. Briefly, QC was performed using FastQC (v0.11.2), and adapter sequences were removed using Trim Galore (v0.4.2), a wrapper script that calls cutadapt (v1.8.1). Reads were then aligned to the genome using bowtie2 (v2.3.4.1) with the following settings: "-D15-R2-L22-iS, 1, 1.15 --score-min L, -0.6, -0.6, -N0." To account for possible cell type contamination in the experiment, reads aligned to the mouse genome (mm9) were first removed. The remaining reads were then aligned to the reference human genome (hg19 / GRCh37). The hg19 aligned reads (BAM format) were then sorted using samtools (v1.8.0) and duplicate reads were removed using picard (v1.89) with the parameters:

[0110] "MAX_SEQUENCES_FOR_DISK_READ_ENDS_MAP=50000, MAX_FILE_HANDLES_FOR_READ_ENDS_MAP=8000, SORTING_COLLECTION_SIZE_RATIO=0. 25, OPTICAL_DUPLICATE_PIXEL_DISTANCE=100, VALIDATION_STRINGENCY=STRICT, COMPRESSION_LEVEL=5, MAX_RECORDS_IN_RAM=500000.

[0111] Finally, ATAC-seq peaks were called using MACS2 (v2.1.0) with the parameter settings “effective genome size = 2.70e+09, bandwidth = 300, model fold = [5, 50], qvalue cutoff = 1.00e-02”.

[0112] To identify regions of differential chromatin accessibility between experimental conditions, MANorm was used with default parameter settings of peak width (1,000) and distance cutoff (500). A p-value cutoff of 0.05 was used to identify peaks unique to each condition. Each resulting peak set was examined for enriched transcription factor binding site motif instances using the HOMER tools suite, modified to use a logarithmic base 2 scoring system, which includes the set of human motifs included in build 2.0 of the Cis-BP database.

[0113] In at least some of the foregoing embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment, except where such substitution is not technically feasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0114] With respect to the use of virtually any plural and / or singular term herein, those skilled in the art can substitute plural for singular and / or singular for plural as appropriate to the context and / or application. For purposes of clarity, various singular / plural permutations may be expressly set forth herein.

[0115] In general, terms used herein, and particularly in the appended claims (e.g., the body of the claims), are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). Where a specific number of introduced claim recitations are intended, such intention will be explicitly set forth in the claim; it will be further understood by those skilled in the art that, in the absence of such recitation, no such intention exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, even if the same claim includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"), the use of such phrases should not be interpreted as meaning that introducing a claim recitation with the indefinite article "a" or "an" limits any particular claim that includes such an introduced claim recitation to embodiments that include only one such recitation. The same applies to the use of a definite article used to introduce a claim recitation. Additionally, even if a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., a minimum recitation of "two recitations" without other modifiers means at least two recitations, or more than two recitations).Furthermore, when a rule similar to "at least one of A, B, C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the rule (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, the following systems: A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a rule similar to "at least one of A, B, or C, etc." is used, such a configuration is generally intended in the sense that one of ordinary skill in the art would understand the rule (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, the following systems: A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those skilled in the art that virtually all disjunctive words and / or phrases presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0116] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual element or subgroup of elements of the Markush group.

[0117] As will be understood by those skilled in the art, for all purposes, including in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges. Any recited range can be readily recognized as fully descriptive and allows for the same range to be divided into at least two, three, four, five, tenths, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, and upper third, etc. Also, as will be understood by those skilled in the art, all language such as "up to," "at least," "greater than," and "less than" refers to a range that is inclusive of the recited number and that can be subsequently divided into subranges as discussed herein. Finally, as will be understood by those skilled in the art, ranges include individual elements. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, etc.

[0118] While various aspects and embodiments are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

[0119] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and made a part of this specification. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over such conflicting material.

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Claims

1. 1. An in vitro method comprising contacting an acceptor cell with a donor cell, said contacting causing transfer of an intracellular component from said donor cell to said acceptor cell; the acceptor cells are human induced pluripotent stem cells (PSCs); the donor cells are PSCs that contain tunneling nanotubes (TNTs) or cytonemes on their surfaces; the donor cells transfer the intracellular components via the TNTs or cytonemes; the donor cells are naive embryonic stem cells and the acceptor cells are primed; and The method of claim 1, wherein the contacting step is carried out until the acceptor cells form dome-shaped naive acceptor cell colonies based on expression of naive stem cell markers including F11R and / or down-regulated expression of primed stem cell markers including SSEA4 in the acceptor cell colonies.

2. The method of claim 1 , wherein the acceptor cells express a primed transcription factor and / or a primed cell surface marker prior to the contacting step.

3. The method of any one of claims 1 to 2, wherein the donor cells express naive transcription factors and / or naive cell surface markers.

4. The method of any one of claims 1 to 3, wherein the donor cells and the acceptor cells are cultured in a ratio of at least 20% donor cells to 80% acceptor cells.

5. The method of any one of claims 1 to 4, wherein the intracellular component is selected from one or more of RNA, protein, and organelle.

6. 6. The method of claim 5, wherein the RNA is mRNA, ncRNA, lncRNA, miRNA, piRNA, siRNA, or shRNA.

7. The method of any one of claims 1 to 6, wherein the donor and acceptor cells are contacted under hypoxic conditions.

8. 8. The method of claim 7, wherein the hypoxic conditions are 5% O ２ Way bigger than that.

9. 9. The method of any one of claims 1 to 8, wherein the donor cells and acceptor cells are contacted under stressor conditions, the stressor conditions being selected from contact with a cytotoxic compound, hypoxia, non-physiological temperature, non-physiological pH, electroporation, or any combination thereof.

10. 10. The method of claim 1, wherein the acceptor cells express primed transcription factors and / or primed cell surface markers, the donor cells express naive transcription factors and / or naive cell surface markers, and the acceptor cells: a) express or upregulate the expression of naive stem cell markers, further comprising one or more of CD130, CD77, CD7, or CD75; and / or b) the acceptor cells form acceptor cell colonies when they exhibit downregulation of primed stem cell markers further comprising one or more of CD90, HLAABC, CD24, or CD57.

11. 11. The method of any one of claims 1 to 10, wherein the acceptor cells express primed transcription factors and / or primed cell surface markers, the donor cells express naive transcription factors and / or naive cell surface markers, and the acceptor cells a) express or upregulate the expression of naive pluripotency markers including one or more of KLF4, KLF5, KLF17, TFCP2L1, DNMT3L, DPPA3, DPPA5, PRDM14, SALL4, ESRRB, TFAP2C, or TBS; b) exhibit downregulation of primed pluripotency markers, including one or more of ZIC2, ZIC3, OTX2, DUSP6, FOXA2, or XIST; and / or c) the acceptor cells form acceptor cell colonies when they exhibit downregulation of primed pluripotency markers, including one or more of DUSP6 and THY1.

12. The method according to any one of claims 1 to 11, wherein one or both of the donor cells and the acceptor cells are treated with one or more of the following: resveratrol, epigallocatechin gallate (EGCG), curcumin, genistein, activin A, Wnt-3a, sodium butyrate, basic fibroblast growth factor (bFGF), oncostatin M (OSM), dexamethasone (DEX), hepatocyte growth factor (HGF), CHIR-9902 1, forskolin, Y-27632 (ROCK inhibitor), (s)-(-)-blebbistatin, IWP2, A83-01, LY294002, SB-431542, NVP-BHG, cyclopamine-KAAD, PD-0325901, FGF4, LDN-193189, insulin-like growth factor (IGF), bone morphogenetic protein 2 (BMP2), transforming growth factor β2 (TGF-β2), BMP4, the method further comprising contacting the patient with at least one agent selected from the group consisting of FGF-7, platelet-derived growth factor (PDGF) β3, epidermal growth factor (EGF), exendin-4, human neuregulin (hHRG) β3, retinoic acid (RA), L-ascorbic acid 2-phosphate (AA2P), ascorbic acid, insulin-transferrin-selenoethanolamine solution (ITS-X), insulin, rifampicin, penicillin, streptomycin, 2-mercaptoethanol, 3-mercaptopropane-1,2-diol (thioglycerol), L-proline, L-glutamine, non-essential amino acid mixture (NEAA), sodium pyruvate, trypsin-EDTA, phosphatidylinositol (PI), interleukin, prostaglandin, and tumor necrosis factor, or any combination thereof.

13. 13. The method of any one of claims 1 to 12, wherein after contacting, the acceptor cell undergoes a change in chromatin accessibility.

14. 14. The method of claim 13, wherein the change in chromatin accessibility comprises increased accessibility to binding motifs for SOX2, or TFAP2C, or both.

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