Methods of reprogramming cell

Abstract

To provide methods for producing an induced pluripotent stem (iPSC).SOLUTION: A method comprises the following steps in order: culturing a somatic cell in a first culture condition adapted to promote the reprogramming of the cell towards a pluripotent state; culturing the cell in a second culture condition adapted to promote a hypomethylated DNA state in the cell; and culturing the cell in a third culture condition adapted to promote a primed pluripotent state, thereby producing an iPSC from the somatic cell. The invention also relates to cells and compositions resulting from those methods.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to improved methods for reprogramming somatic cells to a pluripotent state, and to cells and compositions resulting therefrom. [Background technology]

[0002] Reprogramming somatic cells into pluripotent cells that closely resemble human embryonic stem cells (ESCs) is an area of ​​technology with great potential in biomedical research and regenerative medicine.

[0003] Induced pluripotent stem cell (iPSC) technology typically involves the genetic manipulation of somatic cells or the introduction of defined factors into somatic cells to generate pluripotent cells. The iPSCs generated by these methods can then be used to generate any number of different cell types through the application of specific differentiation conditions to the iPSCs.

[0004] Cellular reprogramming requires resetting the somatic cell epigenome to closely resemble that of ESCs. However, studies have revealed that with current reprogramming approaches, iPSCs typically retain the epigenetic patterns, such as DNA methylation, of the original somatic cells and are therefore epigenetically distinct from ESCs. In other words, iPSCs have the epigenetic memory of the somatic cells from which they were derived.

[0005] Furthermore, iPSC-specific epigenetic abnormalities frequently arise during the reprogramming process. These are abnormal / aberrant epigenetic (e.g., DNA methylation) patterns that are not present in the original cells or ESCs. These aberrant DNA methylation patterns are stable through extensive iPSC passaging, can be transmitted through iPSC differentiation, and are associated with inappropriate transcriptional activity in the induced differentiated cells.

[0006] The epigenetic memory and abnormal epigenetic (e.g., DNA methylation) state of somatic cells functionally affect the resulting iPSCs, altering transcription and biasing iPSC differentiation potential toward the original somatic lineage. Thus, iPSCs possess epigenetic states and epigenetic memories that can affect differentiation and function. Furthermore, incomplete epigenetic reprogramming can be a significant limitation for iPSC technology and therapeutic applications.

[0007] Thus, there is a need for improved methods for reprogramming somatic cells towards a pluripotent state.

[0008] The reference to prior art in this specification is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art could reasonably be expected to be understood, considered relevant, and / or combined with other prior art by a person skilled in the art. Summary of the Invention [Means for solving the problem]

[0009] In a first aspect, the present invention provides a method for producing induced pluripotent stem cells (iPSCs), the method comprising the steps of: (a) culturing somatic cells in first culture conditions adapted to promote reprogramming of the cells towards a pluripotent state; (b) culturing the cells in second culture conditions adapted to promote a hypomethylated DNA state in the cells, wherein the culture in the second culture conditions is for a period of time insufficient to allow the cells to achieve an established naive pluripotent state; (c) culturing the cells in a third culture condition adapted to promote a primed pluripotent state; in turn, thereby producing iPSCs from somatic cells.

[0010] In a second aspect, the present invention provides a method for reprogramming somatic cells into induced pluripotent cells (iPSCs), the method comprising the steps of: (a) increasing the protein expression or amount of one or more factors in a somatic cell, the factors being for reprogramming the somatic cell towards a pluripotent state; (b) culturing the cells in the first culture medium for a time and under conditions sufficient to allow reprogramming of the cells towards a pluripotent state; (c) culturing the cell in a second medium adapted to induce a hypomethylated DNA state for a time and under conditions sufficient to reset the epigenomic profile of the cell; (d) culturing the cells in a third culture medium adapted to induce a primed pluripotent state for a time and under conditions sufficient to convert the cells to a primed pluripotent state; in turn, thereby reprogramming somatic cells into iPSCs.

[0011] In a further aspect, the present invention provides a method for reprogramming somatic cells into induced pluripotent stem cells (iPSCs), the method comprising the steps of: (a) increasing the protein expression or amount of one or more factors in a somatic cell, the factors being for reprogramming the somatic cell towards a pluripotent state; (b) culturing the cells in the first culture medium for a time and under conditions sufficient to allow reprogramming of the cells towards a pluripotent state; (c) contacting the cells with a culture medium adapted to induce a hypomethylated DNA state and culturing the cells for a time and under conditions sufficient to allow reprogramming the cells towards a hypomethylated DNA state; (d) contacting the cells with a priming medium and culturing the cells for a time and under conditions sufficient to allow reprogramming of the cells to a primed pluripotent state; in turn, thereby reprogramming somatic cells into iPSCs.

[0012] The method of the present invention is particularly suitable for minimizing or reducing the possibility of occurrence of abnormal epigenetic patterns in iPSCs.Thus, the iPSCs obtained or obtainable by the method of the present invention have an epigenomic profile that more closely resembles the epigenomic profile of embryonic stem cells (ESCs) than the iPSCs obtained or obtainable by conventional reprogramming methods.

[0013] As used herein, resetting the epigenomic profile of a cell refers to establishing an epigenetic profile similar to that of ESCs.

[0014] In any embodiment, the cells exhibiting a hypomethylated DNA status may be naive pluripotent cells.

[0015] The epigenomic profile of a cell obtained or obtainable according to the present invention is The genomic methylation pattern for at least a portion of the iPSCs obtained according to the method can be determined and compared to the methylation pattern of the ESCs, and thus to the epigenomic profile of the ESCs.

[0016] In any embodiment of the present invention, the iPSCs obtained or obtainable by the method of the present invention are characterized by a non-CG DNA methylation profile similar to that of ESCs. In any embodiment of the present invention, the iPSCs obtained or obtainable by the listed method establish a H3K9me3 methylation state in cells similar to that of ESCs. In any embodiment of the present invention, resetting the epigenetic profile refers to a level of DNA methylation and / or expression of transposable elements similar to that of transposable elements in ESCs.

[0017] In any embodiment of the invention, iPSCs obtained or obtainable by the methods of the invention have levels of CG DNA methylation, and / or levels of CG DNA methylation for transposable elements, similar to the levels of methylation in ESCs at loci as defined in any of Tables 3 or 4 herein.

[0018] In any embodiment of the invention, the somatic cells are human somatic cells and the iPSCs produced according to the method are human iPSCs.

[0019] In certain embodiments of the invention, the culture period during which cells begin to dedifferentiate or reprogram towards a pluripotent state is at least one day after contacting the somatic cells with the first culture medium (according to the first aspect of the invention) or after increasing the protein expression or amount of one or more factors (according to the second or third aspect of the invention). This period may be 2, 3, 4, 5, 6, 7 days or more after contacting the cells with the first culture medium or after increasing the protein expression or amount of one or more factors. In any embodiment, the culture period during which cells begin to dedifferentiate or reprogram towards a pluripotent state may be any period, as long as it allows for the reduction of one or more markers and characteristics associated with somatic cells.

[0020] The second culture medium or second culture condition adapted to induce a hypomethylated state can be any culture condition or medium that promotes global DNA hypomethylation of the cells. In certain embodiments, the culture condition or medium can include any culture medium adapted to promote a naive pluripotent state or a state that exhibits characteristics of a pluripotent state, such as global DNA hypomethylation. Examples of such media are known in the art and further described herein. In certain preferred embodiments, the naive medium comprises a MEK inhibitor, a PKC inhibitor, a GSK3 inhibitor, a STAT3 activator, and a ROCK inhibitor.

[0021] As used herein, global DNA hypomethylation refers to a decrease in the average level of DNA methylation across the genome of a cell compared to the methylation levels observed in somatic (differentiated) cell types, or optionally primed human ESCs.

[0022] The priming medium (or third culture condition or third culture medium) can include any culture medium adapted to promote a primed pluripotent state. Examples of such media are known in the art and are further described herein. In certain embodiments, the priming medium is selected from Essential 8, KSR / FGF2, mTeSR, AKIT, or B8.

[0023] In any embodiment, the culture of the cells in the second culture condition / medium may be for a period of time that is not sufficient for pluripotency to develop.

[0024] More specifically, the timing of step c) in any aspect of the invention may include contacting the cells with a medium for inducing DNA hypomethylation or a naive medium for a period of time that is not sufficient to achieve a naive pluripotent phenotype for the cells, but is sufficient to achieve global DNA hypomethylation in the cells. In other words, the timing of step d), i.e., contacting the cells with a priming medium, may be performed before the cells complete reprogramming and before the cells achieve a naive pluripotent phenotype. In certain embodiments, the timing of step c) is such that the cells have achieved global DNA hypomethylation but do not express one or more markers of naive pluripotency markers, including the markers of naive pluripotency described herein.

[0025] In alternative embodiments of the second and third aspects of the invention, the timing of step c) may be after the cells have become established naive pluripotent cells, including those that express one or more markers of naive pluripotency as described herein.

[0026] As used herein, "naive pluripotent state" or "naive pluripotent phenotype" may also be understood to refer to a cell morphology or phenotype that includes rounded, dome-shaped cells. The naive pluripotent state may also include cells with global DNA hypomethylation. A naive pluripotent state can also include expression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three or all of the markers selected from KLF2, KLF4, TFCP2L1, TBX3, REX1, GBX2, STELLA (DPPA3), KLF17, DPPA5, TFCP2L1, MAEL, UTF1, ZFP57, DNMT3L, FGF4, FOXR1, ARGFX, TRIM60, DDX43, BRDT, ALPPL2, KHDC3L, KHDC1L, and PRAP1, or other markers of naive pluripotency described herein, including in Table 5.

[0027] As used herein, "primed pluripotent state" or "primed pluripotent phenotype" refers to a cell phenotype or morphology that is typically characterized by the presence of flat cell colonies. In certain embodiments, primed pluripotent state refers to pluripotent cells that express one or more mRNAs of post-implantation epiblast-specific transcription factors. For example, primed cells may express one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or all of SFP, EOMES, BRACHYURY, OTX2, ZIC2, ZIC3, ZIC5, DNMT3B, KDR, CDH2, CER1, COL2A1, DAZL, TCF7L1, SOX11, SALL2, or other markers of primed pluripotent state described herein, including Table 5.

[0028] In any embodiment of the invention, the cells undergoing reprogramming are cultured in the second culture condition or second culture medium for a period of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days. The cells are then transferred to a third medium, third culture condition, or priming culture medium.

[0029] In any embodiment of the invention, the cells undergoing reprogramming are cultured in the second culture condition or naive culture medium for a period of at least about 0.5 days, about 1 day, about 2 days, about 3 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days. The cells are then transferred to a third medium, a third culture condition, or a priming culture medium.

[0030] In a preferred embodiment of any aspect of the invention, in the second culture conditions or in the second culture medium The cells are cultured in the naive medium (second culture medium or second culture conditions) for at least 5 days. In a particularly preferred embodiment, the cells are cultured in the naive medium (second culture medium or second culture conditions) for no more than about 21 days.

[0031] In any embodiment of the invention, the cells contacted with the second culture medium, or naive medium, are cultured in the third culture medium, or priming culture medium, for a period of 0.5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days or more.

[0032] In any embodiment of the invention, the cells contacted with the second culture medium or naive medium are cultured in the third culture medium or priming medium for at least about 0.5 days, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days.

[0033] It will be understood that any method for reprogramming somatic cells towards a pluripotent state (i.e. a method for carrying out step a) of the first aspect of the invention or steps a) and b) of the second and third aspects of the invention) may be used according to the method of the invention. Thus, the invention is not limited by specific culture conditions for promoting reprogramming towards pluripotency, or by specific methods for increasing protein expression or amount of relevant factors, or by culture conditions for allowing somatic cells to initiate reprogramming towards pluripotency. Such methods are known in the art and are further described herein.

[0034] In certain embodiments, reprogramming the somatic cells towards a pluripotent state comprises contacting the cells with a combination of small molecules that induce pluripotency.

[0035] In a preferred embodiment, the factors for reprogramming somatic cells towards a pluripotent state are transcription factors.

[0036] Any transcription factor associated with pluripotency may be used according to the method of the present invention. In certain instances, the transcription factor may comprise, consist of, or consist essentially of one or more of the factors OCT4, SOX2, KLF4, and MYC. In a particularly preferred embodiment, the transcription factor comprises all four of the factors OCT4, SOX2, KLF4, and MYC (OSKM), or variants thereof. In a further embodiment, the transcription factor for reprogramming somatic cells, such as fibroblasts, towards a pluripotent state also comprises the factors LIN28 and / or NANOG. In certain embodiments, the protein expression of each of OCT4, SOX2, KLF4, MYC, LIN28, and NANOG is increased in somatic cells.

[0037] Typically, the protein expression or amount of the factors described herein is increased by contacting the cell with an agent that increases the expression of the factor. Preferably, the agent is selected from the group consisting of nucleotide sequences, proteins, aptamers and small molecules, ribosomes, RNAi agents and peptide nucleic acids (PNAs) and analogs or variants thereof. In some embodiments, the agent is exogenous. The present invention also contemplates the use of transcription activation systems (e.g., gRNAs for use in gene regulation systems such as CRISPR / Cas9 or TALEN) to increase the expression of one or more transcription factors.

[0038] Typically, protein expression or amount of a transcription factor described herein is increased by introducing into a cell at least one nucleic acid comprising a nucleotide sequence encoding the transcription factor or a nucleotide sequence encoding a functional fragment thereof.

[0039] In a preferred embodiment of the present invention, the nucleic acid sequence encoding the transcription factor protein is introduced into the cell by a plasmid. One or more nucleic acids encoding one or more transcription factors can be used. It is therefore clear that one or more plasmids can be used for the purpose of increasing the expression or amount of one or more required transcription factors. In other words, the nucleic acid sequence can be in or on a single plasmid, or can be provided to the somatic cell in two or more plasmids.

[0040] In any embodiment of the invention, a plasmid containing a nucleic acid encoding one or more transcription factors for use in accordance with the invention may be an episomal plasmid.

[0041] Preferably, the nucleic acid further comprises a heterologous promoter. Preferably, the nucleic acid is in a vector, such as a viral vector or a non-viral vector. Preferably, the vector is a viral vector that comprises a genome that does not integrate into the host cell genome. The viral vector can be a retroviral vector, a lentiviral vector, an adenovirus, or a Sendai virus.

[0042] In any embodiment of the invention, the protein expression or amount of a factor is increased in a somatic cell by contacting the somatic cell with one or more agents for increasing expression of said factor in the cell. In certain embodiments, the protein expression or amount of a factor is increased in a somatic cell by transducing or transfecting the somatic cell with one or more vectors encoding said transcription factor. The vector may be a viral vector, including an integrative or non-integrative viral vector. In further embodiments, the vector may be an episomal vector.

[0043] In another aspect, the present invention provides an induced pluripotent stem cell (iPSC) obtained or obtainable by any of the methods of the invention described herein.

[0044] In a further embodiment, the present invention provides an isolated induced pluripotent stem cell (iPSC) obtained or obtainable by any of the methods of the invention described herein.

[0045] Still further, the present invention provides a cell population comprising induced pluripotent stem cells (iPSCs) obtained or obtainable by any of the methods of the present invention described herein. Preferably, the present invention provides a cell population, wherein at least 5% of the cells are iPSCs, which are obtained or obtainable by the methods described herein. Preferably, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the population are iPSCs, which are obtained or obtainable by the methods described herein.

[0046] In any of the methods of the invention, the method further comprises differentiating iPSCs obtained or obtainable by any of the methods of the invention described herein. Differentiating the cells may comprise culturing the iPSCs for a time and under conditions sufficient to generate cells having at least one characteristic of a differentiated cell or a cell not in a pluripotent state.

[0047] Thus, the present invention provides differentiated cells generated from induced pluripotent stem cells (iPSCs), obtained or obtainable by any of the methods of the invention described herein. The present invention also provides differentiated cells generated from induced pluripotent stem cells (iPSCs), obtained or obtainable by any of the methods of the invention described herein. The present invention provides an isolated differentiated cell generated from an induced pluripotent stem cell (iPSC), which can be obtained or obtained by any of the methods of the present invention described herein. Still further, the present invention provides a cell population comprising differentiated cells generated from an induced pluripotent stem cell (iPSC), which are obtained or obtainable by any of the methods of the present invention described herein. Preferably, the present invention provides a cell population, wherein at least 5% of the cells are differentiated cells, which are generated from an iPSC obtained or obtainable by the methods described herein. Preferably, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in the population are differentiated cells, which are generated from an iPSC obtained or obtainable by the methods described herein.

[0048] The invention also provides organoids or other organized cell collections derived from a population of iPSCs / differentiated cells generated from iPSCs, where the iPSCs are obtained or obtainable according to the methods of the invention.

[0049] The present invention also provides a pharmaceutical composition comprising: an isolated iPSC, or a population of iPSCs, obtained or obtainable according to the method of the invention; an isolated differentiated cell, or a population of differentiated cells, derived from one or more iPSCs obtained or obtainable according to the method of the invention; or organoids derived from a population of iPSCs / differentiated cells generated from iPSCs, wherein the iPSCs are obtained or obtainable according to the method of the invention; and a pharma- ceutically acceptable excipient.

[0050] Furthermore, the present invention provides a method of treating a disease or condition requiring administration of iPSCs or cell populations, the method comprising administering to a subject in need thereof: an isolated iPSC, or a population of iPSCs, obtained or obtainable according to the method of the invention; an isolated differentiated cell, or a population of differentiated cells, derived from one or more iPSCs obtained or obtainable according to the method of the invention; or An organoid derived from a population of iPSCs / differentiated cells generated from iPSCs, wherein the iPSCs are obtained or obtainable according to the methods of the invention.

[0051] The invention also provides the use of iPSCs obtained or obtainable according to the methods of the invention in the manufacture of a medicament for treating a disease or condition requiring the administration of pluripotent stem cells, or differentiated cells derived therefrom.

[0052] The present invention also provides a kit for producing iPSCs according to the methods described herein. Preferably, the kit also includes instructions for carrying out the methods of the present invention. The kit may also include one or more reagents for carrying out the methods of the present invention, including the first, second and / or third culture medium as defined herein. Still further, the kit may include instructions and / or reagents for differentiating the iPSCs generated according to the methods of the present invention.

[0053] Any of the methods described herein may have one or more, or all, steps performed in vitro, ex vivo, or in vivo.

[0054] As used herein, unless the context specifically requires, the term "comprise" and variations of this term, such as "comprising", "comprises" and "comprised", are not intended to exclude additional additives, components, integers or steps.

[0055] Further aspects of the invention, and further embodiments of the aspects described in the previous paragraphs, will become apparent from the following description, given by way of example only, and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0056] [Figure 1]The acquisition of aberrant DNA methylation occurs 13 days after priming reprogramming and is absent in naïve iPSCs. a) Table showing cell lines used to test for differentially methylated regions (DMRs) between iPSCs and ESCs. Columns indicate the culture medium used for each cell line used in the DMR test, and b) Bar plot shows the number of DMRs detected between iPSCs and ESCs. Memory DMRs are those where no DMRs were detected between fibroblasts and iPSCs. iPSC DMRs are those where the methylation levels are significantly different between ESCs and fibroblasts. Hypermethylated and hypomethylated DMRs are DMRs where the levels are higher and lower iPSC compared to ESCs, respectively. c) Plot shows the average DNA methylation change across all DMRs compared to the progenitor fibroblast state. Each dot represents an individual sample. d) Heatmap showing DNA methylation levels in DMRs across all samples for memory and iPSC DMRs. Rows are ordered by correlation clustering. e) Genome browser plot showing DNA methylation levels adjacent to the transcription start site for TCERG1L and the iPSC DMR. The plot shows that for this iPSC DMR, aberrant DNA methylation accumulates after day 13 of primed reprogramming and does not appear in naive reprogramming. [Diagram 2]Naive-to-priming reprogramming erases somatic memory and generates cells that more closely resemble ESCs. a) Schematic of naive-to-priming reprogramming showing two separate strategies to generate transient naive-treated (TNT)-primed or naive-to-primed iPSCs. b) The number of CG-DMRs corrected by naive-to-priming and TNT-priming reprogramming is segregated by DMR class. c) Histogram showing differences in DNA methylation levels at DMRs between iPSCs and ESCs showing that naive-to-priming and TNT-primed iPSC reprogramming reduces DNA methylation differences between iPSCs and ESCs. The vertical dashed line indicates the 20% delta in mCG levels, which is the magnitude threshold for DMR significance. d) Bar plot shows enrichment z-scores determined by permutation test of enrichment of CG-DMRs in repressive chromatin domains. e) Upset plots show a high degree of CG-DMR correction for DMRs that intersect with fibroblast-specific repressive chromatin. f) The top panel is an aggregate profile plot of CA methylation levels at non-CG DMRs, showing that TNT-primed and naïve-to-primed iPSCs display highly similar non-CG methylation profiles to ESCs. The bottom panel shows less H3K9me3 ChIP-seq enrichment at non-CG DMRs in both TNT-primed and naïve-to-primed iPSCs compared to primed iPSCs. [Diagram 3]Naive-to-primed reprogramming leads to lower transposable element (TE) expression. A) Histogram of DNA methylation levels in TEs shows that DNA methylation is higher in naive-to-primed iPSCs when compared to primed iPSCs, making them more similar to ESCs. Coverage-weighted DNA methylation levels (mCG / CG) were calculated for all copies of each TE. iPSC data shown for cells derived from donor 32F. B) Dendrogram of TE expression shows that naive-to-primed iPSCs more closely resemble ESCs than primed iPSCs. C) Examination of TE differential expression between primed and naive-to-primed iPSCs shows that TE expression is downregulated in naive-to-primed iPSCs. Bar plots show the number of differentially expressed features for genes and TEs. Volcano plots show the magnitude and significance of expression differences between groups. Vertical dashed lines indicate log fold change >1 (i.e., absolute fold change >2). D) The majority of differentially expressed TEs are LINE and LTR elements, which are repressed in naive-to-primed iPSCs. Dots indicate the magnitude of change on the y-axis for TEs in each family of LINE, LTR and SINE TE classes. E) Bar plots show normalized TE expression (counts per million) for TEs labeled in (D). In all cases, naive-to-primed iPSC TE expression is closer to that of H9 ESCs than primed iPSCs. F) Distribution of DNA methylation of transposable element families in DMRs. Methylation was calculated as mCG / CG for each TE for all samples, then grouped by TE family. Corrected DMRs in TEs are shown. [Figure 4] Differentiation scorecard assessment of embryoid bodies derived from primed and naïve-to-primed iPSCs indicates that naïve-to-primed iPSCs exhibit less differentiation bias towards the mesodermal lineage compared to primed iPSCs (fibroblasts are of mesodermal origin and were the somatic cell type of the donor). [Diagram 5]A) Images show the generation of neural stem cells (NSCs) from primed iPSCs (left) and naïve-to-primed iPSCs (right). This shows that differentiation of primed iPSCs into NSCs generates fibroblast-like cells in the spaces between NSC colonies, which are significantly reduced when NSCs are differentiated from naïve-to-primed iPSCs. B) Percentage of cell types detected from single-cell RNA-seq experiments for NSC cultures derived from primed iPSCs, TNT-primed iPSCs, and naïve-to-primed iPSCs. The data demonstrate that both naïve-to-primed and TNT-primed cells exhibit substantially fewer fibroblast-like cells as characterized by marker gene expression. [Figure 6] Flow cytometry analysis shows the percentage of NCAM positive population at day 8 (D8) and day 16 (D16) of cortical neuron differentiation of MEL1 hESCs, MEL1-derived primed iPSCs, MEL1-derived TNT-primed iPSCs, MEL1-derived naive-to-primed iPSCs (p4) and MEL1-derived naive-to-primed iPSCs (p10). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in connection with the embodiments, it will be understood that the invention is not limited to these embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0058] Those skilled in the art will recognize many methods and materials that are similar or equivalent to those described herein. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0059] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0060] According to conventional methods, human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) iPSCs are typically maintained in a pluripotent "primed" state, which resembles post-implantation ESCs. However, these "primed" iPSCs are typically characterized by an epigenomic memory that differs from ESCs. Furthermore, primed iPSCs are characterized by the presence of an iPSC-specific aberrant DNA methylation state, including differentially methylated regions (DMRs).

[0061] We have comprehensively mapped the pathways of DNA methylation dynamics and patterns during reprogramming of somatic cells to either primed or naive iPSCs. Based on this new knowledge, we have developed a novel process to substantially correct epigenetic memory and abnormalities in the methylation profile of iPSCs. Thus, we have developed a novel method to generate iPSCs that more closely resemble ESCs in epigenetic and transcriptional properties and differentiation potential compared to conventional iPSCs. iPSCs generated using this novel method or process can be referred to herein as "naive-to-primed iPSCs" (NtoP iPSCs) or "transient naive-treated primed iPSCs" (TNT-primed iPSCs).

[0062] Regardless of the method of reprogramming source (somatic) cells, the standard approach to generate human iPSCs is by generating iPSCs in primed or naive medium. However, the present inventors have developed a process in which during the reprogramming process, the cells are exposed to naive medium, preferably for a short time, and then the medium is changed and the cells are placed in primed medium. Thus, the methods of the present invention result in iPSCs that are in a primed state (the most commonly used pluripotent state for human iPSCs) but are more similar to true ESCs compared to iPSCs generated using conventional methods. In particular, they may have one or more, or all of the following advantages: A significant reduction in the presence of iPSC-specific DMRs (iDMRs), A significant decrease in memory DMR, Correction of non-CG methylation at non-CG DMRs to levels similar to ESCs; A significant reduction in the presence of aberrant methylation in transposable elements; Non-CG associated with fibroblast layer-associated domains (the original somatic cell type is the fibroblast) Decreased H3K9me3 ChIP-seq signal at DMRs Reduced expression of transposable elements compared to conventionally primed iPSCs; Reduced differentiation bias towards the original somatic cell lineage, Fewer fibroblast-like cells emerged during differentiation of naïve-to-primed and TNT-primed iPSCs into neural stem cells (NSCs) compared with primed cells (whose original somatic cell type is fibroblasts); · Similar differentiation efficiency into different cell types as genetically matched ESCs.

[0063] cell As used herein, the term "pluripotent" or "pluripotency" refers to cells that have the capacity, under appropriate conditions, to give rise to progeny that can differentiate into cell types that collectively exhibit characteristics associated with cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells can contribute to many or all tissues of prenatal, postnatal, or adult animals. Standard tests accepted in the art, such as the ability to form teratomas in 8-12 week old SCID mice, can be used to establish the pluripotency of a cell population, although identification of various pluripotent stem cell characteristics can also be used to detect pluripotent cells.

[0064] As used herein, reference to "pluripotent state," "characteristics of pluripotent stem cells," or "one or more properties of pluripotent cells" refers to cellular features that distinguish pluripotent stem cells from other cells. The ability to give rise to progeny that can differentiate, under appropriate conditions, into cell types that collectively exhibit characteristics associated with cell lineages from all three germ layers (endoderm, mesoderm, and ectoderm) is a hallmark of pluripotent stem cells. Expression or non-expression of specific combinations of molecular markers also characterize pluripotent stem cell lineages. For example, human pluripotent stem cells express at least one, two, or three, and possibly all, of the markers from the following non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, SO2, E-cadherin, UTF-1, OCT4 (POU5F1), REX1, and NANOG.

[0065] Cell morphology associated with pluripotent stem cells is also characteristic of pluripotent stem cells. Pluripotent cells are typically characterized by self-renewal ability, the ability to give rise to cell types of the three germ layers, and the expression of pluripotency markers such as OCT4 (POU5F1), NANOG, and SOX2. Pluripotent cells typically grow in flat colonies with clear borders (if in primed state) or dome shapes (if in naive state). This can be contrasted with the morphology of somatic cells, e.g., large, elongated fibroblasts. Markers expressed by both naive and primed pluripotent cells include OCT4 (POU5F1), SOX2, NANOG, KLF4, EPCAM, and PRDM14.

[0066] Markers that are expressed only by naive pluripotent cells or only by primed pluripotent cells are listed in Table 5 herein.

[0067] As used herein, the term "stem cell" refers to a cell that is not terminally differentiated, i.e., capable of differentiating into other cell types with more specific specialized functions. The term encompasses embryonic stem cells, fetal stem cells, adult stem cells or committed / progenitor cells.

[0068] As used herein, an organoid is a collection of organ-specific cell types that arise from stem cells or organ precursors, and through cell sorting and spatially restricted lineage commitment, self-organize in a manner similar to in vivo, and exhibit properties including: a large number of organ-specific cell types; capable of recapitulating some specific functions of an organ (e.g., contraction, neuronal activity, endocrine, filtration, excretion); and whose cells are grouped together and spatially organized, similar to an organ. Organoids can be used as a tool to study fundamental biological processes, including various disease processes, drug screening, and responses to different environmental stimuli.

[0069] The terms "CG" or "CpG" may be used interchangeably and refer to a region of a DNA molecule where a cytosine nucleotide is present upstream of a guanine nucleotide in a linear sequence of bases (linear strand) in the DNA molecule. The nucleotides forming a linear strand in a DNA molecule are linked via phosphate. Thus, a CG site is also called a CpG site. The CpG notation is further used to distinguish between a linear sequence of cytosine and guanine and a CG base pairing of cytosine and guanine, where the cytosine and guanine are located on opposite strands of the DNA molecule. The cytosine in a CpG dinucleotide can be methylated to form 5-methylcytosine. In mammals, methylating a cytosine in a gene can turn the gene off. The enzyme that adds a methyl group to a cytosine in a DNA molecule is called DNA methyltransferase.

[0070] A non-CpG site is a linear sequence of a DNA molecule in which a cytosine nucleotide is present upstream of a nucleotide that is not guanine. Non-CpG cytosine methylation can occur at cytosine sites where the nucleotide does not form part of a CG dinucleotide sequence.

[0071] DNA methylation is an epigenetic mark associated with gene silencing, occurring primarily at cytosine nucleotides when found adjacent to guanines (CpG sites). CpG sites show clustering in certain genomic regions. These clusters, known as CpG islands, are often found in gene promoter regions. Although the exact mechanism remains to be elucidated, there is evidence that methylation provides a "memory" mark for gene silencing, while transcriptional blockade is achieved by associated histone modifications.

[0072] CpG sites are also found in non-coding genomic regions of DNA and within genes, where methylation plays an important role in stabilizing DNA and preventing the movement of transposable elements such as LINE1. LINE1 is an abundant retrotransposon in the human genome, with approximately 500,000 copies, representing 17.88% of the genome. Due to its abundance in the genome, LINE1 methylation has been validated as a surrogate measure of global DNA methylation.

[0073] Prior art methods for generating iPSCs may result in CpG sites that contain hypermethylated or hypomethylated DMRs compared to the genome of ESCs. A non-CpG hypermethylated DMR refers to a differentially methylated region (DMR) in a primed iPSC genome (iPSCs obtained by prior art methods) that has a higher number of methylated non-CpG sites compared to the corresponding region in human ESCs. A non-CpG hypomethylated DMR refers to a differentially methylated region (DMR) in a primed iPSC genome (iPSCs obtained by prior art methods) that has a lower number of methylated non-CpG sites compared to the corresponding region in human ESCs. A non-CpG hypomethylated or hypermethylated DMR is typically about 100 bp to 4000 kb in length.

[0074] Similarly, prior art methods for generating iPSCs can yield hypermethylated and hypomethylated CpG-DMRs. CpG hypermethylated or hypomethylated DMRs are typically about 100 bp to 4000 kb in length.

[0075] It will be appreciated that the methods of the present invention allow for the correction of at least one or more of the aberrant DMRs observed in iPSCs obtained using conventional methods.

[0076] In any aspect of the invention, iPSCs obtained or obtainable by the methods of the invention have levels of CpG DNA methylation, levels of non-CpG methylation, and / or levels of CpG DNA methylation for transposable elements that more closely resemble the DNA methylation profile at the same loci for ESCs. In some embodiments, the loci are one or more of the regions set forth in any of Tables 3 or 4 herein.

[0077] In any embodiment, the methods of the invention provide iPSCs that have the same, or substantially the same, methylation levels at the LINE1 retrotransposon as ESCs.

[0078] Non-limiting examples of CpG hypermethylated or hypomethylated DMRs associated with incomplete iPSC reprogramming are described in U.S. Patent No. 9,428,811, which is incorporated herein by reference. It will be understood that one or more of the DMRs described in U.S. Patent No. 9,428,811 are corrected by reprogramming somatic cells according to the present invention.

[0079] Methods for identifying the epigenomic signature of a cell relative to the equivalent signature of an ESC are also described in U.S. Pat. No. 9,428,811 and can be used according to the methods of the present invention to confirm that the generated iPSCs (or intermediates obtained during the process of the methods of the present invention) have an epigenomic profile similar to that of an ESC. More specifically, one skilled in the art can identify the epigenomic profile of cells contacted with a second culture medium as described herein. The epigenomic profile of the cells can be determined to confirm that the cells are hypomethylated or transitioned to an epigenomic state similar to that of ESCs and can be transitioned to a third culture medium.

[0080] In a preferred embodiment of the present invention, somatic cells are reprogrammed toward a pluripotent state, such as by culturing the cells in a second culture condition or medium adapted to promote hypomethylation. This condition may include the use of a medium that promotes a naive pluripotent state. However, it will be understood that before the cells reach the naive pluripotent state, the cells are preferably transferred to a third culture condition / medium to promote a primed pluripotent state.

[0081] Thus, in a preferred embodiment, the cells contacted with the second culture medium or exposed to the second culture conditions as described herein have genome-wide hypomethylation prior to contacting with the third culture medium, preferably the genome-wide hypomethylation is less than 70%, 60%, 50%, 40%, or 30%, preferably less than 50%, of the methylation state of the primed pluripotent stem cells.

[0082] Hypomethylation is a unique property of primitive cells in human embryos that is lost in normal (primed) PSCs. The cells may exhibit global demethylation in response to being transiently exposed to naive medium according to the methods of the invention. Alternatively, the cells may exhibit at least a 10-90%, preferably at least about 50%, decrease in genome-wide CpG methylation. Methylation levels may be measured relative to the methylation of primed iPSCs not contacted with naive medium. Methylation levels may be quantified by nucleoside mass spectrometry or by bisulfite conversion coupled to deep sequencing.

[0083] According to the present invention, cells contacted with the second culture medium may also have lower levels of histone modifications associated with gene silencing, such as H3K9me3, compared to primed iPSCs not contacted with the second culture medium. The level of histone modifications can be measured by quantitative immunostaining or chromatin immunoprecipitation coupled to deep sequencing (ChIP-seq).

[0084] Naive female cells may exhibit reversible epigenetic erasure of the X chromosome, more specifically, demethylation of the X chromosome and absence of H3K27me3 foci in XX naive cells, preferably 80% or more of XX cells. In some embodiments, conversion of cells from a naive to a primed state restores H3K27me3 foci in the majority of cells, preferably at least 50%, 60%, or 70% or more.

[0085] Those skilled in the art will be familiar with the terms "naive" and "primed" in relation to stem cells. These terms were identified over a decade ago to describe early and late epiblast ontogeny and to describe ESC and EpiSC derivatives. As used herein, the naive pluripotent state refers to a pluripotent state that more closely resembles a preimplantation embryo. In some circumstances, the term "naive state" is used interchangeably with the term "ground state." Naive state cells are stable self-renewing cultures of homogenous pluripotent stem cells that are substantially epigenetically reset compared to somatic cells and have the developmental identity and functional capacity of preimplantation epiblast.

[0086] In certain embodiments, naive pluripotent cells may express preimplantation epiblast-specific transcription factor mRNA and protein. For example, naive cells may express KLF2, KLF4, TFCP2L1, TBX3, REX1, GBX2, STELLA (DPPA3), KLF 17, DPPA3, DPPA5, TFCP2L1, MAEL, UTF1, ZFP57, DNMT3L, FGF4, FOXR1, ARGFX, TRIM60, DDX43, BRDT, ALPPL2, KHDC3L, KHDC1L and PRAP1. Other markers indicative of a naive pluripotent state are shown in Table 5.

[0087] In certain embodiments, the primed pluripotent cells may express mRNA and protein of transcription factors specific to the post-implantation epiblast. For example, the primed cells may express one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or all of SFP, EOMES, BRACHYURY, OTX2, ZIC2, ZIC3, ZIC5, DNMT3B, KDR, CDH2, CER1, COL2A1, DAZL, TCF7L1, SOX11, SALL2. Other markers indicative of the primed pluripotent state are shown in Table 5.

[0088] Since the identification of the naive and primed pluripotent states, a number of different culture media suitable for maintaining pluripotent cells in either state have been developed, and those skilled in the art will be familiar with such media, examples of which are provided further herein.

[0089] Furthermore, naive cells can be characterized by their morphology. Naive phenotype is typically characterized by a tightly packed round dome-like appearance. Cells can also be described as forming compact refractile colonies. According to a preferred embodiment of the present invention, cells cultured in the second culture medium do not develop the morphology typical of naive cells when they are transferred to the third culture medium.

[0090] As used herein, "naive pluripotent state" or "naive pluripotent phenotype" will be understood to refer to a cell phenotype that includes rounded, dome-shaped cells. As used herein, the naive pluripotent state refers to a pluripotent state that more closely resembles a pre-implantation embryo.

[0091] As used herein, a "primed pluripotent state" or "primed pluripotent phenotype" refers to a cell phenotype that is typically characterized by the presence of flat cell colonies with distinct boundaries. As used herein, a primed pluripotent state refers to a pluripotent state that more closely resembles the epiblast of a post-implantation embryo.

[0092] "Induced pluripotent stem cells" refer to pluripotent stem cells artificially induced from non-pluripotent cells.

[0093] As used herein, "non-pluripotent cells" refer to mammalian cells that are not pluripotent cells. The terms "non-pluripotent cells", "somatic cells" and "differentiated cells" may be used interchangeably. Examples of such cells include differentiated cells and progenitor cells. Examples of differentiated cells include, but are not limited to, cells from tissues selected from bone marrow, skin, skeletal muscle, adipose tissue and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T cells.

[0094] It will be appreciated that any non-pluripotent cell (i.e. any differentiated or somatic cell) may be used as a starting cell for generating iPSCs according to the methods of the present invention. Furthermore, iPSCs obtained or obtainable according to the present invention may be differentiated to a non-pluripotent state.

[0095] A somatic cell may be an adult cell or a cell derived from an adult that exhibits one or more detectable characteristics of an adult or non-embryonic cell. A diseased cell exhibits one or more detectable characteristics of a disease or condition. The cell may also exhibit the following characteristics:

[0096] As used herein, "somatic cells" refer to cells that are finally differentiated. As used herein, the term "somatic cells" refers to any cell that forms the body of an organism, as opposed to a germline cell. In mammals, germline cells (also known as "gametes") are sperm and eggs, which fuse at fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. All other cell types in a mammal's body (except for sperm and eggs, the cells from which they are made (gametocytes), and undifferentiated stem cells) are somatic cells. The organs, skin, bones, blood, and connective tissues in the body are all made of somatic cells. In some embodiments, the somatic cells are "non-embryonic somatic cells," which means somatic cells that are not present in or obtained from an embryo and do not result from the propagation of such cells in vitro. In some embodiments, the somatic cells are "adult somatic cells," which means cells that are present in or obtained from an organism other than an embryo or fetus, or cells that result from the propagation of such cells in vitro. Somatic cells can be immortalized to provide an unlimited supply of cells, for example, by increasing the levels of telomerase reverse transcriptase (TERT). For example, the levels of TERT can be increased by increasing the transcription of TERT from an endogenous gene or by introducing a transgene via any gene delivery method or system.

[0097] Differentiated somatic cells, including cells from fetal, neonatal, juvenile or adult primates, including humans, individuals, are suitable somatic cells in the methods of the present invention. Suitable somatic cells include, but are not limited to, bone marrow cells, epithelial cells, endothelial cells, fibroblasts, hematopoietic cells, keratinocytes, hepatocytes, intestinal cells, mesenchymal cells, bone marrow progenitor cells and spleen cells. Alternatively, somatic cells can be cells that can themselves proliferate and differentiate into other types of cells, including blood stem cells, muscle / bone stem cells, brain stem cells and liver stem cells. Suitable somatic cells are receptive or can be made receptive using methods commonly known in the scientific literature to take up transcription factors, including genetic material encoding the transcription factors. The uptake enhancement methods can vary depending on the cell type and expression system. Exemplary conditions used to prepare receptive cells with suitable transduction efficiency are well known to those of skill in the art. The starting somatic cells can have a doubling time of about 24 hours.

[0098] In a preferred embodiment, the somatic cell is a fibroblast (preferably a skin fibroblast or cardiac), a keratinocyte (preferably an epidermal keratinocyte), a monocyte or an endothelial cell.

[0099] As used herein, the term "isolated cell" refers to a cell that has been removed from the organism in which it was originally found, or the progeny of such a cell. Optionally, the cell has been cultured in vitro, e.g., in the presence of other cells. Optionally, the cell is subsequently introduced into a second organism, or reintroduced into the organism from which it was isolated (or the cell from which it was derived).

[0100] As used herein, the term "isolated population" in reference to an isolated cell population refers to a cell population that is removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure cell population as compared to the heterogeneous population from which the cells are isolated or enriched.

[0101] One of skill in the art would also be able to determine when a somatic cell has begun reprogramming towards a pluripotent state when it has lost morphological characteristics typical of somatic cells, again, one of skill in the art is familiar with the morphological characteristics of somatic cells, including fibroblasts.

[0102] A somatic cell may also be determined to be reprogrammed to a pluripotent state if it exhibits at least one characteristic of a pluripotent cell (e.g., an iPSC or an ESC), such as upregulation of any one or more ESC markers and / or Typically, cells converted into iPSC / ESC-like cells exhibit one, two, three, four, five, six, seven, eight or more characteristics of iPSC / ESC.

[0103] In some embodiments in which an individual is treated with the resulting pluripotent cells, or differentiated cells derived therefrom, the individual's own non-pluripotent cells are used to generate pluripotent cells according to the methods of the invention.

[0104] The cells can be, for example, from a human or a non-human mammal. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, and cows.

[0105] Reprogramming Those of skill in the art will be familiar with standard techniques for promoting the reprogramming of somatic cells towards a pluripotent state.

[0106] Various methods are known in the art for reprogramming somatic cells towards a pluripotent state. Reprogramming of somatic cells typically involves the expression of reprogramming factors (including transcription factors), followed by culture in specific conditions to promote the loss of differentiation markers and the gain of pluripotency markers.

[0107] Examples of suitable methods for reprogramming somatic cells are abundant in the art and are exemplified in WO 2009 / 101407, WO 2014 / 200030, WO 2015 / 056804, WO 2014 / 200114, WO 2014 / 065435, WO 2013 / 176233, WO 2012 / 060473, WO 2012 / 036299, WO 2011 / 158967, WO 2011 / 055851, WO 2011 / 037270, WO 2011 / 090221, the contents of which are incorporated herein by reference.

[0108] Particularly preferred transcription factors, and their nucleic acid sequences, that can be used to reprogram somatic cells (e.g., fibroblasts) according to the methods of the present invention are set forth below in Table 1. However, it will be understood that the present invention is not limited to the use of the transcription factors listed in Table 1 to reprogram somatic cells.

[0109] Transcription factors and other protein factors referred to herein are referred to by HUGO Gene Nomenclature Committee (HGNC) symbols. Table 1 provides exemplary Ensembl Gene IDs and Uniprot IDs for the transcription factors listed herein. Nucleotide sequences are derived from the Ensembl database (Flicek et al. (2014). Nucleic Acids Research Volume 42, Issue D1. Pp. D749-D755) version 83. Any variants, homologs, orthologs or paralogs of the transcription factors referred to herein are also contemplated for use in the present invention.

[0110] Those skilled in the art will also be familiar with the ability to reprogram somatic cells towards a naive pluripotent state (as compared to a primed pluripotent state). It will be understood that the methods of the invention apply to cells that are treated to promote reprogramming towards a naive or primed pluripotent state. In a preferred embodiment of the invention, the methods involve the use of somatic It involves increasing the protein expression of one or more factors in the cell.

[0111] One of skill in the art will appreciate that this information can be used in practicing the methods of the invention, for example, for the purpose of providing increased amounts of a transcription factor in a somatic cell, or for the purpose of providing nucleic acids (including recombinant polynucleotides) for recombinantly expressing a transcription factor in a somatic cell.

[0112] [Table 1]

[0113] The present invention contemplates the use of variants of the transcription factors described herein. The variants may be fragments of the full-length polypeptide or naturally occurring splice variants. The variants may be polypeptides that are at least 70%, 80%, 85%, 90%, 95%, 98%, or 99% identical to a fragment of the polypeptide, where the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99%, so long as the full-length wild-type polypeptide or a domain thereof has a desired functional activity, such as the ability to promote the conversion of a somatic cell type to a target cell type. In some embodiments, the domain is at least 100, 200, 300, or 400 amino acids in length, beginning at any amino acid position in the sequence and extending toward the C-terminus. Variations known in the art to eliminate or substantially reduce the activity of a protein are preferably avoided. In some embodiments, the variants lack the N- and / or C-terminal portions of the full-length polypeptide, for example, up to 10, 20, or 50 amino acids from either end. In some embodiments, the polypeptide has the sequence of a mature (full-length) polypeptide, meaning a polypeptide from which one or more portions, such as a signal peptide, have been removed during normal intracellular proteolytic processing (e.g., during co-translational or post-translational processing). In some embodiments where the protein is produced other than by purifying it from a cell that naturally expresses it, the protein is a chimeric polypeptide, meaning that it contains portions from two or more different species. In some embodiments where the protein is produced other than by purifying it from a cell that naturally expresses it, the protein is a derivative, meaning that the protein contains additional sequences not associated with the protein, so long as they do not substantially reduce the biological activity of the protein. Those skilled in the art will recognize or be able to readily confirm whether a particular polypeptide variant, fragment, or derivative is functional using assays known in the art. For example, the ability of a variant of a transcription factor to convert a somatic cell to a target cell type can be assessed using the assays disclosed in the Examples herein.Other convenient assays include measuring the ability to activate transcription of a reporter construct that contains a transcription factor binding site operably linked to a nucleic acid sequence encoding a detectable marker, such as luciferase. In certain embodiments of the invention, functional variants or fragments have at least 50%, 60%, 70%, 80%, 90%, 95% or more of the activity of the full-length wild-type polypeptide.

[0114] The term "increasing the amount" in relation to increasing the amount of a transcription factor refers to increasing the amount of the transcription factor in a cell of interest (e.g., a somatic cell such as a fibroblast). In some embodiments, the amount of a transcription factor is "increased" in a cell of interest (e.g., a cell into which an expression cassette directing the expression of one or more transcription factor-encoding polynucleotides has been introduced) if the amount of the transcription factor is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control (e.g., a fibroblast cell into which none of the expression cassettes have been introduced). However, any method of increasing the amount of a transcription factor is contemplated, including any method of increasing the amount, rate or efficiency of transcription, translation, stability or activity of a transcription factor (or pre-mRNA or mRNA encoding it). Additionally, downregulation or interference with negative regulators of transcription expression, increasing the efficiency of existing translation (e.g., SINEUP) are also contemplated.

[0115] The present invention relies on routine techniques in the field of recombinant genetics. Essential texts that disclose general methods of use in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0116] It will be understood that where more than one protein is expressed in a cell, one or more expression cassettes may be used, for example, where one expression cassette expresses multiple polypeptides, a polycistronic expression cassette may be used.

[0117] In some embodiments, an expression cassette encoding a reprogramming transcription factor protein may be introduced into a cell as part of a vector. Exemplary vectors include, for example, plasmids and viral vectors (including, but not limited to, adenovirus, AAV, or retroviruses such as lentivirus).

[0118] Nucleic Acids and Vectors The nucleic acids described herein or vectors containing the nucleic acids may include one or more of the sequences referred to in Table 1 above.

[0119] The term "expression", as applicable, refers to the cellular processes involved in the production of RNA and proteins, including, but not limited to, transcription, translation, folding, modification and processing, and, optionally, secretion of proteins.

[0120] The terms "isolated" or "partially purified" as used herein refer to a nucleic acid or polypeptide that has been separated from at least one other component (e.g., a nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide found in its natural source and / or that is present with the nucleic acid or polypeptide when expressed or, in the case of a secreted polypeptide, secreted by a cell. A nucleic acid or polypeptide that is chemically synthesized or synthesized using in vitro transcription / translation is considered "isolated."

[0121] The term "vector" refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host or somatic cell. Preferred vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors." Thus, "expression vectors" refer to vectors that are capable of directing the expression of genes to which they are operatively linked. A "viral vector" is a specialized vector that contains the necessary regulatory regions required for expression of a gene of interest in a host cell. In some embodiments, the gene of interest is operably linked to another sequence in the vector. The vector can be a viral vector or a non-viral vector. When a viral vector is used, it is preferable that the viral vector is replication-deficient, which can be achieved, for example, by removing all viral nucleic acid that codes for replication. Replication-deficient viral vectors still retain their infectivity and enter cells in a similar manner to replicating adenoviral vectors, but once inside a cell, replication-deficient viral vectors do not replicate or grow. Vectors also include liposomes and nanoparticles, as well as other means for delivering DNA molecules to cells.

[0122] The term "operably linked" means that regulatory sequences necessary for expression of a coding sequence are positioned in a DNA molecule in the appropriate position relative to the coding sequence to effect expression of the coding sequence. This same definition may be applied to the positioning of coding sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. The term "operably linked" includes having an appropriate initiation signal (e.g., ATG) in front of the polynucleotide sequence to be expressed, and maintaining the correct reading frame to allow expression of the polynucleotide sequence under the control of the expression control sequences, and production of the desired polypeptide encoded by the polynucleotide sequence.

[0123] The term "viral vector" refers to the use of a virus or virus-associated vector as a carrier of a nucleic acid construct into a cell. The construct can be incorporated and packaged into a non-replicating defective viral genome, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or others, including retrovirus and lentivirus vectors, for infection or transduction into cells. The vector may or may not be integrated into the genome of the cell. The construct can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into a vector capable of episomal replication, such as EPV and EBV vectors.

[0124] As used herein, the term "adenovirus" refers to a virus of the Adenovirida family. Adenoviruses are medium-sized (90-100 nm), non-enveloped (naked), icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome.

[0125] As used herein, the term "non-integrating viral vector" refers to a viral vector that does not integrate into the host genome, and the expression of genes delivered by the viral vector is transient. Since there is little or no integration into the host genome, non-integrating viral vectors have the advantage of not generating DNA mutations by inserting at random points in the genome. For example, non-integrating viral vectors remain extrachromosomal and do not insert their genes into the host genome, potentially disrupting the expression of endogenous genes. Non-integrating viral vectors may include, but are not limited to: adenovirus, alphavirus, picornavirus, and vaccinia virus. These viral vectors are "non-integrating" viral vectors, as the term is used herein, even though any of them may, in some rare circumstances, integrate viral nucleic acid into the genome of a host cell. What is important is that the viral vectors used in the methods described herein do not integrate their nucleic acid into the genome of a host cell as a rule or as a major part of their life cycle under the conditions used.

[0126] The vectors described herein can be modified in any manner generally known in the scientific literature to increase their safety for use in therapy, to include selection and enrichment markers, if desired, and to optimize expression of the nucleotide sequences contained therein. Vectors should contain components that allow the vector to autonomously replicate in somatic cell types. For example, the well-known Epstein Barr method can be used to construct and engineer vectors. The oriP / Nuclear Antigen-1 (EBNA-I) combination (see, e.g., Lindner, SE and B. Sugden, The plasmid replicon of Epstein-Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells, Plasmid 58:1 (2007)) is sufficient to support vector self-replication, and other combinations known to function in mammalian, particularly primate, cells can also be used. Standard techniques for the construction of expression vectors suitable for use in the present invention are well known to those of skill in the art and can be found in publications such as Sambrook J, et al., "Molecular cloning: a laboratory manual", (3rd ed. Cold Spring harbor Press, Cold Spring Harbor, NY 2001) (incorporated herein by reference in its entirety as if set forth herein).

[0127] In the method of the present invention, the genetic material encoding the relevant transcription factors required for conversion is delivered to the somatic cell via one or more reprogramming vectors. Each transcription factor can be introduced into the somatic cell as a polynucleotide transgene encoding the transcription factor operably linked to a heterologous promoter capable of driving the expression of the polynucleotide in the somatic cell.

[0128] Suitable reprogramming vectors are any of those described herein, including episomal vectors (e.g., plasmids) that do not encode all or part of a sufficient viral genome to produce an infectious or replication-competent virus, but the vector may contain structural elements obtained from one or more viruses. One or more of the reprogramming vectors can be introduced into a single somatic cell. One or more transgenes can be provided on a single reprogramming vector. One strong constitutive transcription promoter can provide transcriptional control of multiple transgenes that can be provided as expression cassettes. Separate expression cassettes on a vector can be under the transcriptional control of separate strong constitutive promoters, which may be copies of the same promoter or separate promoters. A variety of heterologous promoters are known in the art and can be used depending on factors such as the desired expression level of the transcription factor. As exemplified below, it may be advantageous to use different promoters with different strengths in the cell to control the transcription of separate expression cassettes. Another point to consider when selecting a transcription promoter is the speed at which the promoter is silenced. Those skilled in the art will appreciate that it may be advantageous to reduce the expression of one or more transgenes or transgene expression cassettes after the product of the gene has completed or substantially completed its role in the reprogramming method. Exemplary promoters are the human EF1α elongation factor promoter, the CMV cytomegalovirus immediate early promoter and the CAG chicken albumin promoter, as well as corresponding homologous promoters from other species. In human somatic cells, both EF1α and CMV are strong promoters, but the CMV promoter is silenced more efficiently than the EF1α promoter, such that expression of transgenes under the control of the former is turned off earlier than expression of transgenes under the control of the latter. Transcription factors can be expressed in somatic cells in relative ratios that can be varied to modulate reprogramming efficiency. Preferably, when multiple transgenes are encoded on a single transcript, an internal ribosome entry site is provided upstream of the transgenes distal from the transcription promoter.The relative ratios of factors can vary depending on the factors being delivered, but the optimal ratios of factors can be determined by one of skill in the art in possession of this disclosure.

[0129] One skilled in the art would be able to introduce all of the factors via a single vector rather than via multiple vectors. Those skilled in the art will also appreciate that the location of a transcription factor on a vector can affect its temporal expression and the resulting reprogramming efficiency. Thus, the inventors have used various combinations of factors for vector combinations. Several such combinations are presented herein to support reprogramming.

[0130] After introduction of the reprogramming vector and while the somatic cell is being reprogrammed, the vector can persist in the target cell while the introduced transcriptional gene is transcribed and translated. The transgene expression can be advantageously downregulated or turned off in the cells reprogrammed to the target cell type. The reprogramming vector can remain extrachromosomal. With very low efficiency, the vector can be integrated into the genome of the cell. The following examples are intended to illustrate the invention but in no way limit it.

[0131] Methods suitable for nucleic acid delivery for transformation of cells, tissues or organisms for use in the present invention are contemplated to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into a cell, tissue or organism, as described herein or known to one of skill in the art (e.g., Stadtfeld and Hochedlinger, Nature Methods 6(5):329-330 (2009); Yusa et al., Nat. Methods 6:363-369 (2009); Woltjen, et al., Nature 458, 766-770 (9 Apr. 2009)). Such methods include, for example, ex vivo transfection, optionally using lipid-based transfection reagents such as Fugene6 (Roche) or Lipofectamine (Invitrogen) (Wilson et al., Science, 244:1344-1346, 1989, Nabel and Baltimore, Nature 326:711-713, 1987) by microinjection (Harland and Weintraub, J. Cell. Biol., 101:1094-1099, 1985; by injection, including U.S. Pat. No. 5,789,215, which is incorporated herein by reference (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, which is incorporated herein by reference; Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al. al., Proc. Nat'l Acad. Sci. USA, 81:7161-7165, 1984); by calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell Biol., 5:1188-1190, 1985); by direct sonication (Fechheimer et al., Proc. Nat'l Acad. Sci. USA, 84:8463-8467, 1987); liposome-mediated transfection (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982; Fraley et al., Proc. Nat'l Acad. Sci. USA, 76: 3348-3352, 1979; Nicolau et al., Methods Enzymol., 149: 157-176, 1987; Wong et a These methods include, but are not limited to, direct delivery of DNA by receptor-mediated transfection (Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987); and any combination of these methods, each of which is incorporated herein by reference.

[0132] A number of polypeptides capable of mediating the introduction of relevant molecules into cells have been previously described and can be adapted for the present invention. See, for example, Langel (2002) Cell Penetrating Peptides: Processes and Applications, CRC Press, Pharmacology and Toxicology Series. Examples of polypeptide sequences that enhance transport across membranes include the Drosophila homeoprotein Antennapedia transcription protein (AntHD) (Joliot et al., New Biol. 3:1121-34, 1991; Joliot et al., Proc. Natl. Acad. Sci. USA, 88:1864-8, 1991; Le Roux et al., J. Immunol. 1999, 11:1121-34, 1991; Le Roux et al., J. Immunol. 1999, 11:1121-34, 1991). al., Proc. Natl. Acad. Sci. USA, 90:9120-4, 1993), herpes simplex virus structural protein VP22 (Elliott and O'Hare, Cell 88:223-33, 1997); HIV-1 transcriptional activator TAT protein (Green and Loewenstein, Cell 55:1179-1188, 1988; Frankel and Pabo, Cell 55:1 289-1193, 1988); Kaposi's FGF signal sequence (kFGF); protein transduction domain-4 (PTD4); Penetratin, M918, Transportan-10; nuclear localization sequence, PEP-I peptide; amphipathic peptides (e.g., MPG peptide); delivery-enhancing transporters as described in U.S. Pat. No. 6,730,293 (including, but not limited to, peptide sequences containing at least 5-25 or more consecutive arginines or 5-25 or more arginines in a contiguous set of 30, 40, or 50 amino acids; including, but not limited to, peptides having sufficient, e.g., at least 5, guanidino or amidino moieties); and the commercially available Penetratin™ 1 peptide; and the Diatos Peptide Vectors ("DPV") of the Vectocell® platform available from Daitos SA, Paris, France. See also WO 2005 / 084158 and WO 2007 / 123667 and the further transporters described therein. Not only are these proteins able to cross the plasma membrane, but the attachment of other proteins, such as the transcription factors described herein, is sufficient to stimulate cellular uptake of these complexes.

[0133] Drugs The term "agent" as used herein refers to any compound or substance, including, but not limited to, small molecules, nucleic acids, polypeptides, peptides, drugs, ions, etc. An "agent" can be any chemical entity or moiety, including, but not limited to, synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is a nucleic acid, a nucleic acid analog, a protein, an antibody, a peptide, an aptamer, an oligomer of nucleic acid, an amino acid, or a carbohydrate, including, but not limited to, a protein, an oligonucleotide, a ribozyme, a DNAzyme, a glycoprotein, an siRNA, a lipoprotein, an aptamer, and modifications and combinations thereof. In certain embodiments, an agent is a small molecule having a chemical moiety. For example, the chemical moiety includes unsubstituted or substituted alkyl, aromatic, or heterocyclic moieties, including macrolides, leptomycin, and related natural products or analogs thereof. The compound is known to have a desired activity and / or property, and / or is capable of being used to treat a variety of conditions, including, but not limited to, ... The compound may be obtained or selected from a library of diverse compounds.

[0134] The term "exogenous" when used in reference to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means; or when used in reference to a cell, refers to a cell that has been isolated and then introduced into another cell or organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or may be one or more additional copies of a nucleic acid that naturally occurs in an organism or cell. An exogenous cell may be from a different organism or may be from the same organism. As a non-limiting example, an exogenous nucleic acid is a nucleic acid that is at a chromosomal location that is different from that of a natural cell, or is adjacent to a nucleic acid sequence that is different from the nucleic acid sequence found in nature. An exogenous nucleic acid may also be extrachromosomal, such as an episomal vector.

[0135] Screening one or more candidate agents for their ability to increase the amount of one or more transcription factors required for reprogramming somatic cell types to a pluripotent state may include contacting a system that allows the product or expression of the transcription factor with the candidate agent and determining whether the amount of the transcription factor is increased. The system may be in vivo, e.g., a tissue or cell in an organism, or in vitro, a cell isolated from an organism, or an in vitro transcription assay, or ex vivo in a cell or tissue. The amount of the transcription factor may be measured directly or indirectly by measuring the amount of protein or RNA (e.g., mRNA or pre-mRNA). The candidate agent functions to increase the amount of the transcription factor by increasing any step in the transcription of the gene encoding the transcription factor, or by increasing the translation of the corresponding mRNA. Alternatively, the candidate agent may reduce the inhibitory activity of a repressor of transcription of the gene encoding the transcription factor, or the activity of a molecule that causes degradation of the mRNA encoding the transcription factor or the protein of the transcription factor itself.

[0136] Suitable detection means include the use of labels such as radionucleotides, enzymes, coenzymes, fluorescers, chemiluminescers, chromogens, enzyme substrates or cofactors, enzyme inhibitors, prosthetic group complexes, free radicals, particles, dyes, etc. Such labeled reagents can be used in a variety of well-known assays, such as radioimmunoassays, enzyme immunoassays (e.g., ELISA), fluorescent immunoassays, etc. See, e.g., U.S. Patent Nos. 3,766,162; 3,791,932; 3,817,837; and 4,233,402.

[0137] The methods of the present invention include high throughput screening applications. For example, high throughput screening assays can be used, including any of the assays according to the present invention, in which an aliquot of a system allowing the production or expression of a transcription factor is exposed to multiple candidate agents in different wells of a multi-well plate. Furthermore, high throughput screening assays according to the present disclosure include aliquots of a system allowing the production or expression of a transcription factor exposed to multiple candidate agents in any kind of miniaturized assay system.

[0138] The disclosed method can be "miniaturized" in an assay system by any acceptable miniaturization method, including but not limited to multi-well plates, microchips or slides, such as 24, 48, 96 or 384 wells per plate. The assay can be reduced in size to be performed on a microchip support, advantageously containing smaller amounts of reagents and other materials. Any miniaturization of the process that leads to high throughput screening is within the scope of the present invention.

[0139] In any of the methods of the invention, the iPSCs, or differentiated cells derived therefrom, can be transferred into the same mammal from which the somatic cells were obtained. The somatic cells to be administered may be autologous, i.e., obtained from the same individual to which the iPSCs or differentiated cells derived therefrom are administered. Alternatively, the iPSC cells may be allogeneically transferred to another individual. Preferably, the cells are autologous to the subject in methods of treating or preventing a medical condition in an individual.

[0140] Cell culture Cells induced to pluripotency can be cultured according to any method known in the art. General guidelines can be found, for example, in Maherali, et al., Cell Stem Cell Culture (2003). Cell 3:595-605(2008).

[0141] In some embodiments, cells are cultured in contact with feeder cells.Exemplary feeder cells include but are not limited to fibroblast cells (e.g., mouse embryonic fibroblast (MEF) cells).Methods for culturing cells on feeder cells are known in the art.

[0142] In some embodiments, the cells are cultured in the absence of feeder cells. The cells can be attached directly to a solid culture surface (e.g., a culture plate), for example, via a molecular tether. The inventors have found that the efficiency of induction of cultured cells into pluripotency is much higher (i.e., a greater proportion of cells achieve pluripotency) when the cells are attached directly to a solid culture surface, compared to the efficiency of otherwise identically treated cells cultured on feeder cells. Exemplary molecular tethers include, but are not limited to, Matrigel, extracellular matrix (ECM), ECM analogs, laminin, fibronectin, or collagen. However, one skilled in the art will recognize that this is a non-limiting list and that other molecules can be used to attach cells to a solid surface. Methods for the initial attachment of the tether to a solid surface are known in the art.

[0143] Those skilled in the art will: Facilitating the reprogramming of cells towards a pluripotent state; Promoting a naive pluripotent state; and Promotes a primed pluripotent state The reader will be familiar with the culture medium compositions and culture conditions for

[0144] In one embodiment, the medium for promoting a hypomethylated DNA state, such as a naive pluripotent state, comprises a MEK inhibitor, a PKC inhibitor, a GSK3 inhibitor, a STAT3 activator and a ROCK inhibitor.

[0145] Reference to MEK inhibitors generally refers to MEK inhibitors. MEK inhibitors can inhibit any member of the MEK family of protein kinases, including MEK1, MEK2, and MEK5. Examples of suitable MEK inhibitors are known in the art and include PD184352 and PD98059, MEK1 and MEK2 inhibitors U0126 and SL327. In particular, PD184352 and PD0325901 have been found to have a high degree of specificity and potency compared to other known MEK inhibitors.

[0146] Examples of protein kinase C (PKC) inhibitors include Goe6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (Gschwendt et al., 1996 FEBS Lett 392:77-80). Another preferred PKC inhibitor is Ro-31-8425. Preferably, the PKC inhibitor is present in the medium at a concentration of 0.01 to 10 μM, 0.1 to 5 μM, preferably 1 to 4 μM. do.

[0147] Reference to GSK3 inhibition refers to the inhibition of one or more GSK3 enzymes.The GSK3 enzyme family is well known, and many variants have been described.In certain embodiments, GSK3β is inhibited.GSK3-α inhibitors can also be used.A wide range of GSK3 inhibitors are known, such as inhibitors CHIR 98014, CHIR 99021, AR-AO144-18, TZD-8, SB21676763 and SB415286.

[0148] The inhibitor can be provided or obtained by a person skilled in the art by conventional means or from conventional sources. The inhibitor can be a small molecule inhibitor or an interfering RNA (RNAi). A person skilled in the art is also familiar with various methods and assays for identifying kinase inhibitors.

[0149] Examples of STAT3 activators include LIF, preferably hLIF.

[0150] The combination of a MEK inhibitor, a GSK3 inhibitor and LIF can be referred to as 2iL.

[0151] As used herein, ROCK inhibitor refers to an inhibitor of Rho-linked kinase. Examples of such inhibitors include ((1R,4r)-4-(((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide, Abcam) (also known as trans-N-(4-(1-aminoethyl)-cyclohexanecarboxamide), 1-(5-isoquinolyl)(sulfonyl)homopiperazine (1-(5-isoquinolinylsulfonyl)homopiperazine). Typically, the amount of ROCK inhibitor is about 0.1-50 μM, preferably about 1-10 μM.

[0152] "Inhibitors", "activators" and "modulators" of expression or activity are used to refer to inhibitory, activating or modulating molecules, respectively, identified using in vitro and in vivo assays for expression or activity of a described target protein (e.g., ligands, agonists, antagonists, and their homologs and mimetics). The term "modulators" includes inhibitors and activators. Inhibitors are, for example, agents that inhibit the expression of or binding to a described target protein (e.g., antagonists), or partially or fully block, reduce, prevent, delay activation of, inactivate, desensitize or downregulate the activity of the protein. Activators are agents that, for example, induce or activate expression of the described target proteins, or stimulate, increase, open, activate, promote, enhance activation or protease inhibitor activity, sensitize or upregulate the activity of the described target proteins (or encoding polynucleotides), e.g., agonists. Modulators include naturally occurring and synthetic ligands, antagonists and agonists (e.g., small chemical molecules, antibodies, etc. that function as either agonists or antagonists). Such assays for inhibitors and activators include, for example, applying a putative modulator compound to cells expressing the described target proteins, and then determining the functional effect on the described target protein activity, as described above. Samples or assays containing the described target proteins that have been treated with potential activators, inhibitors, or modulators are compared to control samples without inhibitors, activators, or modulators to examine the extent of the effect. Control samples (not treated with modulators) are assigned a relative activity value of 100%. Inhibition of the described target protein is achieved when the activity value relative to the control is about 80%, optionally 50% or 25, 10%, 5% or 1%. Activation of the described target protein is achieved when the activity value relative to the control is about 80%, optionally 50% or 25, 10%, 5% or 1%. This is achieved when the activity value relative to the control is 110%, optionally 150%, optionally 200, 300, 400, 500, or 1000-3000% or more.

[0153] As used herein, "inhibit", "prevent" or "reduce", or "inhibiting", "preventing" or "reducing" are used interchangeably herein. These terms refer to a reduction in a measured parameter (e.g., activity, expression, mitochondrial respiration, mitochondrial oxidation, oxidative phosphorylation) in treated cells (tissues or subjects) compared to untreated cells (tissues or subjects). Comparisons can also be made before and after treatment for the same cells or tissues or subjects. This reduction is sufficient to be detectable. In some embodiments, the reduction in treated cells is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or completely inhibited compared to untreated cells. In some embodiments, the measured parameter is undetectable (i.e., completely inhibited) in treated cells compared to untreated cells.

[0154] Examples of suitable culture media are summarized in Table 2 below.

[0155] [Table 2-1]

[0156] [Table 2-2]

[0157] As used herein, hPGCLC induction medium refers to human primordial germ cell-like cell induction medium. It will be understood that this medium can be used to promote a state of global hypomethylation in cells.

[0158] RSeT™ medium is a defined cell culture medium used to maintain naive-like hPSCs under feeder-dependent and hypoxic conditions. RSeT™ medium contains pre-screened quality ingredients and does not contain bFGF or TGFβ. It is compatible with human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells.

[0159] hPSCs cultured in RSeT™ medium exhibit characteristics of a naïve-like state, such as tightly packed domed colonies with crooked edges. Key transcripts associated with naïve-like hPSCs, such as KLF2, KLF4, and TFCP2L1, show increased expression in hPSCs cultured in RSeT™ medium. RSeT™ hPSCs can be reverted to a primed state by culture in mTeSR™1 and then differentiated using standard techniques.

[0160] Exemplary t2iLGoY medium: A 50:50 mixture of DMEM / F-12 (Gibco) and Neurobasal Medium (Gibco) supplemented with 2 mM L-glutamine (Gibco) 0.1 mM 2-mercaptoethanol (Gibco), 0.5% N2 supplement (Gibco), 1% B27 supplement (Gibco), 1% Pen-strep (Gibco), 10ng / ml human LIF (homemade), 250μM L-ascorbic acid (Sigma), 10 μg / ml recombinant human insulin (Sigma), · 1μM PD0325901 (Miltenyi Biotec), · 1μM CHIR99021 (Miltenyi Biotec), 2.5μM Goe6983 (Tocris), 10μM Y-27632 (Abcam).

[0161] differentiation The invention also includes methods of generating differentiated cells from iPSCs produced according to the invention.

[0162] Differentiated cells can be used for cell culture models of human development and disease applicable in drug discovery and development, as well as teratogenicity and toxicology testing; to generate sources of tissue stem cells and more mature cells for applications in clinical cell therapy; to analyze the relative contributions of genetics and epigenetics to developmental disorders, genetic diseases and quantitative traits to facilitate advances in diagnosis, prognosis and patient treatment; to generate tissues and organs for transplantation by in vitro bioengineering or by lineage / organ-specific contributions to human-animal chimeras.

[0163] iPSCs generated according to the present invention can be differentiated into any somatic cell, including, but not limited to, neurons, astrocytes, oligodendrocytes, keratinocytes, epithelial cells, cardiomyocytes, fibroblasts, B cells, T cells, macrophages, hematopoietic stem cells, retinal epithelial cells, smooth muscle cells, skeletal cells, podocytes, kidney cells, hepatocytes, pancreatic beta islets, alveolar cells, adipocytes, chondrocytes, and bone cells.

[0164] Still further, iPSCs generated according to the present invention can be differentiated into primordial germ cells (PGCs) and gametes.

[0165] Those skilled in the art will be familiar with standard methods for differentiating iPSCs generated according to the present invention.There are many documents available to those skilled in the art that provide instructions for the differentiation of iPSCs.Kits that contain differentiation medium for differentiating iPSCs into somatic cell types are also commercially available, such as from Sigma Aldrich and ThermoFisher Scientific.

[0166] Successful differentiation into a somatic cell type can be determined by determining the presence of one or more markers characteristic of somatic cells. Additionally, successful differentiation can be determined by observing a change in morphology from a pluripotent state to a differentiated morphology characteristic of the target cell type.

[0167] The differentiated cells or populations of differentiated cells obtained or obtainable according to the present methods may be characterized by cells having fewer characteristics of somatic cells (i.e., somatic cells subjected to step a)) compared to differentiated cells or populations of differentiated cells obtained by differentiation of iPSCs generated according to conventional methods (i.e., without transient exposure to the second culture medium or second culture conditions described herein). Furthermore, the populations of differentiated cells may have fewer cells having characteristics of somatic cells.

[0168] More specifically, as further demonstrated in the examples herein, a population of cells differentiated from primed iPSCs obtained according to the methods of the prior art typically contains a subset of cells that retain the characteristics of the source cells. In other words, when reprogramming from fibroblasts, any differentiated cells generated from fibroblasts when reprogramming with priming medium typically contain some cells that resemble fibroblasts in both morphology and gene expression characteristics. The iPSCs generated according to the present invention have an epigenetic profile that reduces the possibility of differentiated cells having the characteristics of the original source cells used in reprogramming.

[0169] Pharmaceutical Compositions and Other Uses The present invention, according to certain embodiments, contemplates the use of any cells, tissues and / or organs / organoids generated using iPSCs generated according to the present invention.

[0170] The isolated cells of the present invention can further be used for disease modeling, drug screening and patient-specific cell-based therapy.

[0171] Thus, according to certain aspects of the invention there is provided an isolated iPSC generated according to the method of the invention, or an isolated differentiated cell obtained from an iPSC obtained or obtainable according to the method of the invention.

[0172] Introduction of the cells can be accomplished in vitro, via direct injection into a subject in need of treatment, or ex vivo.

[0173] As used herein, the phrase "subject in need thereof" refers to a mammalian subject (e.g., a human) who has been diagnosed with a pathology. In certain embodiments, the term encompasses individuals who are at risk of developing the pathology. Veterinary uses are also contemplated. Subjects can be of any gender or age, including neonates, infants, juveniles, adolescents, adults, and older adults.

[0174] iPSCs obtained or obtainable according to the present invention, or differentiated cells obtained therefrom, can be transplanted into a subject either on their own or in a pharmaceutical composition in which they are mixed with a suitable carrier or excipient. Similarly, constructs of the present invention can be administered to a subject either on their own or in a pharmaceutical composition.

[0175] As used herein, a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0176] As used herein, the term "active ingredient" refers to a cell of the invention that is responsible for the biological effect.

[0177] Hereinafter, the expressions "physiologically acceptable carrier" and "pharmaceutical acceptable carrier", which may be used interchangeably, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abolish the biological activity and properties of the administered compound.

[0178] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Non-limiting examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0179] The pharmaceutical compositions of the present invention can be manufactured by processes that are well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0180] Thus, pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants that facilitate processing of the active ingredients into medicamentously usable preparations. Appropriate formulations will depend on the route of administration selected.

[0181] For injection, the active ingredients of the pharmaceutical composition are preferably in an aqueous solution, such as Hanks' solution, Ringer's solution, or the like. The compositions may be formulated in aqueous solutions of 0.1% to 10% by weight, or in physiologically compatible buffers such as physiological salt buffer.

[0182] Typically, the pharmaceutical compositions are administered in a local rather than systemic manner, for example, by injecting the pharmaceutical composition directly into a tissue area of ​​the patient.

[0183] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion.The preparations for injection can be provided in unit dosage form, for example, in ampoules or in multi-dose containers, optionally with the addition of preservatives.The compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending agents, stabilizing agents and / or dispersing agents.

[0184] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredient can be prepared as suitable oily or aqueous injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredient to allow for the preparation of highly concentrated solutions.

[0185] Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve its intended purpose, Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0186] The compositions of the present invention may be provided in a pack or dispenser device (e.g., an FDA approved kit) that may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be a syringe. The syringe may be pre-filled with cells. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be contained with a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects approval by the agency of the composition or form for human or veterinary administration. Such notice may, for example, be of a label approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. Compositions containing the preparations of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for treatment of an indicated condition, as further detailed above.

[0187] The present invention includes the following non-limiting examples.

Example

[0188] Example 1: Reprogramming of somatic cells into iPSCs (the "transient naive treatment" protocol) For nuclear reprogramming experiments, primary adult human dermal fibroblasts (HDFa, Life Technologies) were grown in medium 106 (Life Technologies) supplemented with LSGS (Gibco).

[0189] Fibroblasts at the early passage stage (<P6) were seeded into 6-well plates at 50,000 - 70,000 cells per well, and then transfected in fibroblast medium containing DMEM (Gibco), 10% FBS (Hyclone), 1% non-essential amino acids (Gibco), 1 mM GlutaMAX (Gibco), 1% Pen-strep (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), and 1 mM sodium pyruvate (Gibco). After 48 hours, the cells in one well were trypsinized for counting to determine the volume of virus (MOI) required for transfection. Transfection was performed using the CytoTune 2.0 iPSC Sendai Reprogramming Kit (Invitrogen) consisting of four transcription factors, OCT4, SOX2, cMYC, and KLF4.

[0190] After 24 hours, the virus was removed and the medium was changed every other day. Seven days after transfection, the cells were harvested using TrypLE express (Life tech), re-seeded onto a layer of irradiated MEF feeders in fibroblast medium, and then the next day, the medium was changed to naive medium (2iLGoY, Guo et al., see Table 2).

[0191]

[0192] ​After 5 days, when dome-shaped colonies became apparent, intermediate cells were harvested using Accutase (Stem Cell Technologies) and re-seeded onto vitronectin (Life tech)-coated plates under naive conditions. The next day, the medium was changed to priming medium (Essential 8 (Life tech)). Alternatively, without splitting the cells, the medium can be changed from naive to priming. When the culture became confluent, the cells were harvested using 0.5 mM EDTA / PBS solution (Invitrogen), and a human pluripotent stem cell culture system without vitronectin / E8 feeder was maintained. The cells were cultured in a 37 °C, 5% O2, and 5% CO2 incubator, and the medium was changed daily. The cells were passaged every 4 - 5 days.

[0193] Example 2: Epigenomic correction in established iPSCs (“naive-to-priming protocol”) For nuclear reprogramming experiments, primary adult human dermal fibroblasts (HDFa, Life Technologies) were grown in Medium 106 (Life Technologies) supplemented with LSGS (Gibco).

[0194] Fibroblasts at the early passage stage (<P6) were seeded at 50,000 - 70,000 cells per well in a 6-well plate and then transduced in fibroblast medium containing DMEM (Gibco), 10% FBS (Hyclone), 1% non-essential amino acids (Gibco), 1 mM GlutaMAX (Gibco), 1% Pen-strep (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), and 1 mM sodium pyruvate (Gibco).

[0195] After 48 hours, the cells in one well were trypsinized for counting to determine the volume of virus required for transduction (MOI). Transduction was performed using the CytoTune 2.0 iPSC Sendai Reprogramming Kit (Invitrogen), which consists of four transcription factors: OCT4, SOX2, cMYC, and KLF4.

[0196] After 24 hours, the virus was removed and the medium was changed every other day. Seven days after transduction, cells were harvested using TrypLE express (Life tech) and reseeded onto a layer of irradiated MEF feeders in fibroblast medium, then the medium was changed to naive medium (2iLGoY) the next day.

[0197] 16-18 days after transduction (i.e., 8-10 days in naive conditions), naive iPSCs were harvested using Accutase (Stem cell technologies) and passaged more than 10 times to obtain established naive iPSCs.

[0198] The naive phenotype of the established naive iPSCs was confirmed by flow cytometry and immunostaining for naive pluripotency-associated markers.

[0199] Naive iPSCs were then harvested using Accutase (Stem Cell Technologies) and reseeded on vitronectin (Life tech) coated plates in naive conditions. The next day, the medium was replaced with Essential 8 (Life tech) medium. When the cultures became confluent, the cells were harvested using 0.5 mM EDTA / PBS solution (Invitrogen) to maintain the vitronectin / E8 feeder-free human pluripotent stem cell culture system. The cells were cultured at 37°C, 5% O2, and 5% CO2.

[0200] Example 3: Epigenetic profiling of iPSCs at different stages of reprogramming Acquisition of abnormal DMR during reprogramming 1. Whole-genome bisulfite sequencing (by MethylC-seq) and DNA methylation analysis DNA methylation analysis was performed using FACS-isolated cells. Genomic DNA was isolated using the DNeasy Blood and Tissue Kit according to the manufacturer's instructions. Libraries were prepared by shearing genomic DNA to an average length of 200 bp in a Covaris S2 sonicator with 0.5% (w / w) unmethylated lambda phage DNA as described (Urich et al. (2015) Nature Protocols 10:3 p475-483). Fragments were end-repaired, A-tailed, and ligated to methylated Illumina TruSeq adapters or BIOO Nextflex adapters, followed by bisulfite conversion using the EZ DNA-Methylation Gold Kit (Zymo Research). Library fragments were then subjected to 4-9 cycles of PCR amplification with KAPA HiFi Uracil+ DNA polymerase (KAPA Biosystems). Sequencing was performed on an Illumina HiSeq 1500 or NovaSeq 6000. Sequencing adapters were trimmed with BBduk with options mink=3, qtrim=r, trimq=10 minlength=20, and then aligned to hg19 with Bowtie2 and BSseeker2 with option -n1 (Chen et al. (2010) BMC Bioinformatics 11 p203; Langmead et al. al.(2009)Genome Biology 10:3 R25). PCR duplicates were removed using Sambamba prior to methylation quantification with BSseeker2 (Chen et al.2010;Tarasov et al.(2015)Bioinformatics 31:2 p2032-3024). Read counts of CG dinucleotides were aggregated for both strands before downstream analysis. Differentially methylated regions (DMRs) were determined statistically using the DMRseq algorithm (Korthauer et al.(2018)Biostatistics 20:3 p367-383), and DMRs were considered significant if the methylation difference was >20% and the DMR P value was <0.05. PMD was determined using fibroblast methylation profiles and a hidden Markov model as implemented in MethylSeekR (Burger et al. (2013) Nucleic Acids Research 41:16 e155). All non-CG DNA methylation values ​​were corrected for non-conversion rates determined by spiked lambda phage DNA methylation levels.

[0201] The acquisition of aberrant DMRs during reprogramming was identified by first calling DMRs between primed iPSC and ESC lines, including four ESC replicates (32F passage 3 in KSR medium; 32F passage 10 in KSR medium; 32F passage 26 in E8 medium; 38F passage 20 in E8 medium) and five ESC replicates (H1 in mTeSR1 medium; The DMRs were then called between the primed iPSC lines and progenitor fibroblasts (two replicates; 1x32F fibroblasts and 1x38F fibroblasts). The DMRs were classified into two classes. The first class was designated as "memory DMRs" and were regions determined to be significantly different between iPSCs and ESCs, and did not overlap with the DMRs called between fibroblasts and iPSCs. In these regions, the methylation levels in iPSCs were more similar to fibroblasts than to ESCs. The second class of DMRs was designated as "iPSC DMRs" and were regions determined to be significantly different between iPSCs and ESCs, which also overlap with the DMRs determined to be significantly different between iPSCs and fibroblasts. In these regions, the methylation levels in iPSCs were different from both ESCs and progenitor fibroblasts.

[0202] Naive-to-primed reprogramming erases somatic memory and generates cells that more closely resemble ESCs With regard to DNA methylation, DMRs in naive-to-primed or TNT-primed iPSCs more closely resemble ESCs if the average methylation level in the region is less than 20% compared to ESC lines. With regard to gene and transposable element expression, the difference between naive-to-primed cells and ESC lines is less than two-fold.

[0203] RNA sequencing (RNA-seq) Using the RNeasy Micro Kit (Qiagen, Cat#74004) with the QIAcube (Qiagen), approximately 2–20 × 10 4 RNA extraction was performed from cells. RNA concentration was measured with Qubit RNA HS Assay Kit (ThermoFisher, Cat#Q32855) on a Qubit 2.0 Fluorometer (ThermoFisher). Approximately 25ng of RNA was used for library construction using the SPIA kit (NuGen) according to the manufacturer's instructions, followed by HiSeq. The genomes were sequenced by a 1500 or HiSeq 3000 sequencer (Illumina). Sequencing libraries were sequenced in single-end format with 50 nt reads and a target read number of 20-30M. RNA-seq reads were aligned to hg19 using HISAT2 (Kim et al., (2015) Nature Methods 12:4 357-360) and gene and transposable element quantification was performed using TEtranscripts using the parameters recommended for the human genome (Jin et al., (2015) Bioinformatics 31:22 p3593-3599). Differential expression of genes and transposable elements was calculated using edgeR (Robinson et al., (2010) Bioinformatics 26:1 139-140) with TMM normalization. Differential expression was defined as a gene or transposable element with a >2-fold expression difference and a false discovery rate <0.01.

[0204] Figure 3A shows a histogram of DNA methylation levels at TEs in naive-to-primed cells compared to ESCs and iPSCs generated according to conventional methods, and Figure 3B shows a dendrogram showing that TE expression indicates that naive-to-primed iPSCs more closely resemble ESCs than primed iPSCs.

[0205] Differentiation Scorecard Assessment Embryoid body differentiation was performed according to the manufacturer's instructions for the Scorecard assay (Invitrogen). Briefly, pluripotent cells were dissociated into cell clumps and then transferred to non-adherent dishes containing EB differentiation medium. EBs were harvested after 7 days, lysed, and total RNA was extracted with a QIAcube (Qiagen) / RNeasy micro kit (Qiagen) and reverse transcribed using a SuperScript III cDNA synthesis kit (Invitrogen). cDNA was then synthesized using TaqMan Gene Expression Master Mix (Thermo Fisheye r) into a 384-well scorecard plate (Invitrogen), and RT-PCR was performed using a 7900HT real-time PCR system (Thermo Fisher). The data obtained were analyzed using hPSC scorecard analysis software (Thermo Fisher).

[0206] The data shown in Figure 4 depicting the differentiation scorecard for embryoid bodies derived from primed and naïve-to-primed iPSCs indicates that naïve-to-primed iPSCs exhibit less differentiation bias towards the mesodermal lineage compared to primed iPSCs (fibroblasts are of mesodermal origin and are a somatic cell type prior to reprogramming to pluripotency).

[0207] Example 4: Differentiation of Naïve-to-Primed iPSCs Cortical neuron differentiation from MEL1-hESC-fibroblast-derived iPSCs Two days before differentiation (day 2), hiPSCs were cultured at low density (0.5–1 × 10 4 cells / cm 2) onto Matrigel (BD Biosciences)-coated culture vessels. On day 0, E8 medium was changed to DDM medium (DMEM / F12+GlutaMax) (Life Technologies) supplemented with 2% B27 (without vitamin A) (Life Technologies) and 100 ng / ml LDN (Stem Cell Technologies) and replenished every 2 days. The percentage of NCAM (Miltenyi Biotec) positive cells was assessed on days 8 and 16 by flow cytometry analysis.

[0208] Neural stem cell (NSC) generation from iPSCs iPSCs were cultured in E8 medium (Life Technologies) on Cultrex (R&D Systems) coated TC dishes and split 1:10 every 5 days. Colonies were mechanically disaggregated with 0.5 mM EDTA in PBS (Sigma). 10 μM ROCK inhibitor (Selleckchem) was added 24 h after splitting.

[0209] After splitting, colonies were collected by sedimentation, resuspended in E8 medium containing ROCK inhibitor, and cultured in petri dishes to form embryoid bodies (EBs) in suspension. After 24 hours, the medium was changed to Knockout DMEM (Life Technologies) containing 20% ​​Knockout Serum Replacement (Life Technologies), 1 mM β-mercaptoethanol (Sigma), 1% non-essential amino acids (NEAA, Life Technologies), 1% penicillin / streptomycin (Life Technologies), and 1% Glutamax (Life Technologies) supplemented with 10 μM SB-431542 (Selleckchem), 1 μM dorsomorphin (Selleckchem) for neural induction, and 3 μM CHIR 99021 (Cayman Chemical) and 0.5 μM PMA (Sigma). The medium was changed on day 3 to N2B27 medium (50% DMEM-F12 (Life Technologies), 50% Neurobasal (Life Technologies) with 1:200 N2 supplement (R&D Systems), 1:100 B27 supplement lacking vitamin A with 1% penicillin / streptomycin (Life Technologies) (Miltenyi Biotec), and 1% Glutamax (Life Technologies) supplemented with the same small molecule supplements). On day 4, SB -431542 and dorsomorphin were withdrawn, and 150 μM ascorbic acid (AA; Sigma) was added to the medium. On day 6, EBs were triturated into smaller fragments with a 1,000 μL pipette and plated onto Cultrex-coated 12-well plates at a density of approximately 10–15 per well in NSC growth medium (N2B27 with CHIR, PMA, and AA). After another 5 days, cells were split at a 1:5 ratio onto new Cultrex-coated wells using Trypsin-EDTA (Life Technologies) and Trypsin inhibitor (Sigma). After another 5 days, cells were harvested by trypsinization for 10 min at 37 °C to generate single-cell suspensions for the 10×3′ single-cell RNA-seq v3 workflow (10× Genomics).

[0210] Figure 5A shows images of NSCs from primed and naïve-to-primed iPSCs. These images show that differentiation of primed iPSCs into NSCs generates fibroblast-like cells in the spaces between NSC colonies. When naïve-to-primed iPSCs are differentiated into NSCs, the presence of fibroblast-like cells is significantly reduced.

[0211] Figure 6 shows flow cytometry results of MEL1-derived iPSCs generated according to the present invention (naive-to-primed and TNT-primed), MEL1-derived hESCs, and MEL1-derived iPSCs generated from primed iPSCs. The results show that the percentages of NCAM-positive cells at days 8 and 16 of cortical neuron differentiation from TNT-primed and naive-to-primed iPSCs more closely resemble those obtained from ESCs than from primed iPSCs.

[0212] Single-cell RNA-seq and hashtagging Single cell suspensions were counted using a hemocytometer and 200,000 cells per sample were used for incubation with hashtag antibodies. Cells were filtered through a 40 μm cell strainer, centrifuged at 800 g for 5 min, resuspended in a total volume of 46 μl cell staining buffer (2% BSA (Sigma), 0.01% Tween (Sigma)) in 1xDPBS (Life Technologies) containing 4 μl Fc blocking reagent (Biolegend) and incubated on ice for 10 min. Each sample was then given 0.2 μg of a different TotalSeq-A anti-human hashtag antibody (Biolegend) and incubated on ice for 30 min for antibody binding. After incubation, 1 ml of cell staining buffer was added and samples were centrifuged at 300 g for 3 min. The supernatant was removed and the wash was repeated a total of three times to remove all unbound antibodies. Cells were counted again and equal cell numbers were combined for each sample to obtain a suitable cell concentration for loading onto a 10x Chromium controller intended to represent 10,000 cells. The mixed cell suspension was filtered once more using a 40 μm cell strainer and processed for single-cell RNA-seq with 10x 3'v3 chemistry according to the manufacturer's instructions.

[0213] Libraries for single-cell RNAseq were made according to the standard workflow, while HTO libraries for hashtag information were generated as follows: HTO primers were added during the cDNA amplification step to allow amplification of HTO barcodes, and the supernatant from the first step of the post-cDNA amplification PCR cleanup was not discarded but used to prepare the HTO library. The HTO product was purified using 2x SPRI and amplified for 8 PCR cycles using 10x SI-PCR oligos and TruSeq Small RNA RPIx primers to generate libraries of approximately 180 bp size.

[0214] Single-cell RNA-seq (scRNA-seq) analysis Sequencing was performed on a NovaSeq 6000 sequencer, yielding approximately 420 million reads for the single-cell RNA-seq library and approximately 40 million reads for the HTO library. 0 million reads were generated. RNA-seq fastq files were processed using CellRanger count 3.1.0, while HTO fastq files were processed using CITE-seq-Count 1.4.3. Both datasets were loaded into Seurat 3.1 and combined by intersecting them based on cell barcodes. RNA data were log-normalized and variable features were detected by mean variance, while HTO data were normalized by centered log-ratio transformation. HTODemux was used to assign single cells to their sample origin and doublets and negatives were excluded from further analysis. The top 1000 most variable features were used for scaling and PCA of RNA data using 10 dimensions with a resolution of 0.6 for clustering and tSNE.

[0215] Cluster identity was defined based on expression of markers of mesoderm (BMP1, BMP4, HAND1, SNAI1, TGFB1, TGFB2), endoderm (CLDN6, FOXA1, NODAL), neural stem cells (NES, RBFOX3, PAX3, PAX6, SOX1, SOX2) or mitochondria (low UMI numbers and enriched for mitochondrial transcripts). The HTO identity of each cell can be used to define the percentage of cell identity within each sample used.

[0216] Data shown in Figure 5B indicate that both naïve-to-primed and TNT-primed cells exhibit substantially fewer fibroblast-like cells as characterized by marker gene expression.

[0217] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of which different combinations constitute various alternative aspects of the invention.

[0218] [Table 3-1]

[0219] [Table 3-2]

[0220] Table 4: Summarized CG DNA methylation levels for transposable elements (TEs) intersecting with the corrected CG-DMR in primed iPSCs (Primed), naive-to-primed iPSCs (NtoP), TNT-primed iPSCs (TNT), and ESCs. The genomic coordinates of each TE are listed, as well as the family, group (class) and individual TE type. Methylation level values ​​are calculated as mCG / CG (range: 0-1) of the whole TE for the listed coordinates for TEs with overlap with the corrected CG-DMR. (Table starts on the next page).

[0221] [Table 4-1]

[0222] [Table 4-2]

[0223] [Table 4-3]

[0224] [Table 5-1]

[0225]

Table 5-2

[0226]

Table 5-3

[0227]

Table 5-4

Claims

1. A method for producing induced pluripotent stem cells (iPSCs), comprising the steps of: (a) culturing said cells in first culture conditions adapted to promote reprogramming of said cells towards a pluripotent state; (b) culturing the cells in second culture conditions adapted to promote a hypomethylated DNA state, wherein culturing in the second culture conditions is for a period of time insufficient to allow the cells to achieve an established naive pluripotent state; (c) culturing the cells in a third culture condition adapted to promote a primed pluripotent state; in order to produce iPSCs from somatic cells.

2. 1. A method for reprogramming a somatic cell into a pluripotent cell, comprising the steps of: (a) increasing the protein expression or amount of one or more factors in the somatic cell, the factors being for reprogramming the somatic cell towards a pluripotent state; (b) culturing the cells in a first culture medium for a time and under conditions sufficient to allow said reprogramming of the cells towards a pluripotent state; (c) culturing the cells in a second medium adapted to induce a hypomethylated DNA state for a time and under conditions sufficient to reset the epigenetic profile of the cells; (d) culturing the cells in a third culture medium adapted to induce a primed pluripotent state for a time and under conditions sufficient to convert the cells to a primed pluripotent state; thereby reprogramming said somatic cell into a pluripotent cell.

3. 1. A method for reprogramming a somatic cell into a pluripotent cell, comprising the steps of: (a) increasing the protein expression or amount of one or more factors in said somatic cells, said factors for reprogramming said somatic cells towards a pluripotent state; (b) culturing the cells in a first culture medium for a time and under conditions sufficient to allow reprogramming of the cells towards a pluripotent state; (c) contacting the cells with a culture medium adapted to induce a hypomethylated DNA state and culturing the cells for a time and under conditions sufficient to allow reprogramming of the cells towards a hypomethylated DNA state; (d) contacting the cells with a priming medium and culturing the cells for a time and under conditions sufficient to allow reprogramming of the cells to a primed pluripotent state; thereby reprogramming said somatic cell into a pluripotent cell.

4. 4. The method of any one of claims 1 to 3, wherein the iPSCs have an epigenomic profile that is at least 75% similar, 80% similar, or 90% or more similar to an epigenomic profile of an embryonic stem cell, and optionally the epigenomic profile is determined by reference to non-CG methylation levels.

5. The method according to any one of claims 1 to 4, wherein the somatic cells are human somatic cells and the iPSCs produced by the method are human iPSCs.

6. 6. The method of any one of claims 1 to 5, wherein the culture time at which the cells begin reprogramming towards a pluripotent state is at least 1 day after contacting the somatic cells with the first culture medium or after increasing the protein expression or amount of the one or more factors.

7. 7. The method of claim 6, wherein the time is 2, 3, 4, 5, 6, 7 days or more after contacting the cells with the first culture medium or increasing the protein expression or amount of the one or more factors.

8. 8. The method of any one of claims 1 to 7, wherein the culture time during which the cells initiate reprogramming towards a pluripotent state can be any period that promotes a reduction in markers and characteristics associated with the somatic cells.

9. The method of any one of claims 1 to 8, wherein the second culture condition or second culture medium is any culture condition or medium adapted to promote a naive pluripotent state.

10. 10. The method of claim 9, wherein the medium comprises a MEK inhibitor, a PKC inhibitor, a GSK3 inhibitor, a STAT3 activator, human leukemia inhibitor factor (hLIF) and a ROCK inhibitor.

11. 10. The method of claim 9, wherein the medium is selected from the group consisting of T2iLGoY, 5iLAF, RSeT, PXGL and NHSM as described herein.

12. The method of any one of claims 1 to 11, wherein the third culture condition or third culture medium comprises any culture medium difference adapted to promote a primed pluripotent state.

13. 13. The method of claim 12, wherein the medium is selected from the group consisting of Essential 8, KSR / FGF2, mTeSR, AKIT, or B8 as described herein.

14. The method of any one of claims 1 to 13, wherein culturing the cells under the second culture conditions or in the second culture medium is for a period of not more than 21 days.

15. 15. The method of claim 14, wherein the period is 13 days or less, optionally 10 days or less, or 5 days or less.

16. 16. The method of any one of claims 2 to 15, wherein culturing the cells in the second culture medium is performed for a period and under conditions that are not sufficient to achieve a naive pluripotent state of the cells, and step d) is performed before the cells have completed reprogramming and before the cells have achieved a naive pluripotent state.

17. 16. The method according to any one of claims 2 to 15, wherein culturing the cells in the second culture medium is performed for a period and under conditions to achieve a naive pluripotent state of the cells, and step d) is performed after the cells have achieved a naive pluripotent state.

18. 18. The method of any one of claims 1 to 17, wherein the naive pluripotent state comprises a cell phenotype that comprises rounded, dome-shaped cells.

19. The method of any one of claims 1 to 18, wherein the primed pluripotent state comprises a cell phenotype characterized by the presence of flat cell colonies.

20. 20. The method of any one of claims 1 to 19, wherein the naive pluripotent state further comprises expression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, nine, twenty, twenty-one, twenty-two, twenty-three or all markers selected from KLF2, KLF4, TFCP2L1, TBX3, REX1, GBX2, STELLA (DPPA3), KLF17, DPPA5, TFCP2L1, MAEL, UTF1, ZFP57, DNMT3L, FGF4, FOXR1, ARGF, TRIM60, DDX43, BRDT, ALPPL2, KHDC3L, KHDC1L and PRAP1.

21. 21. The method of any one of claims 1 to 20, wherein the primed pluripotent state further comprises expression of one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or all of the markers selected from SFP, EOMES, BRACHYURY, OTX2, ZIC2, ZIC3, ZIC5, DNMT3B, KDR, CDH2, CER1, COL2A1, DAZL, TCF7L1, SOX11 and SALL2.

22. 22. The method of any one of claims 1-14 or 16-21, wherein the cells are cultured in the second culture conditions or second culture medium for a period of 0.5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days prior to contacting with the third culture medium or third culture conditions.

23. 23. The method of any one of claims 1 to 22, wherein the cells are cultured in the third culture medium or third culture conditions for at least about 0.5 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days.

24. 24. The method of any one of claims 1 to 23, wherein step a) comprises contacting said cells with an agent for increasing the expression or amount of one or more of said factors for reprogramming said somatic cells towards a pluripotent state.

25. 25. The method of claim 24, wherein the agent is selected from the group consisting of nucleotide sequences, proteins, aptamers and small molecules, ribosomes, RNAi agents and peptide nucleic acids (PNAs) and analogs or modifications thereof.

26. 26. The method of claim 25, wherein the agent is a nucleic acid, and the nucleic acid is provided to contact the cell in the form of a vector, preferably the vector is a viral vector.

27. The method according to any one of claims 24 to 26, wherein the factor is selected from the following transcription factors: OCT4, SOX2, KLF4 and MYC in their variants.

28. The method according to any one of claims 24 to 27, wherein the factor comprises all four of the transcription factors OCT4, SOX2, KLF4 and MYC (OSKM), or variants thereof.

29. 29. The method of claim 27 or 28, wherein the transcription factors further comprise factors LIN28 and / or NANOG.

30. 30. An induced pluripotent stem cell (iPSC) or isolated iPSC obtained or obtainable by a method according to any one of claims 1 to 29.

31. Obtained or obtainable according to the method of any one of claims 1 to 29 30. A population of cells comprising induced pluripotent stem cells (iPSCs), preferably wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in said population are iPSCs, and are obtained or obtainable by the method of any one of claims 1 to 29.

32. A differentiated cell or isolated differentiated cell, primordial germ cell or gamete derived from an iPSC obtained or obtainable according to the method of any one of claims 1 to 29.

33. 30. A population of differentiated cells, primordial germ cells or gametes derived from iPSCs obtained or obtainable according to the method of any one of claims 1 to 29, preferably wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cells in said population are differentiated cells, and differentiated cells derived from iPSCs obtained or obtainable according to the method of any one of claims 1 to 29.

34. 34. An organoid or organized collection of cells derived from a population of iPSCs or a population of differentiated cells according to claim 32 or 33.

35. - iPSCs or isolated iPSCs according to claim 30, A population of iPSCs according to claim 31 , - A differentiated cell or an isolated differentiated cell according to claim 32, - a population of differentiated cells according to claim 33, or - The organoid or organized cell collection or part thereof according to claim 34, and a pharma- ceutically acceptable excipient.

13. A pharmaceutical composition comprising:

36. 1. A method of treating a disease or condition requiring administration of iPSCs or a cell population to a subject in need thereof, comprising: - iPSCs or isolated iPSCs according to claim 30, A population of iPSCs according to claim 31 , - A differentiated cell or an isolated differentiated cell according to claim 32, A population of differentiated cells according to claim 33, - a collection of organoids or organized cells according to claim 34 or a part thereof; or The pharmaceutical composition according to claim 35. The method of claim 1, further comprising administering

37. 35. The iPSC or isolated iPSC of claim 30, the population of iPSCs of claim 31, the differentiated cell or isolated differentiated cell of claim 32, the population of differentiated cells of claim 33, the organoid or organized cell collection or part thereof of claim 34, or the pharmaceutical composition of claim 35, for use in the treatment of a disease or condition requiring administration of iPSCs or a cell population.

38. A kit for producing iPSCs according to any one of claims 1 to 29, optionally comprising reagents for culturing somatic cells and instructions for carrying out the method according to any one of claims 1 to 29.

Images