A method for reprogramming somatic cells to a different cell fate or primitive cell state.

Abstract

Induction of overexpression of defined exogenous transcription factors (TFs) or treatment with specific pathway-modulating cocktails can reprogram somatic cells to pluripotency or another cell state. A barrier to initiating reprogramming lies in the molecular identity of the starting cell, which is enforced by lineage-directing TFs. However, it remains unclear whether suppression of somatic lineage-defining TFs in the starting cell in the absence of such exogenous TFs is sufficient to induce cellular reprogramming. Using an intraspecific somatic cell hybrid model, we identify SNAI2 and PRRX1 as the most important determinants of mesenchymal commitment in rat fetal fibroblasts (REFs) and demonstrate that siRNA-mediated transient knockdown of these individual factors is sufficient to convert REFs into functional adipocytes, chondrocytes, or osteocytes without the need for exogenous TFs. In addition, siRNA-mediated transient knockdown of SNAI2 alone is sufficient to transform REFs into a dedifferentiated pluripotent stem-like cell (dPSC) state that forms embryoid bodies in the absence of exogenous TFs, demonstrating that triple germ layer differentiation is possible. These results establish for the first time that transient inhibition of a single somatic lineage-specifying TF can effectively induce transdifferentiation to another somatic state or dedifferentiation to dPSCs in the absence of exogenous TFs or small molecule cocktails.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application No. 62 / 877,798, entitled "Methods for Reprogramming Somatic Cells to Another Cell Fate or Primitive Cell State," filed on July 23, 2019. The entire content of this provisional patent application is incorporated as part of the disclosure of this application.

Background Art

[0002] In 2006, researchers Takahashi and Yamanaka revolutionized stem cell research by showing that the forced expression of just four transcription factors (Oct4, Sox2, Klf4, and c - Myc (OSKM)) was sufficient to convert fibroblasts into embryonic stem cell (ESC) - like cells, named induced pluripotent stem cells (iPSCs). Induced pluripotent stem cells (iPSCs) can be generated in vitro, can differentiate into any cell type, are patient - specific, and thus can solve the problems of tissue compatibility and rejection that have hindered the success of cell transplantation and tissue transplantation. Therefore, they have great potential for pharmaceutical development, disease modeling, and regenerative medicine. Furthermore, since iPSCs are induced from somatic cells rather than developing germ cells, unlike ESCs, there are no ethical concerns.

[0003] However, for these applications, the generated iPSCs must be pure, homogeneous, and chromosomally stable, and must be produced in considerable yield and purity. The use of viral vectors or oncogenes such as Myc as factors for OSKM-mediated cell reprogramming has raised reasonable concerns regarding the safe use of generated iPSC cells in clinical settings. Currently, most iPSCs are produced by either ectopic expression of reprogramming factors delivered via viral vectors, or by non-viral methods such as transfection with messenger RNA (mRNA) instead of DNA (Warren et al. 2010) or treatment with small molecule cocktails / chemical reprogramming (Huangfu et al. 2008, Xu et al. 2008, Ichida et al. 2009).

[0004] The main drawback of TF-mediated and chemical reprogramming is its extremely low reprogramming efficiency. Previous studies using the OSKM factor demonstrated that the average reprogramming efficiency of somatic cells in colony-forming pluripotent stem cells is approximately 0.2%–0.8% (Anokye-Danso et al., 2011). In recent years, the field of cell reprogramming has shifted towards non-viral chemical / small molecule-based reprogramming strategies that reprogram somatic cells to become pluripotent by treatment with small molecule cocktails that modulate pathways. In 2013, Hou et al. were the first to demonstrate successful reprogramming of mouse cells into iPS cells using a combination of seven small molecules: VPA (HDAC inhibitor), CHIR99021 (GSK3 inhibitor), RepSox E616452 (ALK5 inhibitor of the TGFβ pathway), tranylcypromine (lysine-specific demethylase inhibitor), forskolin (cAMP signaling activator), 3-deazanepranosin A DZNep (comprehensive histone methylation inhibitor), and TTNPB (activates the retinoic acid receptor RAR). Despite being pioneering, the yield of iPSCs generated using these small molecules was still very low (approximately 0.2% efficiency).

[0005] Due to current limitations in this field, it is desirable to develop methods for generating reprogrammable somatic cells with high efficiency without requiring ectopic expression of reprogramming factors or other cellular reprogramming pathway modulators. [Disclosure of the Invention]

[0006] In some embodiments, somatic fibroblasts are provided that are capable of transdifferentiating into somatic cells of another lineage without viral or nonviral delivery of exogenous transcription factors and / or small molecule modulators, wherein the somatic fibroblasts contain an siRNA or shRNA molecule that inhibits the expression of a single lineage-defining transcription factor. In some aspects, the siRNA or shRNA molecule inhibits SNAI2, PRRX1, HAND1, CDX2, or a combination thereof. In some aspects, the somatic cells of another lineage are somatic cells of an adipocyte lineage, somatic cells of an osteogenic cell lineage, or somatic cells of a chondrogenic cell lineage.

[0007] In another embodiment, somatic fibroblasts are provided that can dedifferentiate into multipotent or pluripotent stem cells without viral or nonviral delivery of exogenous transcription factors and / or small molecule modulators, wherein the somatic fibroblasts are treated with an siRNA or shRNA molecule that inhibits the expression of a single lineage-defining transcription factor. In some aspects, the siRNA or shRNA molecule inhibits SNAI2, PRRX1, HAND1, CDX2, or a combination thereof. In some aspects, the multipotent or pluripotent stem cells are dedifferentiated multipotent mesenchymal stem cells or dedifferentiated pluripotent stem cells.

[0008] In some embodiments, a population of transdifferentiated somatic cells is provided. The population of transdifferentiated somatic cells is generated from a population of lineage-constrained fibroblasts with an efficiency of greater than 1%. In some aspects, the population of transdifferentiated somatic cells is a population of adipocyte lineage somatic cells, a population of osteogenic cell lineage somatic cells, or a population of chondrogenic cell lineage somatic cells. In other aspects, the population of transdifferentiated somatic cells can be used in tissue reconstruction procedures. For example, the population of transdifferentiated somatic cells can be used in reconstructive plastic surgery or reconstructive orthopedic surgery.

[0009] In other embodiments, a population of dedifferentiated stem cells is provided. This population of dedifferentiated stem cells includes a population of reprogrammed fibroblasts without permanent genetic modification, which are induced from a population of adult fibroblasts into a population of dedifferentiated stem cells with an efficiency of greater than 1%. In some aspects, the population of dedifferentiated stem cells is a population of dedifferentiated multipotent mesenchymal stem cells or a population of dedifferentiated pluripotent stem cells. In other aspects, the population of dedifferentiated stem cells can be used in tissue reconstitution procedures, wound treatment, or transplantation surgery.

[0010] In certain embodiments, a cell culture system is provided for producing a population of transdifferentiated somatic cells. The cell culture system may comprise a population of lineage-constrained somatic cells, an siRNA or shRNA molecule that inhibits the expression of a single lineage-defining transcription factor, and a culture medium specific to the population of transdifferentiated somatic cells, and the cell culture system does not include exogenous transcription factors and / or small molecule modulators (chemical reprogramming). In certain aspects, the population of lineage-constrained somatic cells in the cell culture system is fibroblasts. In certain aspects, the single lineage-defining transcription factor is SNAI2, PRRX1, HAND1, CDX2, or a combination thereof. In certain aspects, the population of transdifferentiated somatic cells is a population of adipocyte lineage, osteogenic cell lineage, or chondrogenic cell lineage.

[0011] In another embodiment, a cell culture system for producing a population of dedifferentiated stem cells is provided. The cell culture system may comprise a population of lineage-constrained somatic cells, an siRNA or shRNA molecule that inhibits the expression of a single lineage-defining transcription factor, and a culture medium comprising a stem cell medium specific to the population of dedifferentiated stem cells, and the cell culture system does not comprise exogenous transcription factors and / or small molecule modulators. In a particular aspect, the population of somatic cells in the cell culture system is fibroblasts. In a particular aspect, the single lineage-defining transcription factor is SNAI2, PRRX1, HAND1, CDX2, or a combination thereof. In a particular aspect, the population of dedifferentiated stem cells is a population of dedifferentiated multipotent mesenchymal stem cells or a population of dedifferentiated pluripotent stem cells.

[0012] In another embodiment, a method for producing transdifferentiated somatic cells is provided. The method may include the steps of introducing an siRNA or shRNA molecule that transiently inhibits the expression of a single lineage-defining transcription factor of lineage-constrained somatic cells; and incubating the somatic cells in a culture medium specific to transdifferentiated somatic cells, wherein the method for producing transdifferentiated somatic cells is carried out without the delivery of exogenous transcription factors and / or small molecule modulators (chemical reprogramming) with or without viral delivery. In a particular aspect, the population of somatic cells in the cell culture system is lineage-constrained fibroblasts. In a particular aspect, the single lineage-defining transcription factor is SNAI2, PRRX1, HAND1, CDX2, or a combination thereof. In a particular aspect, the transdifferentiated somatic cells are cells of an adipocyte lineage, an osteogenic cell lineage, or an chondrogenic cell lineage.

[0013] In another embodiment, a method for producing dedifferentiated stem cells is provided. The method may include the steps of introducing an siRNA or shRNA molecule that transiently inhibits the expression of a single lineage-defining transcription factor of lineage-constrained somatic cells; and incubating the siRNA / shRNA-treated cells in a culture medium specific to dedifferentiated stem cells (mesenchymal stem cells or embryonic stem cells), wherein the method for producing dedifferentiated stem cells is carried out without the delivery of exogenous transcription factors and / or small molecule modulators (chemical reprogramming) with or without viral delivery. In a particular aspect, the lineage-constrained population of somatic cells in the cell culture system is fibroblasts. In a particular aspect, the single lineage-defining transcription factor is SNAI2, PRRX1, HAND1, CDX2, or a combination thereof. In a particular aspect, the dedifferentiated stem cells are dedifferentiated multipotent mesenchymal stem cells or dedifferentiated pluripotent stem cells.

[0014] In some embodiments, autologous tissue grafts are provided. The autologous tissue grafts comprise a population of transdifferentiated somatic cells, (i) the population of transdifferentiated somatic cells is derived from a population of somatic cells obtained from a subject for use in a tissue reconstitution procedure, and (ii) the population of transdifferentiated somatic cells is generated from a population of siRNA-treated fibroblasts without permanent genetic modification (as described in paragraph

[0011] ), the population of reprogrammed fibroblasts being induced to become a population of transdifferentiated somatic cells with an efficiency of greater than 1%. In some aspects, the population of transdifferentiated somatic cells is produced using the method described in the above embodiments.

[0015] In another embodiment, an autologous tissue graft is provided. The autologous tissue graft comprises a population of dedifferentiated stem cells, (i) the population of dedifferentiated stem cells is derived from a population of somatic cells obtained from a subject for use in tissue reconstruction procedures, wound treatment, or transplant surgery, and (ii) the population of dedifferentiated stem cells is generated from a population of reprogrammed fibroblasts without permanent genetic modification (as described in paragraph

[0012] ), the population of reprogrammed fibroblasts being induced to become a population of dedifferentiated stem cells with an efficiency of greater than 1%. In some aspects, the population of dedifferentiated stem cells is produced using the method described in the above embodiment.

[0016] In some embodiments, methods are provided for treating conditions in a subject. These methods may include the step of grafting or transplanting a population of reprogrammed somatic cells, dedifferentiated stem cells, or transdifferentiated somatic cells into or onto the surface of a tissue or organ of the subject. Examples of cells and conditions that can be treated include, but are not limited to, those described throughout this disclosure. In some embodiments, the population of reprogrammed somatic cells, dedifferentiated stem cells, or transdifferentiated somatic cells used in the method for treating a condition is part of an autologous tissue graft.

[0017] This application includes at least one drawing produced in color. A copy of this application, including the color drawing, will be provided by the Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0018] [Figure 1]Figures 1A-D illustrate the identification of core transcription factors that define the identity of mesenchymal fibroblasts. Figure A shows a Venn diagram representation of a two-step genetic information science strategy for identifying core transcription factors that contribute to the identity of mesenchymal fibroblasts. By selecting transcription factors (TFs) that showed more than 5-fold expression in mesenchymal-derived rat embryonic fibroblasts (REF) compared to endodermal-derived rat hepatocarcinoma cells (RH), 149 TFs were grouped as mesenchymal fibroblast-enriched TFs (MFEFs). Of these 149 MFEFs, 86 TFs that showed more than 2.5-fold suppression in hepatocarcinoma cell-fibroblast fusion nuclear cell hybrids (HF) of the same species compared to REF were identified as mesenchymal fibroblast-specific TFs (MFSFs). Figure B shows gene expression microarray heatmaps of REF, RH, and HF cell lines, showing all genes that showed more than 5-fold overexpression in REF compared to RH cells. The TF gene members were selected as final candidates for MFEFs. C shows gene expression microarray heatmaps of HF and REF cell lines, showing all genes that showed more than 2.5-fold repression in HF compared to REF. The TF gene members were identified as MFSFs. D is a table showing the top 10 MFEFs and MFSFs. Note that the ranking of the top MFEFs changed in the core MFSFs after applying a two-step selection strategy. [Figure 2A] Figures 2A–2E illustrate the effects of engineered re-expression of repressed fibroblast-specific transcription factors (TFs) in somatic cell hybrids. Figure 2A is a schematic diagram depicting morphological transformation by cell fusion between endodermal-derived rat hepatocellular carcinoma (RH) cells and mesenchymal-derived rat embryonic fibroblast (REF) cells, followed by transformation of HF cells by engineered overexpression of PRRX1 and SNAI2, respectively, to generate stable, intraspecific hepatocellular carcinoma cell-fibroblast (HF) fusion nuclear hybrid cells. [Figure 2B]Figure 2B shows a series of photographs as indicated. Starting from the top row, the first row of Figure 2B is a series of bright-field micrographs showing the round morphology of RH clusters, the spindle-shaped morphology of REF cells, and the hybrid morphology of HF cells. Reacquisition of the elongated spindle morphology in G418-resistant PRRX1-overexpressing HF clones (HF-PRRX1) and G418-resistant SNAI2-overexpressing HF clones (HF-SNAI2). (Scale bar, 10 μm). The second row of Figure 2B is a series of immunofluorescence staining images of the prototype fibroblast-specific marker Collagen 1a1 (Col1a1) in rat hepatocellular carcinoma cells (RH), rat embryonic fibroblasts (REF), rat hepatocellular carcinoma cell-fibroblast hybrid (HF) cells, G418-resistant PRRX1-overexpressing HF clones (HF-PRRX1), and G418-resistant SNAI2-overexpressing HF clones (HF-SNAI2). Col1a1 expression is significantly higher in (REF) cells compared to RH and HF cells. In HF cells, Col1a1 expression is significantly increased upon manipulated re-expression of SNAI2 and PRRX1 (Col1a1 stained red and DAPI stained blue) (scale bar, 10 μm). The third row of Figure 2B shows a series of immunofluorescence staining images illustrating PRRX1 expression in RH cells, REF cells, HF cells, HF-PRRX1 cells, and HF-SNAI2 cells (scale bar, 10 μm). The fourth row of Figure 2B shows a series of immunofluorescence staining images illustrating SNAI2 expression in RH cells, REF cells, HF cells, HF-PRRX1 cells, and HF-SNAI2 cells (SNAI2 is stained green and DAPI is stained blue) (scale bar, 10 μm). [Figure 2C] Figure 2C is a series of bar graphs showing the repression ratio of PRRX1, SNAI2, and Col1a1 in HF hybrids compared to REF validated by qRT-PCR (top), the reconstruction of prototype mesenchymal fibroblast TFs of the repressed fibroblast-specific TFs PRRX1 and SNAI2, and gene expression during manipulated re-expression (middle and bottom, respectively). [Figure 2D]Figure 2D is a bar graph illustrating the TGFβ-responsive cell migration ability of different cell lines. (REF) cells showed the highest migration ability in response to TGFβ stimulation compared to (RH) hepatocellular carcinoma or (HF) hybrid cells, and G418-resistant PRRX1-overexpressing HF clones (HF-PRRX1) and G418-resistant SNAI2-overexpressing HF clones (HF-SNAI2) showed remarkable reacquisition of TGFβ-responsive cell migration ability, a functional trait specific to the prototype fibroblasts. [Figure 2E] Figure 2E shows a schematic diagram illustrating the transdifferentiation of REF cells into adipocytes, osteocytes, and chondrocytes, and their dedifferentiation into mesenchymal stem cells, in individual treatments with siSnai2 or siPRRX1. dPSCs were generated only in the siSnai2 group. [Figure 3A] Figures 3A–3J show that siRNA-mediated transient repression of SNAI2 or PRRX1 in rat embryonic fibroblasts (REFs) induces ex vivo transdifferentiation of adipocytes, osteocytes, or chondrocytes. Figure 3A shows a series of images as shown. Starting from the top row, the left column of the first row in Figure 3A shows bright-field micrographs of REFs transfected with siCntrl and incubated in adipogenic medium for 14 days. The left column of the second row in Figure 3A shows bright-field micrographs of REFs transfected with siPRRX1 and incubated in adipogenic medium for 14 days. The left column of the third row in Figure 3A shows bright-field micrographs of REFs transfected with siSnai2 and incubated in adipogenic medium for 14 days. In each row, adjacent IF micrographs evaluate the expression of the adipogenic TF Cepba in red and the DAPI nuclear counterstaining in blue in all three groups (siCntrlREF, siPRRX1REF, and siSnai2REF) (scale bar, 10 μm). The siSnai2REF group showed the highest adipogenesis, with lipid droplet accumulation filling vacuoles, as confirmed by oil red O staining. [Figure 3B]Figure 3B shows a series of images as indicated. Starting from the top row, the left column of the first row in Figure 3A shows bright-field micrographs of REF transfected with siCntrl and incubated in osteogenic medium for 14 days. The left column of the second row in Figure 3B shows bright-field micrographs of REF transfected with siPRRX1 and incubated in osteogenic induction medium for 14 days. The left column of the third row in Figure 3B shows bright-field micrographs of REF transfected with siSnai2 and incubated in osteogenic medium for 14 days. Adjacent IF micrographs in each row evaluate the expression of the osteogenic TF Runx2 in all three groups (siCntrlREF, siPRRX1REF, and siSnai2REF) (in red, with DAPI nuclear counterstaining in blue). (Scale bar, 10 μm). The siPRRX1REF group showed the highest levels of osteogenicity and calcified bone deposition, as confirmed by alizarin red staining. [Figure 3C] Figure 3C shows a series of images as indicated. Starting from the top row, the left column of the first row in Figure 3C shows bright-field micrographs of REF cells transfected with siCntrl for 14 days in chondroplastic medium. The left column of the second row in Figure 3C shows bright-field micrographs of REF cells transfected with siPRRX1 for 14 days in chondroplastic medium. The left column of the third row in Figure 3C shows bright-field micrographs of REF cells transfected with siSnai2 and then incubated in chondroplastic medium for 14 days. Adjacent IF micrographs in each row evaluate the expression of the chondroplastic TF Sox9 in red and DAPI nuclear counterstaining in blue in all three groups (siCntrlREF, siPRRX1REF, and siSnai2REF). (Scale bar, 10 μm). siSnai2REF cells showed the highest levels of chondrogenesis and aggrecan formation, as confirmed by Alcian blue staining. [Figure 3D] Figure 3D is a bar graph showing the percentage intensity of oil red O staining to confirm lipogenesis, as quantified by ImageJ software analysis. [Figure 3E]Figure 3E is a bar graph showing the % intensity of alizarin red staining for confirmation of osteogenesis when quantified by Image J software analysis. [Figure 3F] Figure 3F is a bar graph showing the % intensity of alcian blue staining for confirmation of chondrogenesis when quantified by Image J software analysis. [Figure 3G] Figure 3G is a series of bar graphs showing the results of qRT-PCR analysis, which shows the fold increase in the expression of the mRNA of Cebpa, an adipocyte-specific TF, in siPRRX1 and siSnai2 REF incubated in adipogenic medium for 14 days, represented as a bar graph. The expression of each target gene was calculated as the relative expression to the housekeeping gene peptidylprolyl isomerase (Ppia) and represented as the induction fold relative to the control REF cells treated with siCntrl. The data are presented as the mean ± standard deviation (SD) of three independent experiments. [Figure 3H] Figure 3H is a series of bar graphs showing the results of qRT-PCR analysis, which shows the fold increase in the expression of the mRNA of Runx2, an osteocyte-specific TF, in REF treated with siPRRX1 and siSnai2 and then incubated in osteogenic medium for 14 days (left). The expression of each target gene was calculated as the relative expression to the housekeeping gene secreted phosphor protein 1 (Spp1) and represented as the induction fold relative to the control cells treated with siCntrl. The data are presented as the mean ± SD of three independent experiments. [Figure 3I]Figure 3I is a series of bar graphs showing the results of qRT-PCR analysis, which shows the fold increase in the expression of mRNA of Sox9, a chondrocyte-specific TF, in REF cells treated with siPRRX1 and siSnai2 and incubated in chondrogenic medium for 14 days (left). The expression of each target gene was calculated as the relative expression to peptidylprolyl isomerase (Ppia), a housekeeping gene, and represented as the induction fold relative to control cells treated with siCntrl. Data are represented as the mean ± SD of three independent experiments. [Figure 3J] Figure 3J is a schematic diagram illustrating the process of siRNA-mediated cell reprogramming in the absence of exogenous factors. [Figure 4A] Figure 4 shows the dedifferentiation of rat fetal fibroblasts (REF) into dMSCs and dPSCs by transient suppression of SNAI2 and PRRX1. Figure 4A is a series of bright-field microscope photographs of REF cells transfected with siCntrl (row 1), siPrrxi (row 2), and siSnai2 (row 3). Incubation of siPRRX1 REF and siSnai2 REF for 14 days in mesenchymal stem cell medium resulted in the generation of dedifferentiated mesenchymal stem-like cells (dMSCs). The adjacent IF microscope photographs evaluate the expression of Myc, a mesenchymal stem cell-specific TF, in red and DAPI nuclear counterstaining in blue in the three groups (scale bar, 10 μm). [Figure 4B] Figure 4B is a bar graph showing MSC generation confirmed using an alkaline phosphatase (ALP) assay. siSnai2 REF cells showed the highest mesenchymal stem cell activity. [Figure 4C] Figure 4C is a series of bar graphs showing the results of real-time qPCR analysis of the steady-state gene expression of MSC / dMSC genes. The expression of Myc, a characteristic MSC TF, was calculated as the relative expression to β-2-microglobulin (B2M) and represented as the induction fold relative to cells transfected with siCntrl. Data are represented as the mean ± SD of three independent experiments. [Figure 4D]Figure 4D shows bright-field micrographs of a series of REF cells transfected with siCntrl (row 1), siPrrxi (row 2), and siSnai2 (row 3). REF cells underwent 14-day incubation in rat ESC medium, resulting in the generation of dedifferentiated pluripotent stem-like cells (dPSCs). Bright-field micrographs show the formation of suspended embryoid bodies when dPSCs were suspended in differentiation medium for 8 days in low-adhesion plates. Only REF cells transfected with siSnai2 underwent effective nuclear reprogramming to dPSC cells, as demonstrated by their ability to form embryoid bodies in their suspension culture (row 3). Adjacent IF micrographs evaluate the expression of the ESC TFs Sox2 and Nanog in red and DAPI nuclear counterstaining in blue for all three groups (scale bar, 10 μm). [Figure 4E] Figure 4E is a series of bar graphs showing the results of qRTPCR analysis of the steady-state gene expression of characteristic ESC TFs, Sox2 and Nanog, where Klf4 is calculated as relative expression to β-2-microglobulin (B2M) and siCntrl is expressed as the induction factor relative to transfected cells. Only REF cells transfected with siSnai2 showed expression of the pluripotent TFs Sox2 and Nanog. Data are expressed as the mean ± SD of three independent experiments. [Figure 4F] Figure 4F shows a series of micrographs of 8-day-old embryoid bodies derived from dPSCs, immunostained for ectoderm TF. Ectoderm-differentiated cells were stained simultaneously with DAPI (blue) nuclear counterstaining using Northern Lights® (NL) 557 conjugate OTX-2 (red) and NL493 conjugate SOX2 (green). [Figure 4G] Figure 4G shows a series of micrographs of 8-day-old embryoid bodies derived from dPSCs, immunostained for mesoderm TFs. Mesoderm-differentiated cells were stained simultaneously with DAPI (blue) nuclear counterstaining using NL557 conjugate Brachyury (green) and NL637 conjugate Hand1 (red). [Figure 4H]Figure 4H shows a series of micrographs of 8-day-old embryoid bodies derived from dPSCs, immunostained for endodermal TF. Endoderm-differentiated cells were simultaneously stained with NL637 conjugate SOX17 (red) and NL493 conjugate Gata-4 (green). All nuclei were stained with DAPI (blue). [Figure 5] Figure 5 is a flowchart showing the generation of somatic cell hybrids. [Figure 6] Figure 6 shows a model for generating somatic cell hybrids. The cell types shown were fused using polyethylene glycol, and hybrids (FR) were selected using a medium that allowed only hybrid cells to survive. [Figure 7A] Figure 7 shows a series of data illustrating a comparative analysis of the degree of co-localization of SNAI2, HAND1, and CDX2 binding to MFSG activity enhancers and ESCG repressors at the genome level in human mesoderm. Figure 7A is a Venn diagram representation of the overlap of activity enhancers (defined by co-localization of H3K4Me1 and H3K27Ac histone marks), SNAI2, HAND1, and CDX2 genomic targets co-localizing with the defined activity enhancers, and MFSG activity enhancer targets in human mesoderm. [Figure 7B] Figure 7B is a bar graph showing the combined and individual percentages of colocalization of SNAI2, HAND1, and CDX2 genomic targets with MFSG activity enhancer targets in human mesoderm. [Figure 7C] Figure 7C is a Venn diagram representation of the overlap between activity enhancers (defined by the co-localization of H3K4Me1 and H3K27Ac histone marks) in human mesoderm, SNAI2, HAND1, and CDX2 genomic targets that co-localize with the defined activity enhancers, and MFSF activity enhancer targets. [Figure 7D] Figure 7D is a bar graph showing the combined and individual percentages of colocalization of SNAI2, HAND1, and CDX2 genomic targets with MFSF activity enhancer targets in human mesoderm. [Figure 7E]Figure 7E is a Venn diagram representation of the overlap between repressive enhancers (defined by the co-localization of H3K4Me1 and H3K27me3 histone marks) in human mesoderm, SNAI2, HAND1, and CDX2 genomic targets that co-localize with the defined repressive enhancers, and ESCG activity repressive enhancer targets. [Figure 7F] Figure 7F is a bar graph showing the combined and individual percentages of colocalization of SNAI2, HAND1, and CDX2 genomic targets with ESCG repression enhancer targets in human mesoderm. [Figure 7G] Figure 7G is a Venn diagram representation of the overlap between repressive enhancers (defined by the co-localization of H3K4Me1 and H3K27me3 histone marks) in human mesoderm, SNAI2, HAND1, and CDX2 genomic targets that co-localize with the defined repressive enhancers, and ESCF activity repressive enhancer targets. [Figure 7H] Figure 7H is a bar graph showing the combined and individual percentages of colocalization of SNAI2, HAND1, and CDX2 genomic targets with ESCF repression enhancer targets in human mesoderm. [Figure 8] Figure 8 shows the consistent shifts in enhancer chromatin modifications that occur at SNAI2 activity enhancer targets during mesodermal lineage progression. Figure 8A shows the H3K4me1, H3K27ac, and H3K27me3 ChIP-Seq tracks and matched RNA-Seq tracks at the prototype SNAI2-binding activity enhancer MFSG target PRRX1 across a range of mesodermal lineage progression in 21 different human cells and tissues from ESCs to differentiated adult fibroblasts, osteoblasts, chondrocytes, and adipocytes. Figure 8B shows the H3K4me1, H3K27ac, and H3K27me3 ChIP-Seq tracks and matched RNA-Seq tracks at the prototype SNAI2-binding activity enhancer MFSG target FOXF1 across a range of mesodermal lineage progression in 21 different human cells and tissues from ESCs to differentiated adult fibroblasts, osteoblasts, chondrocytes, and adipocytes. [Figure 9]Figure 9 shows the consistent shifts in enhancer chromatin modifications that occur at SNAI2 repressive enhancer targets during mesodermal lineage progression. Figure 9A shows the H3K4me1, H3K27ac, and H3K27me3 ChIP-Seq tracks and matched RNA-Seq tracks at the prototype SNAI2-binding repressive enhancer ESCG target SOX2 across the range of mesodermal lineage progression in 21 different human cells and tissues from ESCs to differentiated adult fibroblasts, osteoblasts, chondrocytes, and adipocytes. Figure 9B shows the H3K4me1, H3K27ac, and H3K27me3 ChIP-Seq tracks and matched RNA-Seq tracks at the prototype SNAI2-binding repressive enhancer ESCG target ZIC2 across the range of mesodermal lineage progression in 21 different human cells and tissues from ESCs to differentiated adult fibroblasts, osteoblasts, chondrocytes, and adipocytes. [Figure 10A] Figure 10 shows a series of data demonstrating that SNAI2 enhancer binding in human mesoderm modulates the transcriptional expression of MFSG activity enhancer targets and the repression of ESCG repression enhancer targets. The data shown in Figure 10 were visualized using the Washington University Genome Browser. Figure 10A is a Venn diagram representation of the overlap between activity enhancers (as defined by the colocalization of H3K4Me1 and H3K27Ac histone marks), SNAI2-binding activity enhancers, and MFSG targets. [Figure 10B] Figure 10B is a Venn diagram representation of the overlap between the repression enhancer (as defined by the colocalization of H3K4me1 and H3K27me3 histone marks), the SNAI2 binding repression enhancer, and the ESCG target. [Figure 10C] Figure 10C is a heatmap showing hierarchical clustering of RNA-Seq expression profiles of SNAI2-binding MFSG activity enhancer targets across the progression of mesodermal lineages in 21 different human cell and tissue types from ESCs to differentiated adult fibroblasts. [Figure 10D]Figure 10D is a heatmap showing hierarchical clustering of RNA-Seq expression profiles of SNAI2-binding ESCG repressive enhancer targets across the progression of mesodermal lineages in 21 different human cell and tissue types from ESCs to differentiated adult fibroblasts. [Figure 10E] Figure 10E shows aggregate plots of H3K4me1, H3K27ac, H3K27me3, and H3K9me3 ChIP-Seq signals centered on the midpoint of the SNAI2 peak at the identified active enhancer MFSG target. [Figure 10F] Figure 10F shows an example of the ChIP-Seq profile of PRRX1, a prototype SNAI2-binding activity MFSG enhancer target gene in human mesoderm. The data were visualized using the University of Washington Genome Browser. [Figure 10G] Figure 10G shows aggregate plots of H3K4me1, H3K27ac, H3K27me3, and H3K9me3 ChIP-Seq signals centered on the midpoint of the SNAI2 peak at the identified inhibitory enhancer ESCG targets. [Figure 10H] Figure 10H shows an example of the ChIP-Seq profile of SOX2, a prototype SNAI2-binding repressing ESCG enhancer target gene in human mesoderm. [Best Mode for Carrying Out the Invention]

[0019] This specification provides transdifferentiated and dedifferentiated cells and populations, reprogrammed cells, and methods for producing and using such cells. Direct reprogramming of cells from readily available somatic cells to desired lineages holds great potential in cell-based regenerative therapy. Since the discovery of transcription factor (TF)-mediated reprogramming of differentiated somatic cells to induced pluripotent stem cells (iPSCs) by forced overexpression of Oct4-Sox2-Klf4-Myc (OSKM) (Takahashi & Yamanaka 2006), the field of somatic cell reprogramming has rapidly developed into a thriving area of ​​research, with several studies further expanding this work (Graf 2011; Srivastava & DeWitt 2016). Many groups have also reported the direct programming of somatic cells into other differentiated cell types (i.e., transdifferentiation) through the forced expression of lineage-instructive TFs (Davis et al. 1987; Vierbuchen et al. 2010; Xu et al. 2015). More recently, chemical reprogramming of somatic cells promoted by small molecules has been demonstrated as an effective non-viral reprogramming strategy (Hou et al. 2013, Liu et al. 2016; Xie et al. 2017). However, despite significant progress, current methods for reprogramming lineage-constrained cells into iPSCs or other cell types often produce poorly transformed cells by preserving the transcriptome and epigenetic state of the starting cells.

[0020] Some studies have enhanced the efficiency of reprogramming induced by exogenous TFs using alternative methods (Weltner et al. 2018; Kogut et al. 2018).

[0021] However, the difference between the current approach and the method described herein lies in the fundamental strategy supporting cell reprogramming. The current approach focuses on the characteristics of target cells in an attempt to induce or "force" lineage-constrained somatic cells to become a different cell type, whereas the method described herein focuses on starting cells for passively inducing reprogramming. To achieve this passive reprogramming, the method described herein employs a process to increase cell plasticity by suppressing lineage-inducible TFs specific to the starting cells or parent cells (these cells are also referred herein to as "parent TFs" or "lineage-determining transcription factors") alone, enabling transdifferentiation to a different cell fate or dedifferentiation to a primitive cell state in the absence of target transdifferentiated or dedifferentiated cells-specific transcription factors (TFs) or small molecule modulators (referred herein to as "exogenous TFs and / or modulators"). No studies have reported to date investigated the potential of this strategy. Furthermore, no method has been reported to date in which disrupting a single gene in differentiated cells, such as fibroblasts, can produce any selected cell type simply by changing the culture conditions after the single gene disruption.

[0022] The methods described herein are based on releasing the restrictions imposed by lineage-inducible TFs. Lineage-specific TFs in differentiated cells strictly enforce cellular identity, inhibit plasticity acquisition, and contribute to the low reprogramming efficiency associated with extrinsic TF induction and / or small molecule induction of cellular reprogramming. Because the presence of residual epigenetic memory imposed by lineage-specific TFs in starting cells hinders nuclear reprogramming, releasing these restrictions imposed by such TFs allows reprogramming to proceed without interference, even in the absence of Yamanaka factors or pathway regulatory cocktails.

[0023] In studies described later, it was also shown for the first time that transient suppression of TFs defining a single somatic cell lineage is sufficient to effectively overcome safeguards of molecules that preserve cellular identity and induce dedifferentiation into a pluripotent state. Using this differential approach, we generated pluripotent stem cells and cells of other lineages from lineage-constrained cells. This contribution is significant because it establishes a novel and effective in vitro reprogramming technique for generating dedifferentiated stem cells (including iPSCs) with superior yield and purity by overcoming molecular barriers that limit reprogramming efficiency and contribute to the emergence of partially reprogrammed cell populations, without requiring ectopic overexpression of pluripotency factors. Since this strategy does not require forced overexpression of ectopic genes such as Oct4, Sox2, Klf4, and c-Myc, the dedifferentiated stem cells generated by the method described herein are expected to be free from harmful tumorigenic mutations. Furthermore, because the method is nonviral, these are expected to be free from the risks associated with genomic integration of lentiviral or retroviral vectors. In addition, the methods described herein are expected to help improve the efficiency of existing iPSC generation protocols. Improving the efficiency of existing cell reprogramming techniques remains an important and unaddressed issue. The methods for generating efficiently dedifferentiated stem cells described herein are ultimately expected to help advance the use of iPSCs for therapeutic purposes.

[0024] Identification of transcription factors that determine the generation and lineage of somatic cell hybrids. The embodiments described herein relate to methods for reprogramming lineage-constrained cells into a dedifferentiated or transdifferentiated state without introducing any exogenous TFs and / or modulators. These methods first involve identifying critical, germ-layer-specific, lineage-inducing intrinsic factors (also referred to herein as “gatekeeper transcription factors,” “parental TFs,” “lineage-determining transcription factors,” or “LDTFs”) that are involved in maintaining lineage constraint in specific cells. To be an LDTF or gatekeeper transcription factor (GTF), targeted inhibition of the factor should be capable of reversing lineage preservation and maintenance, as well as restoring the ability to differentiate into multipotency and / or pluripotency. Furthermore, targeted siRNA-mediated transient inhibition of identified LDTFs in differentiated cells should be a practical alternative to forced overexpression of pluripotency factors in such cells, as a method for generating multipotency and / or pluripotent stem cells from them without requiring permanent genetic modification. As disclosed below, LDTFs whose physiological expression is important for inhibiting cell plasticity and pluripotency, and which protect the processes of lineage specialization, constraint, and preservation in differentiated cells, have been identified.

[0025] LDTFs can be identified using an unbiased, comprehensive experimental discovery approach that includes the generation of stable, same-species somatic cell fusion nucleus hybrids (HFs). The advantage of this technique is that in the generated hybrid cells, phylogenetic factors of the parental cells are suppressed, thus giving the inventors a functional readout of the importance of these factors in determining the fate of the parental cells. The generation of HFs used for identifying LDTFs in the embodiments described herein is detailed below.

[0026] In certain embodiments, LDTFs that can be used or modulated to reprogram lineage-constrained fibroblasts according to the embodiments described herein include, but are not limited to, SNAI2, PRRX1, HAND1, CDX2, or combinations thereof. Other LDTFs can be identified for other types of lineage-constrained somatic cells using desired starting cells and employing the methods described herein.

[0027] Therefore, a method is provided to identify important germ-layer-specific LDTFs that are involved in maintaining lineage constraints of specific cells, and whose transient suppression can effectively influence the nuclear reprogramming of such cells back to their pre-lineage-constrained state of cellular plasticity. The method comprises the step of generating a somatic cell hybrid by fusing parental cells of two identical species: primary and secondary parental cells. With respect to the purpose of this method, primary parental cells refer to cells for which LDTF identification is desired and for which nuclear reprogramming is desired. In a particular embodiment, the primary parental cells are lineage-constrained fibroblasts and the secondary cells are lineage-constrained hepatocellular carcinoma cells, but any suitable somatic cells can be used as primary and secondary parental cells.

[0028] To facilitate the transition between lineage-constrained somatic cells and transdifferentiated cells, it is sometimes desirable to identify LDTFs in primary parental cell types derived from each primary germ layer. It is also desirable to use cells that can be obtained non-invasively as primary cell types. For example, in a certain embodiment, somatic cell hybrids can be generated using mesoderm-derived fibroblasts as primary parental cells to identify LDTFs in the fibroblasts. The LDTFs identified in the fibroblasts can then be targeted to reprogram the fibroblasts and generate mesoderm-derived transdifferentiated somatic cell types. In another embodiment, somatic cell hybrids can be generated using ectoderm-derived keratinocytes as primary parental cells to identify LDTFs in the keratinocytes. The LDTFs identified in the keratinocytes can then be targeted to reprogram the keratinocytes and generate ectoderm-derived transdifferentiated somatic cell types. Furthermore, in another embodiment, somatic cell hybrids are generated to identify LDTFs in endothelial cells or hepatocytes of the endothelium lining, using endothelial cells derived from the endoderm or hepatocytes as primary parent cells. The LDTFs identified in the endothelial cells or hepatocytes of the endothelium lining can then be targeted to reprogram the endothelial cells or hepatocytes of the endothelium lining and generate a somatic cell type that has undergone transdifferentiation derived from the endoderm.

[0029] Somatic cell hybrids and parental cells are subjected to genome-wide transcriptome profiling techniques to identify LDTFs that enforce cell fate in each. Genome-wide transcriptome profiling techniques may include, but are not limited to, whole-genome microarrays, DNA sequencing, RNA sequencing, chromatin profiling and chromatin immunoprecipitation (ChIP) assays, and any other next-generation sequencing (NGS) techniques known in the industry.

[0030] The identification of candidate LDTFs involves selecting a combination of primary TFs that show more than 5-fold overexpression in primary parental cells compared to secondary parental cells. The primary TFs are then screened to identify a combination of hybrid TFs that show more than 2.5-fold suppression in somatic cell hybrids compared to primary parental cells. The hybrid TFs are then used as candidate LDTFs for targeting in the primary parental cell type. In a particular embodiment, the hybrid TF showing the greatest suppression is selected as the target LDTF. When the primary parental cells are lineage-constrained fibroblasts, the selected target LDTFs were SNAI2, PRRX1, HAND1, CDX2, or a combination thereof, according to the specific embodiments described herein. However, this method can be applied to any primary parental cells intended for use as starting cells for the reprogramming methods described herein.

[0031] Generation of differentiated cells According to the embodiments described herein, the identification of LDTFs and treatment with LDTF-specific siRNA described above and further described in the examples enable the generation of transdifferentiated or dedifferentiated cells without genetic modification of the starting cells, by forced expression of reprogramming factors without nonviral delivery of exogenous reprogramming factors and without treatment with small molecule pathway modulators. In other words, the methods described herein provide the generation of transdifferentiated or dedifferentiated cells without viral or nonviral delivery of exogenous reprogramming factors and small molecule pathway modulators.

[0032] In one embodiment, a method for producing transdifferentiated somatic cells is provided. In another embodiment, a method for producing dedifferentiated stem cells is provided. Both of these methods may include a step of transiently inhibiting (or suppressing) LDTFs in lineage-constrained somatic cells. In some embodiments, the transient inhibition step is performed in lineage-constrained fibroblasts. In such embodiments, the LDTF or LDTF that is transiently inhibited may be (i) SNAI2, (ii) PRRX1, (iii) HAND1, (iv) CDX2, (v) SNAI2 and PRRX1, (vi) SNAI2 and HAND1, (vii) SNAI2 and CDX2, or (viii) SNAI2, HAND1 and CDX2.

[0033] Transient inhibition of LDTF is achieved by introducing any molecule that can transiently suppress its expression. In one embodiment, transient inhibition of LDTF is achieved by the use of siRNA or shRNA molecules. While other types of molecules and systems (e.g., antisense oligonucleotides, miRNAs) can be used to transiently inhibit LDTF, siRNA and shRNA molecules are advantageous because they are designed to target and inhibit specific genes, can reduce gene expression at the mRNA level, and can be delivered to cells nonvirally. In other words, siRNA and shRNA molecules can selectively and transiently inhibit a single target gene with minimal off-target effects using RNA interference mechanisms within the cell. Other small molecules such as miRNAs have limited selectivity because they have multiple targets. Permanent solutions for silencing a single gene (e.g., CRISPR-Cas9) are also undesirable because permanent gene knockout can be detrimental to the dynamic process of cellular reprogramming and may not function. In this embodiment, a method for producing transdifferentiated somatic cells includes the step of introducing an siRNA or shRNA molecule that transiently inhibits the expression of LDTFs in lineage-constrained somatic cells, or in a specific aspect inhibits the expression of a single LDTF. In other words, lineage-constrained somatic cells are treated with an siRNA or shRNA molecule that targets LDTFs and incubated in a culture medium of a desired target cell to transdifferentiate them to a different cell fate or to dedifferentiate them to a multipotent or pluripotent primitive cell state.

[0034] In embodiments where the lineage-constrained somatic cells are fibroblasts, the LDTFs transiently inhibited by siRNA or shRNA molecules are (i) SNAI2, (ii) PRRX1, (iii) HAND1, (iv) CDX2, (v) SNAI2 and PRRX1, (vi) SNAI2 and HAND1, (vii) SNAI2 and CDX2, or (viii) SNAI2, HAND1 and CDX2. In specific aspects, the LDTFs transiently inhibited by siRNA or shRNA molecules are SNAI2 alone, PRRX1 alone, HAND1 alone, or CDX2 alone (i.e., transient inhibition of a single LDTF).

[0035] In one embodiment, the siRNA molecule used to transiently inhibit LDTF may be derived from shRNA that has been delivered to a cell and subsequently cleaved by an intracellular dicer. In other words, an shRNA molecule may be delivered to a cell, cleaved intracellularly, and then form an siRNA that transiently inhibits LDTF, or the siRNA molecule itself may be delivered to the target cell. In both scenarios, inhibition of LDTF is ultimately achieved by the siRNA.

[0036] The siRNA or shRNA molecules that can be used in accordance with the embodiments described herein may be single-stranded siRNA molecules containing an siRNA sequence (antisense strand), or double-stranded siRNA molecules containing an siRNA sequence (antisense strand) and a complementary passenger strand (sense strand). The shRNA molecules that can be used in accordance with the embodiments described herein may include an siRNA sequence (antisense strand), a complementary passenger strand (sense strand), and an intervening loop sequence that combines the siRNA and the passenger strand. Representative siRNA sequences, complementary passenger strands, and loop sequences that can be used to form the siRNA or shRNA molecules used in the embodiments described herein include, but are not limited to, the sequences listed in Tables 1 and 2 below.

[0037] [Table 1-1]

[0038] [Table 1-2]

[0039] [Table 1-3]

[0040] [Table 2-1]

[0041] [Table 2-2]

[0042] [Table 2-3]

[0043] siRNA and shRNA molecules can be delivered to target somatic cells via non-viral delivery methods, such as liposome delivery, targeted liposome delivery, nanoparticle delivery, or other methods known in the art, but are not limited to these. Transient inhibition of a single LDTF as described above can effectively influence the nuclear reprogramming of lineage-constrained somatic cells back to their pre-lineage-constrained state of cellular plasticity. Once the state of cellular plasticity is achieved, cells treated with siRNA / shRNA can be directed to differentiate in the direction of a desired specific lineage trajectory, provided appropriate in vitro culture conditions or a specific in vivo microenvironment is provided, to form either differentiated cells of a different lineage than the starting cell lineage (i.e., transdifferentiated somatic cells) or dedifferentiated stem cells with the ability to differentiate into multipotent or pluripotent lines.

[0044] Therefore, a method for producing transdifferentiated somatic cells or dedifferentiated stem cells according to the embodiments described herein also includes the step of exposing reprogrammed, lineage-constrained somatic cells to an environment specific to a desired transdifferentiated somatic cell (i.e., a desired cell lineage or cell type) or a desired stem cell (e.g., pluripotent or multipotent stem cell). For example, reprogrammed somatic cells (i.e., somatic cells treated with siRNA or shRNA) may be incubated under in vitro culture conditions specific to transdifferentiated somatic cells.

[0045] According to embodiments described herein, lineage-restricted somatic cells can be reprogrammed to become cells of any desired lineage or any type of terminally differentiated cell. In one embodiment, the starting cell is a lineage-restricted fibroblast. Examples of transdifferentiated somatic cells that can be produced by the methods described herein include, but are not limited to, cells of the adipocyte lineage, such as adipocytes; cells of the osteogenic cell lineage, such as osteocytes; cells of the chondrogenic cell lineage, such as chondrocytes; cells of the cardiovascular lineage, such as cardiomyocytes; cells of the pancreatic lineage, such as pancreatic β-cells; cells of the ophthalmic lineage, such as retinal pigment epithelial cells; cells of the hepatic lineage, such as hepatocytes; cells of the lung lineage, such as lung cells; and cells of the renal lineage, such as nephron cells. In other embodiments, lineage-restricted somatic cells can be reprogrammed to become a desired type of dedifferentiated stem cell. Examples of dedifferentiated stem cells that can be produced by the methods described herein include, but are not limited to, dedifferentiated pluripotent stem cells and dedifferentiated mesenchymal stem cells.

[0046] Table 3 below shows examples of reprogrammed somatic cells and the corresponding in vitro environments to which they may be exposed in order to produce the desired type or lineage of differentiated or dedifferentiated cells.

[0047] [Table 3]

[0048] ( ** Any suitable culture medium for the desired transdifferentiated or dedifferentiated cells can be used as an environment for inducing transdifferentiation or dedifferentiation of cells treated with the appropriate siRNA / shRNA. Mediums specific to the target transdifferentiated or dedifferentiated cells may be obtained from commercially available suppliers (e.g., Gibco, PromoCell, HyClone®, and other suppliers known in the industry) or may be generated using suitable components known in the industry to create conditions for growing the desired transdifferentiated or dedifferentiated cells.

[0049] The methods described herein are advantageous over current techniques for several reasons. First, the methods for producing dedifferentiated or transdifferentiated cells described herein are carried out without the delivery of exogenous TFs and / or small molecule modulators, either virally or nonvirally. Instead, the methods rely solely on the transient suppression of targeted LDTFs using siRNA or shRNA molecules described herein according to certain embodiments. By removing molecular obstacles to reprogramming, the methods described herein reset cells to an early point in their developmental timeline, thereby potentially desuppressing plasticity-maintaining factors and allowing the cells to proceed with their intrinsic differentiation mechanisms as guided by the components supplied to their growth medium.

[0050] In addition, the methods for producing dedifferentiated or transdifferentiated cells described herein also significantly improve reprogramming efficiency compared to current methods that use miRNA or small molecule modulators to reprogram cells. As previously described, current methods yield a reprogramming efficiency of less than 1%, even when 100% of the starting cell population is exposed to the treatment. Such low efficiency inevitably suggests that a significant proportion of cells are resistant to reprogramming, despite overexpression of transcription factors or very high exposure to small molecules that modulate the pathway. This may be due to insurmountable barriers to reprogramming, often resulting from the retention of the starting cell's transcriptional regulatory network (TRN) and epigenetic state. Therefore, nonspecific pathway inhibition itself is not sufficient to overcome the starting cell's epigenetic memory. To make the somatic cell reprogramming cascade more efficient, it is first necessary to neutralize the effects of the starting cell's TRN and residual epigenetic state through the use of transient repression as described herein. By suppressing important, lineage-inducible TFs in parental cells, lineage-bound cells gradually lose the transcriptional and epigenetic environment that constrains them, thus reducing the interference of residual transcriptional and epigenetic memory from parental cells on the cell reprogramming cascade, and thus making the reprogramming process more efficient. As a result, the reprogramming efficiency of lineage-bound somatic cells converted to a desired transdifferentiated or dedifferentiated cell type via the use of siRNA or shRNA molecules to transiently suppress one or more major LDTFs can be greater than 1%. The efficiency may vary depending on the desired target transdifferentiated or dedifferentiated cell type. Therefore, in certain embodiments, the reprogramming efficiency may be 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or approximately 100%.In other embodiments, the reprogramming efficiency is 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100%.

[0051] Cells and populations of cells Cells and populations of cells produced using the methods for generating or producing the differentiated or dedifferentiated cells described above are provided herein.

[0052] In a particular embodiment, reprogrammed somatic cells or populations of cells are provided. In one embodiment, the reprogrammed somatic cells are reprogrammed somatic fibroblasts treated with an siRNA or shRNA molecule that transiently inhibits the expression of LDTF.

[0053] In certain aspects of this embodiment, the LDTFs transiently inhibited by siRNA or shRNA molecules are (i) SNAI2, (ii) PRRX1, (iii) HAND1, (iv) CDX2, (v) SNAI2 and PRRX1, (vi) SNAI2 and HAND1, (vii) SNAI2 and CDX2, or (viii) SNAI2, HAND1 and CDX2. In certain aspects, the LDTFs transiently inhibited by siRNA or shRNA molecules are SNAI2 alone, PRRX1 alone, HAND1 alone, or CDX2 alone (i.e., transient inhibition of a single LDTF). Through transient suppression of LDTFs and incubation with the target medium of the desired target cells, the reprogrammed somatic cells, in some embodiments, can be transdifferentiated into another lineage of somatic cells when placed in a suitable environment without the delivery of exogenous TFs and / or small molecule modulators (chemical reprogramming) via viral or nonviral delivery. In another embodiment, siRNA / shRNA-treated somatic cells can be dedifferentiated into multipotent or pluripotent stem cells without the delivery of exogenous TFs and / or modulators via viral or nonviral means.

[0054] In other embodiments, populations of transdifferentiated cells or transdifferentiated somatic cells are provided. The types of transdifferentiated somatic cells that may be provided are as described above. In one embodiment, the population of transdifferentiated somatic cells is a population of reprogrammed somatic cells (e.g., reprogrammed fibroblasts) without permanent genetic modification by using siRNA or shRNA to temporarily suppress LDTF rather than permanently, as described above. Furthermore, the temporary suppression of LDTF without requiring the introduction of any exogenous TF and / or small molecule modulators (chemical reprogramming) significantly increases the efficiency of reprogramming and conversion from lineage-constrained somatic cells (e.g., fibroblasts) to desired transdifferentiated cells, overcoming a significant barrier that currently hinders the clinical application of iPSCs. In a particular embodiment, a population of reprogrammed somatic cells (e.g., reprogrammed fibroblasts) was converted from a population of lineage-constrained somatic cells (e.g., fibroblasts) to a population of transdifferentiated somatic cells with an efficiency of greater than 1%. In other embodiments, the reprogramming efficiency may be 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or approximately 100%.

[0055] In other embodiments, dedifferentiated stem cells or populations of dedifferentiated stem cells are provided. Possible types of dedifferentiated stem cells provided include the pluripotent and multipotent stem cells described above. In one embodiment, the population of dedifferentiated stem cells includes, for example, a population of reprogrammed somatic cells (e.g., reprogrammed fibroblasts) by the use of siRNA or shRNA to transiently suppress LDTF rather than permanently (without requiring permanent genetic manipulation), as described above. Furthermore, transient suppression of LDTF without requiring the introduction of any exogenous TF and / or small molecule modulators (chemical reprogramming) significantly increases the efficiency of reprogramming and conversion from lineage-constrained somatic cells (e.g., lineage-constrained fibroblasts) to desired dedifferentiated cells, overcoming a significant barrier that currently hinders the clinical application of iPSCs. In a particular embodiment, a population of reprogrammed somatic cells (e.g., reprogrammed fibroblasts) was converted from a population of lineage-constrained somatic cells (e.g., lineage-constrained fibroblasts) to a population of dedifferentiated stem cells with an efficiency of greater than 1%. The efficiency may vary based on the desired target type of converted or dedifferentiated cell. In other embodiments, the reprogramming efficiency may be 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or approximately 100%.

[0056] Cell culture system A cell culture system is used in a particular manner to produce cells or populations of cells using the methods described herein. The cell culture system comprises materials necessary to produce the desired transdifferentiated or dedifferentiated cells.

[0057] In one embodiment, a cell culture system for producing a population of transdifferentiated somatic cells is provided. The types of transdifferentiated somatic cells that can be produced are as described above. Examples of cell culture systems for producing transdifferentiated somatic cells include, but are not limited to, a lineage-constrained population of somatic cells (e.g., fibroblasts), an siRNA or shRNA molecule that inhibits LDTF expression (as described above), and a culture medium specific to the population of transdifferentiated somatic cells, examples of which are shown in Table 3 above.

[0058] In another embodiment, a cell culture system for producing a population of dedifferentiated stem cells is provided. Possible types of dedifferentiated stem cells to be provided include the pluripotent and multipotent stem cells described above. Examples of cell culture systems for producing dedifferentiated stem cells include, but are not limited to, a population of lineage-constrained somatic cells (e.g., fibroblasts), siRNA or shRNA molecules that inhibit LDTF expression (as described above), and culture media such as cell culture media specific to the population of dedifferentiated stem cells, examples of which are shown in Table 3 above.

[0059] How to use Transdifferentiated or dedifferentiated cells produced by the methods described above can be used in many medical applications to treat a variety of conditions. In certain embodiments, a population of reprogrammed somatic cells (i.e., somatic cells treated with siRNA or shRNA as described above and incubated with the culture medium of the desired target cell) can be used to treat a condition. In other embodiments, a population of transdifferentiated somatic cells can be used to treat a condition. In other embodiments, a population of dedifferentiated stem cells can be used to treat a condition. Non-limiting examples of conditions that can be treated using reprogrammed somatic cells, transdifferentiated somatic cells, or dedifferentiated stem cells include acute or chronic wounds that persist as a result of trauma or as a result of a chronic condition (e.g., bedsores) or a disease (e.g., wounds resulting from late-stage diabetes); osteoarthritis; osteoporosis; loss of bone structure due to trauma or surgical removal, injury or loss of soft tissue due to trauma or surgical removal; macular degeneration; diabetes mellitus; fibrosis (e.g., fibrosis of the lung, kidney, or liver); myocardial infarction; chronic or acute heart failure; liver failure, lung failure, and kidney failure.

[0060] In some embodiments, methods for treating a condition may include transplanting or grafting a population of autodifferentiated transformant cells (e.g., those described above) onto or inside the surface of a target tissue or organ. “Treating” or “curing” a condition may also mean preventing the condition, reducing its severity, slowing the onset or progression of the condition, reducing the risk of progression, preventing or delaying the progression of symptoms associated with the condition, reducing or ending symptoms associated with the condition, achieving complete or partial relief of the condition, or a combination of these. In embodiments described herein, treatment of a condition may also include engraftment or transplantation of a population of cells. Appropriate grafting or transplantation methods can be selected depending on where the population of cells is to be transplanted or grafted (i.e., what type of tissue or organ). For example, implantable or injectable grafts can be used to treat a condition.

[0061] Implantable grafts may contain a solid matrix that allows cells to be implanted and cultured with the necessary growth factors (i.e., an environment specific to the population of cells to be implanted), and then implanted into the affected tissue or organ of the target. Injectable grafts can fill any defective shape or space in a damaged organ or tissue. Injectable grafts involve the injection of dedifferentiated stem cells or reprogrammed somatic cells in the form of a cell suspension containing a biomaterial that coagulates in situ by various cross-linking methods known in the art. The mixture may be injected directly into the tissue or organ, exposed to the surface of the tissue or organ, or attached thereto.

[0062] Non-limiting examples of biomaterials usable in injectable grafts include, but are not limited to, inorganic substances, chitosan, alginates, hyaluronic acid, fibrin, gelatin, and natural materials such as many synthetic polymers. Such materials are often solidified by methods such as thermal gelation, photocrosslinking, or chemical crosslinking. Cell suspensions may also be supplemented with soluble signals or specific matrix components. These grafts can be injected relatively easily into the target area, eliminating (or minimizing) the need for invasive surgery, reducing costs, patient discomfort, infection risk, and scar formation. Chemically modified HA can also be used as injectable materials for tissue engineering because its effects are long-lasting while maintaining biocompatibility. Crosslinking methods also maintain the biocompatibility of the material, and its presence over a wide area of ​​regenerative or stem / progenitor cell niches makes it an attractive injectable material.

[0063] In certain embodiments, a population of dedifferentiated stem cells or reprogrammed somatic cells is delivered to a target tissue or organ for the purpose of inducing differentiation of the stem cells or reprogrammed somatic cells into native cells normally present in the tissue or organ, based on the environment present in the tissue or organ in vivo, and the population of dedifferentiated stem cells or reprogrammed somatic cells may be delivered via an injectable graft. The injectable graft can be delivered by injection of a cell suspension containing dedifferentiated stem cells or reprogrammed somatic cells into the target tissue or organ.

[0064] In certain embodiments, transdifferentiated or dedifferentiated cells can be used in medical procedures to regenerate tissue in wounds or degenerative diseases. For example, in one embodiment, dedifferentiated cells can be used in wound healing. In that embodiment, fibroblasts from the wound surface obtained from a wounded subject may be subjected to the above-described method for producing dedifferentiated stem cells to generate a population of autologous dedifferentiated mesenchymal stem cells that can be applied topically to the wound of the subject. When exposed to the in vivo microenvironment of the wound, the autologous mesenchymal stem cells are expected to influence tissue regeneration, tissue remodeling, and wound healing without the need for cell transplantation. This technology offers the advantages of easy implementation, high cost-effectiveness, and low barriers to entry, even in resource-constrained environments, such as combat zones or frontline hospitals.

[0065] In other embodiments, the population of transdifferentiated somatic cells can be used in graft or transplant surgery for the treatment of degenerative diseases such as osteoarthritis or osteoporosis. For example, in one embodiment, fibroblasts obtained from a subject suffering from osteoarthritis can be subjected to the transdifferentiation method described above to generate a population of autologous chondrocytes (i.e., chondrocytes) that can be grafted or transplanted into or on the surface of the joint of the subject suffering from osteoarthritis. In another embodiment, fibroblasts obtained from a subject suffering from osteoporosis can be subjected to the transdifferentiation method described above to generate a population of autologous osteocytes (i.e., bone cells) that can be grafted or transplanted into or on the bone of the subject suffering from osteoporosis.

[0066] In other embodiments, the population of transdifferentiated somatic cells can be used in graft or transplant surgery for reconstructive surgery. For example, in one embodiment, fibroblasts obtained from a subject requiring orthopedic or maxillofacial reconstructive surgery due to trauma or loss of other bone structure can be subjected to the transdifferentiation method described above to produce a population of autologous osteocytes (i.e., bone cells) that can be used to reconstruct the bone structure of the subject. In another embodiment, fibroblasts obtained from a subject requiring or having chosen reconstructive plastic surgery can be subjected to the transdifferentiation method described above to produce a population of autologous adipocytes (i.e., adipocytes) that can be used in reconstructive plastic surgery.

[0067] In other embodiments, the methods described herein can be employed in vivo to perform differentiation of scar tissue in affected tissue. For example, in one embodiment, therapeutic siRNA or shRNA may be administered to a subject with scar tissue resulting from pulmonary fibrosis, which is caused by changes including fibroblast proliferation in the lung. Any suitable delivery method, though not limited to these, can be used, including injection, intravenous infusion, and inhalation. The siRNA or shRNA may be designed to inhibit LDTFs, such as SNAI2, PRRX1, HAND1, or CDX2 (or a combination thereof), and if the delivery of siRNA targets fibrotic cells in the lung, the siRNA or shRNA transiently inhibits LDTFs, and upon exposure to a lung-specific microenvironment in vivo, reprograms the fibrotic cells into lung cells, thereby reversing the pathological effects of fibrosis. In another embodiment, therapeutic siRNA or shRNA may be administered to a subject with scar tissue in the liver due to cirrhosis. siRNA or shRNA can be designed to inhibit LDTFs, such as SNAI2, PRRX1, HAND1, or CDX2 (or a combination thereof), and if the delivery of siRNA or shRNA targets fibrotic cells in the liver, the siRNA or shRNA transiently inhibits LDTFs, reprogramming fibrotic cells into hepatocytes upon in vivo exposure to a liver-specific microenvironment, thereby reversing the pathological effects of fibrosis.

[0068] In other embodiments, the methods described herein can be employed in vivo to perform tissue redifferentiation in subjects suffering from chronic diseases. For example, in one embodiment, therapeutic siRNA or shRNA may be administered to subjects with diabetes. The siRNA or shRNA may be designed to suppress LDTF, and if the delivery of the siRNA or shRNA targets the islets of the pancreas, the siRNA or shRNA transiently inhibits LDTF and induces pancreatic cells to regenerate normal pancreatic β-cells. In another embodiment, therapeutic siRNA or shRNA may be administered to subjects with heart failure. The siRNA or shRNA may be designed to suppress LDTF, and if the delivery of the siRNA or shRNA targets the cardiomyocytes, the siRNA or shRNA transiently inhibits LDTF and induces cardiomyocytes to regenerate normal cardiomyocytes. The therapeutic siRNA or shRNA may be administered to subjects with diabetes. In another embodiment, therapeutic siRNA or shRNA may be administered to subjects with macular degeneration. siRNA or shRNA can be designed to suppress LDTF, and when siRNA or shRNA delivery targets the retina, it transiently inhibits LDTF, inducing retinal cells to regenerate normal retinal pigment epithelial cells.

[0069] In another embodiment, dedifferentiated cells can be used in the regeneration of tissues or organs to be transplanted to patients in need of transplantation. In such an embodiment, fibroblasts obtained from subjects in need of tissue or organ transplantation may be subjected to the dedifferentiation method described above to generate a population of dedifferentiated autologous pluripotent stem cells. Such pluripotent stem cells can be used to generate cells or tissues of any germ layer origin for use via cell and / or tissue transplantation to replace diseased cells or organs. Since the origin of the cells / tissues / organs is autologous, the risk of tissue or organ rejection is expected to be eliminated. [Examples]

[0070] The following examples are intended to illustrate various aspects of the invention. Therefore, the specific aspects discussed are not to be construed as limitations on the scope of the invention. It will be obvious to those skilled in the art that various equivalents, variations, and modifications can be made without departing from the scope of the invention, and it will be understood that such equivalent aspects are included herein. Furthermore, all references cited herein are incorporated herein in their entirety as if they were described in detail herein.

[0071] Example 1: Differentiation and dedifferentiation of somatic cells from lineage-restricted fibroblasts. Using the aforementioned somatic cell hybrid model between the same species, we identified SNAI2 and PRRX1 as key TFs that preserve mesenchymal fate in rat embryonic fibroblasts (REFs), demonstrating that siRNA-mediated transient knockdown of individual factors results in the direct conversion of REFs to functional adipocytes, chondrocytes, or osteocytes. In addition, the following studies demonstrate that siRNA-mediated transient knockdown of SNAI2 alone is sufficient to transform REFs into dedifferentiated pluripotent stem cells (dPSCs) that form embryoid bodies, and that triple germ layer differentiation is possible in the absence of exogenous TFs and / or small molecule modulators (chemical reprogramming). These results establish for the first time that transient suppression of TFs that define a single somatic cell lineage can effectively overcome safeguards of molecules that preserve cellular identity and is sufficient to induce differentiation to a different cell fate or dedifferentiation to a primitive cell state.

[0072] Materials and methods Cell lines and culture conditions The rat hepatocarcinoma cell line FTO2B is a clone derived from ouabain-resistant thymidine kinase TK-deficient H4IIEC3 (Killary & Fournier 1984). RAT-1 is an SV40-transformed rat embryonic fibroblast (REF) cell line expressing functional thymidine kinase (Botchan et al. 1976). HF is a hybrid cell line generated by fusing FTO2B and RAT-2 cells (Bulla et al. 2012). All cell lines were maintained at 37°C in a humidified water-jacketed 5% CO2 incubator in 1:1 Ham's F12 / Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (GIBCO BRL) and 5 μg / 100 ml penicillin-streptomycin (GIBCO).

[0073] Generation of liver cancer X fibroblast hybrids FTO2B cells, RAT-1 cells (10 6Each cell type was plated together. After 24 hours, the hybrid cell monolayers were fused and FR(2) hybrid cell lines were created using 50% polyethylene glycol (50% w / v) for 1 minute. The following day, cells were separated in a 1:20 ratio into a medium containing 3 mM ouabain and hypoxanthine-aminopterin-thymidine (HAT) medium and selected for parental fibroblasts and hepatocellular carcinoma cells, respectively. After 2-3 weeks, no parental cells survived the selection strategy, but a large number of viable cells were observed. These were pooled (more than 100 clones per pool) and expanded in hypoxanthine-aminopterin-thymidine (HAT) medium. These survived because they were superficially hybrid cells, were ouabain-resistant, and possessed functionally active thymidine kinase enzymes. All cell lines were maintained at 37°C in a humidified water-jacketed 5% CO2 incubator in 1:1 Ham's F12 / Dulbeccoo's modified Eagle medium (DMEM) containing 5% fetal bovine serum (FBS) (GIBCO BRL) and 5 μg / 100 ml penicillin-streptomycin (GIBCO). A cell monolayer density of 70% was achieved. Chromosome spreads and RNA isolation were performed for each cell line.

[0074] Chromosome spread To analyze cell hybrids for complete karyotype, subconfluent cell monolayers were exposed to 0.05 μM colsemid (a mitotic inhibitor) for 40 minutes, and the cells were harvested. The cells were incubated in 0.075 M KCl for 10 minutes and then treated with methanol / acetic acid (3:1). The cells were dropped onto cold, moistened slides, and the slides were stained with crystal violet. 10–15 spreads were scored for the number of chromosomes per spread. To confirm that the parental cells had not survived the selection strategy, viable cells were pooled in HAT medium and tested to determine whether these presumed FR(2) hybrid cells were actually true hybrids by determining the number of chromosomes present in the cells. Metaphase chromosome spreads were prepared for each parental cell line and somatic cell hybrid, and the chromosomes were quantified.

[0075] Microarray analysis RNA was extracted using the RNA Easy Mini Kit (Qiagen) according to the manufacturer's protocol. RNA purity and concentration were determined using an Epoch Nano-drop spectrophotometer at 260 and 280 nm. After RNA extraction, RNA samples from all cell lines were microarray analyzed. The RNA was hybridized to an Affymetrix GeneChip® rat genome 230 2.0 expression array (Affymetrix, Inc., Santa Clara, CA) using GeneChip (according to the manufacturer's instructions for the Expression 3' Amplification One-Cycle Target Labeling and Control Reagents kit). The chip was scanned with a GeneChip scanner model 3000 7G Plus. Gene expression was detected and normalized.

[0076] cDNA synthesis and qRT-PCR analysis RNA was extracted from a monolayer with a concentration of 70-80% using the RNA Easy Mini Kit (Qiagen) according to the manufacturer's protocol. RNA purity and concentration were determined using an Epoch Nano-drop spectrophotometer at 260 and 280 nm. Reverse transcription was performed using a thermal cycler (Life Technologies, USA) with the Superscript III First Strand cDNA Synthesis Kit (Invitrogen) according to the manufacturer's protocol. The resulting cDNA was used for qRT-PCR. Quantitative real-time RT-PCR (qRT-PCR) was performed using a Step One Plus real-time PCR system (Life Technologies, USA) in 20 μl of reaction mixture [2 μl cDNA (5 ng / μl), 6.75 μl sterile nuclease-free water, 10 μl Fast SYBR® Green Master Mix (Life Technologies, USA), and 1.25 μl gene-specific primers (0.5 μm, from IDTDNA)]. The PCR parameters consisted of heating at 95°C for 10 seconds, followed by heating at 95°C for 5 seconds and 60°C for 30 seconds for 40 cycles. The amplification signal of the target gene mRNA was normalized to the mRNA of endogenous housekeeping genes in the same reaction. The primers used for q-RT-PCR are as follows:

[0077] [Table 4]

[0078] Overexpression of candidate genes (PRRX1 and SNAI2) in HF hybrids Expression vectors containing the full-length rat genes SNAI2 and PRRX1 (Origene, Inc.) were introduced into hybrid cell lines HF by lipofection using Lipofectamine Plus reagent (Invitrogen, Inc.) according to the manufacturer's protocol. After 2-3 weeks, stable G418-resistant clones (HF-SNAI2 and HF-PRRX1, respectively) were selected, expanded, and used in subsequent experiments.

[0079] Knockdown of candidate genes (PRRX1 and SNAI2) in REF cells RAT-2REF (obtained from ATCC) was placed in a 6-well cell culture plate (Nunc) containing 2 × 10 cells. 6 Cells were planted at a density of cells / well (with 4 repetitions), incubated in a CO2 incubator at 37°C, and transfection was performed 1 day later using 50 nM (final concentration) of pooled short interfering RNA (siRNA, Santa Cruz Biotech), 2.5 μl of Lipofectamine RNA iMAX (Invitrogen, USA), and Opti-MEM (Invitrogen, USA), according to the manufacturer's instructions. siRNA transfection was calibrated using RNAi-negative universal control MED (Invitrogen). Repeat transfections were performed on day 4 to achieve sustained inhibition before using the cells in subsequent experiments on day 7 (Figure 3J).

[0080] Cell migration assay Transwell cell migration assays of REF, HF, HF-SNAI2, and HF-PRRX1 cells were performed using the Trevigen 24-well cell migration assay (catalog no. 3465-024-K) according to the manufacturer's protocol (n=3 biological replicas per experimental group). FTO2-B rat hepatocarcinoma (RH) cells, RAT-2 fibroblast (REF) cells, hybrid (HF) cells, and SNAI2 and PRRX1 overexpressing HF clones (HF-SNAI2 and HF-PRRX1, respectively) were transferred to a migration buffer consisting of serum-free FDV medium and 0.1% BSA, with 1 × 10⁶ cells per well. 6 The cells were resuspended at cells / ml. Tempered FDV medium containing 10% fetal bovine serum was used as a chemoattractant. 100 μl of cells were added to the transwell membrane in the upper chamber, and 600 μl of FBS containing (chemoattractant) FDV medium was added to the lower chamber. The same volume of serum-free FDV medium was added to the control well as a negative control. The cells were incubated in a CO2 incubator at 37°C for 30 hours. After incubation, the medium in the upper chamber was carefully aspirated without rupturing the membrane, and each well was washed with 100 μl of warm (37°C) 1× wash buffer provided in the kit. The lower chamber was aspirated and washed twice with 500 μl of warm (37°C) 1× wash buffer. Next, 12 μl of calcein AM solution (included in the kit) was added to 12 mL of 1× cell dissociation solution (included in the kit). 500 μl of cell dissociation solution / calcein AM was added to the chamber at the bottom of each well. The chamber was reassembled and incubated in a CO2 incubator at 37°C for 1 hour. The chamber was then disassembled by removing the insert, and the assay chamber solution at the bottom of the assay plate was read using a Biotek Synergy HT plate reader at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The experimental data was converted to cell number in units of relative fluorescence units (RFU) to determine the number of migrated cells.

[0081] adipogenesis induction REF cells were cultured in DMEM medium containing 10% FBS and 1% Pen-Strep. Once the cells reached a 70% density, they were trypsinized, pelletized, and counted. 30,000 cells were plated into four wells for each experiment (siCntrlREF, siPrrx1REF, and siSnai2REF) in 96-well plates (n=4 biological replicas per experiment). The wells were transfected with control, PRRX1, and SNAI2 siRNAs. Seven days after the initial siRNA transfection, the transfected cells were treated with adipogenesis-inducing medium (A) for 3 days (Cyagen Biosciences, catalog no. GUXMX-90031), followed by adipogenesis-maintaining medium (B) for 11 days. On day 14, oil red staining was performed to assess adipogenic activity, and the cell pellets were saved for further analysis.

[0082] Oil Red O-Dye Two weeks after lipogenesis induction, cells were rinsed with phosphate-buffered salt solution (PBS) and fixed with 4% paraformaldehyde (Sigma Aldrich) in PBS. The formaldehyde solution was removed by tilting the plate, and the cells were rinsed with sterile water. Each well was then covered with 60% isopropanol and incubated for 5 minutes. The isopropanol solution was pipettered, and 2 ml of Oil Red O (Sigma, catalog no. O1391) solution was added to each well, and the cells were incubated at room temperature for 20 minutes. The wells were then washed with water until the running water was clear. All wells were kept humidified with water and observed under a microscope. Lipid droplets were stained red with Oil Red O. To record the OD, the isopropanol solution was pipettered into a new 96-well microtiter plate (Corning NBS Microplate), and the OD was recorded at 490 / 500 / 510 nm.

[0083] osteogenic induction REF cells were cultured in DMEM medium containing 10% FBS and 1% Pen-Strep. When the cells reached a 70% density, they were trypsinized, pelletized, and counted. 30,000 cells were plated into four wells for each experiment (siCntrlREF, siPrrx1REF, and siSnai2REF) in 96-well plates (n=4 biological replicas per experiment). The wells were transfected with control, PRRX1, and SNAI2 siRNA. Seven days after the first siRNA transfection, the transfected cells were placed in osteogenic medium (DMEM, 10% FBS, 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate, 10 nM dexamethasone). Every three days, the old medium was aspirated and fresh osteogenic medium was added. After 14 days, the cells were washed in cold PBS, fixed with 4% PFA in PBS, stained with 40 mM alizarin red to stain calcium, and their osteogenic activity was confirmed. The cell pellet was then saved for further analysis.

[0084] Alizarin Red dye Two weeks after osteogenic induction, the cells were rinsed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma Aldrich) in PBS at room temperature for 15 minutes. After removing the formaldehyde solution, the cells were rinsed with sterile diH2O. After completely removing the water, 1 mL of 40 mM Alizarin Red S (ARS) was added, and Alizarin Red staining was performed according to the manufacturer's protocol (ScienCell, ARS staining quantification assay ARed-Q catalog number 8678).

[0085] Induction of cartilage formation REF cells were cultured in DMEM medium containing 10% FBS and 1% Pen-Strep. Once the cells reached a 70% density, they were trypsinized, pelletized, and counted. 30,000 cells were plated into four wells for each experiment (siCntrlREF, siPrrx1REF, and siSnai2REF) in 96-well plates (n=4 biological replicas per experiment). The wells were transfected with control, PRRX1, and SNAI2 siRNAs. Seven days after the initial siRNA transfection, the transfected cells were placed in chondrogenic medium (MSC go Chondrogenic XF®, Biological Industries) and incubated in an incubator (37°C, 5% CO2) for 14 days. The differentiation medium was changed every three days for 14 days, after which chondrogenic activity was assessed using Alcian blue staining, and the cell pellets were saved for further analysis.

[0086] Alcian Blue dyeing Two weeks after osteogenic induction, cells were rinsed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS (Sigma Aldrich) for 15 minutes at room temperature. After removing the formaldehyde solution, cells were rinsed with sterile diH2O. After completely removing the water, 0.2 ml of 1% Alcian blue solution (Sigma; A-3157) was added to each well, and Alcian blue staining was performed according to the kit manufacturer's protocol (MSC go Chondrogenic XF®, Biological Industries).

[0087] dMSC generation REF cells were cultured in DMEM medium containing 10% FBS and 1% Pen-Strep. Once the cells reached a 70% density, they were trypsinized, pelletized, and counted. 30,000 cells were plated into four wells for each experiment (siCntrlREF, siPrrx1REF, and siSnai2REF) in a 96-well plate (n=4 biological replicas per experiment). The wells were transfected with control, PRRX1, and SNAI2 siRNAs. Seven days after the initial siRNA transfection, the transfected cells were placed in rat MSC medium (Rat MSC Growth Medium Kit, Cell Applications, Inc.) and incubated in an incubator (37°C, 5% CO2) for 14 days. The MSC medium was changed every three days for 14 days, and then mesenchymal stem cell activity was evaluated using an alkaline phosphatase assay (Anaspec EGT, catalog number AS-72146) according to the manufacturer's instructions. The cell pellet was then saved for further analysis.

[0088] dPSC and embryoid body formation All three groups of rat embryonic fibroblast (REF) cells (siCntrlREF, siPrrx1REF, siSnai2REF) were transferred to a REF feeder layer (n=4 biological replicas per experiment) inactivated with mitomycin C (Sigma) in 12-well plates (4 duplicates) (7 days after initial siRNA transfection) and maintained in an incubator (37°C, 5% CO2) for 14 days in rat ESC medium (DMEM / F12 supplemented with 20% knockout serum substitute (KSR) and 8 ng / mL fibroblast growth factor (FGF-2)) (Gomes et al. 2010). After 14 days, the generated dPSCs were transferred to non-adherent culture dishes and suspended in differentiation medium for 8 days to induce embryoid body (EB) formation. Cell pellets from the matching experiments were saved for further analysis. dPSCs were differentiated into each of the three germ layers according to the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems®, catalog number SC027B).

[0089] Fixation and cryopreservation of embryoid bodies EB cells were collected from the culture dish using a pipette and transferred to a 15 mL conical tube until all material settled at the bottom of the tube. After gently removing the culture medium, the EB cells were fixed in a 4% paraformaldehyde (PFA) solution in PBS at room temperature for 30 minutes. The PFA solution was then removed, and the cells were washed with PBS for 5 minutes.

[0090] Immunofluorescence staining Immunofluorescence staining was performed on liver cancer (FT02B), rat embryonic fibroblast (REF), liver cancer cell-fibroblast hybrid (HF) cells, and PRRX1 and SNAI2 overexpressing HF clones (HF-PRRX1 and HF-SNAI2) grown on 12 mm glass coverslips. The coverslips were removed at appropriate time points, washed in ice-cold BSA / PBS, and then fixed in 4% paraformaldehyde (PFA). The cells were then permeableized with 0.1% Triton X-100 and incubated with PBSX containing 1% (w / v) donkey serum for 2 hours to block all existing rat binding sites. They were then incubated overnight in a humidified chamber at 4°C with appropriate monoclonal primary antibodies [PRRX1 (Novus Biologicals), SNAI2 (SantaCruz Biotechnologies), Col1a1 (Origene), Sox2 (Invitrogen USA), Nanog (Invitrogen USA), Myc (SantaCruz Biotechnologies) 5 ug / ml], followed by labeling with Alexafluor conjugate secondary antibody (1:100 dilution). The coverslips containing the cells were then incubated in the dark at room temperature for 10 minutes with 4',6-diamidino-2-phenylindole (DAPI) (1 μg / mL) solution and washed twice with deionized water to remove any remaining salts. Subsequently, the samples were placed on droplets of Prolong Antefade gold mounting medium (Life Technologies) and analyzed using a fluorescence microscope (Leica DMIL LED) with 20x magnification and an appropriate wavelength setting.

[0091] Immunofluorescence detection of the three germ layers The fixed specimens were embedded in agarose to trap and fix the embryoid bodies. Once the agarose solidified, it was maintained at 4°C for hardening. The specimens were then sent for further processing. The specimens were embedded in paraffin wax and sectioned to a thickness of 5–6 μm using a microtome. The sections were then attached to positively charged slides, the wax was removed in Histoclear, and the slides were rehydrated according to the protocol (Sivaguru et al., 2013). The rehydrated slides were made permeable with 0.5% Triton X-100 in phosphate-buffered saline and labeled with a three-color immunocytochemistry kit of three human germ layers from R&D Biosystems according to the manufacturer's protocol (catalog number SC022, R&D Systems, Inc., MN, USA). Briefly, the sections were blocked with 10% normal donkey serum (Jackson Laboratories, USA) containing 1% BSA (Thermofisher, USA). After blocking, sections were incubated with two conjugated primary antibody combinations for each strain of interest, e.g., ectoderm (anti-Sox1 conjugated with Northern Lights 493 and anti-Otx-2 conjugated with Northern Lights 557), mesoderm (anti-Brachyury Northern Lights 557 and anti-Hand1 conjugated with Northern Lights 637), and endoderm (anti-Gata-4 conjugated with Northern Lights 493 and anti-Sox17 conjugated with Northern Lights 637). Each combination was incubated in the dark at room temperature for 3 hours, washed with PBS containing BSA, counterstained with Hoechst nuclear dye (Thermofisher, USA), placed on Prolong Gold antifade mounting medium (Thermofisher, USA), left at room temperature for 24 hours, sealed with nail polish, and stored at 4°C.

[0092] Airyscan Super-Resolution Imaging Labeled sections were imaged under a Zeiss LSM880 laser scanning microscope equipped with the Airyscan Super-Resolution system. Collected excitation and emission wavelengths included 405 nm excitation (emissions collected between 410 and 460 nm), 488 nm excitation (emissions collected between 500 and 550 nm), 561 nm excitation (emissions collected between 570 and 615 nm), and 633 nm excitation (emissions collected between 650 and 700 nm). The entire stone thin section was scanned using a 63× plan apochromatic (NA 1.4 oil immersion objective) with an internal GaAsP photomultiplier tube detector. Numerous images were taken for each sample at different sections and locations. Images were recorded at a super-resolution sampling frequency of 40 nm per pixel. The raw data was processed for super-resolution using the same program's Zeiss Zen software and pseudo-colored to visualize the four channels.

[0093] Statistics and Reproducibility All in vitro differentiation and reprogramming experiments were independently repeated at least three times to obtain similar results. Details regarding sample size and reproducibility are provided in the chapter headings for each method. Where appropriate, data are presented as mean ± SEM. Where appropriate, group comparisons were performed using Student's t-tests assuming a two-tailed distribution.

[0094] Identification of transcription factors that determine the generation and lineage of somatic cell hybrids. Next-generation sequencing (NGS), including powerful techniques such as whole-genome microarrays (Shendure & Aiden 2012), DNA sequencing (Ozsolak & Milos 2011), RNA sequencing (Zhao et al. 2008), chromatin profiling (Furey 2012), and chromatin immunoprecipitation (ChIP) assays (Carey et al. 2009), is increasingly being used to map transcriptional regulatory circuits that mediate cell differentiation and gene loss. In addition, somatic cell hybridization, discovered by Barski et al. (Barski et al. 1960; Sorieul & Ephrussi 1961), was among the first techniques employed to investigate the molecular mechanisms underlying tissue-specific gene expression (see Figure 5). Because spontaneous cell fusion occurs at a very low frequency, Sendai virus or polyethylene glycol (PEG) is used as a fusion agent (Kao et al. 1974). The initial product of fusion is a heteronuclear symbiosis containing two or more distinct nuclei from both parents within a common cytoplasm. Only a small fraction of these heteronuclear symbiosis proceed to nuclear fusion and mitosis. The most well-known medium for selecting hybrids is hypoxanthine + aminopterin + thymidine (HAT) medium. Aminopterin uses simple sugars and amino acids to halt the biosynthesis of purines and pyrimidines. Hypoxanthine present in the medium is converted to guanine with the help of an enzyme known as hypoxanthine guanine phosphoribosyltransferase (HGPRT), while the phosphorylation of thymidine is catalyzed by thymidine kinase (TK). Only cells with active HGPRT and TK enzymes can survive and proliferate in HAT medium. For HAT to be used as a selective medium, parental cells must be deficient in one of these two enzymes, while somatic cell hybrid cells should have both of these enzymes in an active state, which is expected to help the hybrid survive in HAT medium.

[0095] Because hybrid systems take into account the net effects of the gene regulatory mechanisms of each parental cell line and present activated or repressed states of lineage-specific genes from particular parental cells, somatic hybridization may be a useful approach to understanding the molecular mechanisms of appropriate gene repression in lineages. Currently, there are no other approaches that can be used to simultaneously study the end consequences of multiple gene regulatory mechanisms on a global scale without introducing genetically modified alterations.

[0096] Using somatic cell hybridization techniques, the loss of lineage-specific genes has been studied in liver, muscle, bone marrow, and pituitary cells (Ringertz & Savage 1976). In particular, the loss of liver-specific gene expression in hepatocellular carcinoma X fibroblast hybrids has been extensively characterized (Killary & Fournier 1984; Killary et al. 1984) (see Figure 6). Numerous important regulatory transcription factors that play a contributing role in maintaining liver-specific cell lineages have been identified in hepatocellular carcinoma cells (Bulla & Fournier 1994; Bulla et al. 2004; Bulla et al. 2010).

[0097] In contrast, there has been relatively little research evaluating the gene regulatory networks involved in the loss of lineage-specific genes in fibroblasts. Fibroblasts are one of the heterogeneous mesenchymal progenitor cells widely found alongside almost all cell types as the primary stromal cell support system (Strutz et al. 1995). In addition to their supportive role, these cells play a role in structural organization, migration, and epithelial-mesenchymal transition (Acloque et al. 2009). Collagen, the connective tissue of fibroblasts, accounts for 25–35% of total protein in the normal mammalian body. The origin of fibroblasts is generally known to occur during the developmental transition from epithelial to mesenchymal phenotype induced by transforming growth factor (TGF) (Willis et al. 2005).

[0098] To study gene silencing, somatic cell hybridization techniques were employed to generate (hepatoma X fibroblast) FR(2) hybrids. Instead of using interspecies hybrids, which are typically used to generate cell hybrids (to facilitate the distinction of chromosomes derived from each parental cell), rat hepatoma and rat fibroblasts were used to generate FR(2) hybrids. This type of intraspecies hybrid has been proven to have a highly characterized and stable genotype that rarely exhibits chromosomal loss, making it a useful model for applying whole-genome microarray analysis to study tissue-specific gene loss phenomena.

[0099] To identify the most important lineage-defining TFs of REF, an unbiased and comprehensive experimental discovery approach was employed, including the generation of stable, same-species somatic fusion nuclear hybrids (HFs) created by fusing mesenchymal-derived REF with endodermal-derived rat hepatocellular carcinoma (RH) cells after genetically manipulating each cell line to possess unique, but distinct, xenobiotic resistance mechanisms (Bulla et al. 2012). Only true fusion nuclear hybrid cells that survived exposure to xenobiotics were selected, while the remaining parental cells that did not survive exposure to either xenobiotics were eliminated. By subjecting the resulting hybrid cells, in addition to the starting parental cells, to genome-wide transcriptome profiling, key master regulatory TFs that enforce the fate of mesenchymal and fibroblast cells were identified. These TFs are crucial for lineage preservation and maintenance, and their downregulation is also a necessary condition for the regaining of cell plasticity by these cells. Initially, 149 TFs showing more than 5-fold overexpression in REF compared to RH cells were selected. These are referred to as mesenchymal fibroblast-rich TFs (MFEFs) (Figure 1A). Next, MFEFs were screened, and 86 TFs that showed suppression greater than 2.5 times compared to REFs in the hybrid were identified. These are referred to as mesenchymal fibroblast-specific TFs (MFSFs) (Figure 1A). SNAI2 and PRRX1 were the top MFSFs that were suppressed compared to REFs in the hybrid (Figure 1D).

[0100] Next, the expression of candidate MFSFs identified by microarray analysis was verified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Suppression of fibroblast-specific genes (PRRX1, SNAI2, and Col1a1) was also confirmed in somatic cell hybrids (Figure 2C). To confirm the robustness of these identified MFSFs, the top two MFSFs (SNAI2 and PRRX1) were individually ectopically overexpressed in somatic cell hybrids. The generation of hybrid clones overexpressing G418-resistant SNAI2 and PRRX1, respectively, resulted in the reacquisition of fibroblast characteristics (morphology, structure, and functionality) in the hybrid cells. SNAI2 and PRRX1 clones, as confirmed by IF and qRT-PCR, achieved fibroblast-like spindle morphology (Figures 2A, 2B), reexpressed the fibroblast prototype gene Col1a1 (Figures 2B, 2C), and, in particular, exhibited significantly increased motility compared to hybrids in response to TGFβ stimulation, a characteristic fibroblast-specific functional trait (Acharya et al. 2008) (Figure 2D). On the other hand, individual siRNA-mediated knockdown of SNAI2 and PRRX1 in REF cells resulted in siSnai2REF and siPrrx1 REF cells, respectively, which showed loss of spindle morphology, loss of Col1a1 expression, and reduced motility compared to the parental REF cells.

[0101] Suppressing a transcription factor that defines a specific lineage in isolation makes somatic cell reprogramming possible. After confirming the essential importance of SNAI2 and PRRX1 in preserving the structural and functional identity of mesenchymal-derived REF cells, we then determined whether the forced transient suppression of either of these two critical TF cells (utilizing a sequential transfection protocol using siRNA specific to either SNAI2 or PRRX1) could release the limitations on cell fate bound by mesenchymal and fibroblast identity, making these cells more plastic and easier to reprogram. To test this, siSnai2REF and siPrrx1REF cells were incubated in the respective growth media of closely related mesenchymal cell lines. After culturing in adipogenesis medium for 3 days, followed by adipogenesis maintenance medium for 11 days, siSnai2REF and siPRRX1 REF cells achieved a spherical morphology filled with lipid droplets similar to adipocytes. Lipid cells were visualized using light microscopy and stained red with Oil Red O (Figures 3A, 3D). These cells expressed Cebpa, a TF appropriate for adipocyte lineages, as evaluated by qRT-PCR (Figure 3G) and immunofluorescence (IF) (Figure 3A). Similarly, knockdown of individual SNAI2 or PRRX1 in parenteral REF cells by siRNA transfection, and maintenance of siSnai2REF and siPRRX1REF cells for 14 days in either osteogenic or chondrogenic medium resulted in functional osteocytes and chondrocytes, respectively. Lineage-appropriate function, which involves bone formation, was demonstrated by positive staining with Alizarin Red (Figures 3B, 3E), while chondrogenesis was confirmed by positive staining with Alcian Blue (Figures 3C, 3F). The expression of osteocyte and chondrocyte lineage-inducing TFs Runx2 and Sox9, respectively, was also confirmed using qRT-PCR (Figures 3H, 3I) and immunofluorescence (IF) (Figures 3B, 3C).

[0102] Next, we determined whether transient suppression of either SNAI2 or PRRX1 and incubation under suitable cell culture conditions could effectively transform parental fibroblasts into a more primitive precursor cell state before developmentally differentiating them into mesenchymal cells. When siSnai2REF and siPRRX1REF cells were cultured in rat mesenchymal stem cell (rMSC) medium for 14 days, they resulted in transformation into dedifferentiated pluripotent stem cells (dMSCs) with altered morphology and enhanced expression of prototype MSC TF Myc (verified by both IF (Figure 4A) and qRT-PCR (Figure 4C)). The dMSCs showed significantly enhanced expression of alkaline phosphatase, an indicator of undifferentiated stem cell activity (Figure 4B). When siSnai2REF and siPRRX1REF cells were cultured for 14 days on mitomycin C-inactivated REF in rat embryonic stem cell (rESC) medium (containing 2i / LIF) (Jackson et al. 2010), dedifferentiated pluripotent stem cell characteristics were achieved only in the siSnai2REF group (Figure 4D), and characteristic pluripotency factors such as Sox2, Klf4, and Nanog were expressed (Figures 4E, 4F). Since these cells were obtained by removing the reprogramming barrier (training-preserving TF) that inhibits dedifferentiation, the inventors refer to these cells as dedifferentiated pluripotent stem cells (dPSCs). When dPSC suspension cultures were placed in rESC differentiation medium for 8 days, they formed embryoid bodies composed of all three germ layers. When examined by IF staining and confocal microscopy, they stained positively for triple germ layer-specific TFs, namely Sox2 and Otx2 (ectoderm) (Figure 4F), Brachyury and Hand1 (mesoderm) (Figure 4G), and Gata4 and Sox17 (endoderm) (Figure 4H).

[0103] Cell-specific identity is determined by the expression of cell type-specific transcription factors and the underlying epigenetic state. Both TF-mediated cell reprogramming (where target cell-specific TFs are ectopically overexpressed) and small molecule-driven reprogramming (where pathway modulators induce changes in the epigenetic environment) are effective means employed to generate iPSCs or differentiated cells of a desired lineage. However, a fundamental drawback of such protocols remains the low reprogramming efficiency and the generation of partially transformed cells, primarily due to the retention of the starting cell's transcriptional network and residual epigenetic memory (Nashun et al. 2015). Studies in such moderately transformed cells have shown that some specific genes in the starting cells are persistently expressed, and their siRNA-mediated knockdown significantly improves reprogramming efficiency (Mikkelsen et al. 2008, Ebrahimi et al. 2015). Multiple gene knockdowns in the transcriptional regulatory network (TRN) of starting fibroblasts resulted in differentiated adipocytes (Tomaru et al. 2014), but transdifferentiation was not achieved via the repression of a single key, lineage-inducible transdifferentiating factor (TF) in the starting cells. Similarly, while TFs that interfere with dedifferentiation have been demonstrated to induce cell type-specific transcriptional profiles, repression of identified TFs did not allow for dedifferentiation (Hikichi et al. 2013).SNAI2 was one of the earliest genes to be repressed in conventional OSKM-mediated cell reprogramming efforts (Mikkelsen et al. 2008, Polo et al. 2012, Cachiarelli et al. 2015), and it is noteworthy that the repression of PRRX1 or SNAI2 enhanced both OSKM (Yang et al. 2011) and Nanog (Gingold et al. 2014), respectively, in the absence of any exogenous transdifferentiation TF or pluripotency TF (e.g., OSKM) or exogenous small molecule cocktails, resulting in successful transdifferentiation to an alternative cell fate or dedifferentiation to a pluripotent / pluripotent primitive cell state. This is because it is known that lineage constraint, preservation, and maintenance are regulated by the starting cell TRN, which consists of multiple lineage-specific TFs that work together to enforce and maintain the cell lineage through transcriptome and epigenetic control. As a result, the existence of a single key "gatekeeper" TF (GTF) within the lineage-constrained cell TRN that can independently regulate the switch between lineage preservation and cell plasticity has never been demonstrated, and the idea that important biological functions such as the enforcement and preservation of lineage (after lineage constraint has already occurred) can be disrupted by manipulating a single major TF member of the starting cell TRN was considered impossible.

[0104] Using REF as a model system, these results demonstrate that transient suppression of TF, which preserves a single key lineage of starting cells (in this case, SNAI2 or PRRX1), can overcome the lineage-preserving or reprogramming barriers imposed by TRN in starting cells, thereby making the cells more plastic and, when incubated in appropriate media, can lead to direct lineage conversion of the starting cells to another cell type (e.g., adipocyte, chondrocyte, or osteocyte). Suppression of either PRRX1 or SNAI2 allowed REF to dedifferentiate into pluripotent dMSCs, while SNAI2 likely exerts its control at a more primitive stage in the hierarchy of mesenchymal cell development from pluripotent precursors than PRRX1, as demonstrated by the ability of siSnai2 to dedifferentiate REF into dPSCs. This may be due to Snai2 first exerting transcriptional regulatory control in CD326-CD56+ embryonic mesoderm progenitor cells (expression 160-fold more than ESCs) on day 3.5, which occurs before the initial expression of Prrx1 (a TF more specific to mesenchymal sublineages) in the mesodermal phylogenetic hierarchy (Evseenko et al. 2010). The described method is thought to reset cells to an early point in their developmental timeline, and its transient nature is thought to lead to the desuppression of plasticity-maintaining factors (e.g., pluripotency TFs), and then allow their intrinsic differentiation mechanisms to proceed as guided by the components provided to their growth medium. The detailed molecular mechanisms underlying the cell transformation brought about by the transient suppression of SNAI2 and PRRX1 remain unclear, as all possible chromatin environmental changes that may have occurred by such RNAi manipulation remain unknown. In conclusion, siRNA-mediated transient repression of TFs that define somatic cell lineage is a controversial strategy for facilitating the switching of cellular identity to a different transdifferentiated cell fate, or to a dedifferentiated multipotent or pluripotent cell state.This could potentially enhance the reprogramming efficiency of TF / small molecule-driven protocols in terms of both cell yield and process time, ultimately helping to achieve the goal of reliably obtaining clinically important somatic or pluripotent stem cells for therapeutic use.

[0105] Example 2: The development of human mesoderm lines is validated in a rat model, and SNAI2 is established as a gatekeeper LDTF. Among the TRNs that preserve the cell lineage of TFs, there may be specific "gatekeeper" TFs that play a crucial role, acting as molecular wedges, and their inhibition alone may allow cell reprogramming on its own. It is important to note that lineage-specific TFs involved in early cell fate determination and lineage constraint, such as Eomes and Brachyury for mesoderm constraint, are known to suppress pluripotency and neuroectoderm gene programs (Tosic et al. 2019). However, they are active at the endomesoderm stage (Tsankov et al. 2015) and subsequently show low or undetectable expression in downstream mesoderm-derived cells such as fibroblasts. Therefore, they cannot be used for siRNA-mediated inhibition as a method to desuppress pluripotency factors and allow the acquisition of plasticity. The desired lineage-specific "gatekeeper" TFs must be expressed in differentiated cells, be usable for siRNA-mediated inhibition, and serve the purpose of lineage preservation and maintenance after lineage constraint has already occurred.

[0106] Confirmation of the rat model To assess whether the above findings in the rat in vitro model are related to and thus confirmed by human cell development and differentiation, we used RNA-Seq analysis to examine the expression trends of mesenchymal fibroblast-specific genes (MFSGs) (SNAI2, PRRX1) and the repression trends of embryonic stem cell genes (ESCG, e.g., POU5F1, SOX2, NANOG, LIN28A) (Ben-Porath et al. 2008) across the range of mesodermal lineage progression in 21 different human cell and tissue species obtained from the development of the publicly available ENCODE (Consortium 2012; Davis et al. 2018) and ROADMAP (Roadmap Epigenomics et al. 2015) databases.

[0107] Most members of the ESCG signature showed strong expression in ESCs, weak to undetectable expression in fibroblasts, and a sudden shift in their expression profiles during developmental mesoderm. Most members of the MFSG signature showed strong expression in fibroblasts, weak to undetectable expression in ESCs, and similarly a sudden shift in their expression profiles during developmental mesoderm. As expected, SNAI2 and PRRX1 first showed a significant increase in their expression during mesoderm. Based on the sudden shift in gene expression profiles, we focused our efforts on the mesoderm stage to test and clarify the mechanistic criteria for these observations.

[0108] SNAI2 is the gatekeeper. Enhancers are known to play an important regulatory role in both pluripotency and lineage-specific gene regulation (Gokbuget & Blelloch 2019). Therefore, we conducted a study to identify the crossover between enhancer chromatin modifications occurring at the mesodermal stage and their potential SNAI2 genomic targets in regulating ESCG repression and MFSG expression.

[0109] First, using publicly available histone-modified ChIP-Seq (H3K4me1, H3K4me3, H3K27ac, H3K27me3, H3K9me3) data from the ENCODE (Consortium 2012; Davis et al. 2018) and ROADMAP (Roadmap Epigenomics et al. 2015) databases, we defined specified active enhancers based on overlapping H3K4me1 and H3K27ac peaks, as well as suppressive enhancers based on overlapping H3K4me1 and H3K27me3 peaks in ChIP-Seq analysis, using established methods (Creyghton et al. 2010; Zentner et al. 2011).

[0110] Next, ChIP-Seq data from human HUES64ESC-derived CD56+ mesoderm were available for mesoderm TFs (SNAI2, HAND1, CDX2) and ESC TFs (POU5F1, SOX2, NANOG) (Tsankov et al. 2015), and these were analyzed. The SNAI2 peak was found to co-localize more broadly with both active and repressive enhancers than the HAND1 or CDX2 peak, as elucidated by TF ChIP-Seq analysis (Figures 7A-7H). This suggests that SNAI2 plays a dominant role in mesoderm in the enhancement of transcription of observed major lineage-specific genes (see Figures 8, 10A-10H; PRRX1, FOXF1) and the repression of transcription of observed major ESC genes (see Figures 9, 10A-10H; SOX2, ZIC2), as confirmed by matching RNA-Seq analysis. A significant increase in H3K27 acetylation was observed in the SNAI2-binding active MFSG enhancer (Figure 8), while a significant decrease in H3K27 acetylation and a corresponding significant increase in H3K27 trimethylation were observed in the SNAI2-repressed ESCG enhancer (Figure 9). RNA-Seq profiling confirmed that the transcriptional effects on the SNAI2-binding MFSG-activating enhancer and the ESCG-repressing enhancer observed in human mesoderm were sustained, and that mesodermal phylogenetic development became more pronounced and established as it progressed from the fetal stage to differentiated adult fibroblasts (Figures 8 and 9). Since SNAI2 binds to both the active enhancer and the repressor at the mesodermal stage, it acts as a gatekeeper LDTF by regulating both phylogenetic gene expression and plasticity gene repression.

[0111] Furthermore, of the 38 ESC gene targets that have repressors at their respective loci in human mesoderm, the combination of SNAI2, HAND1, and CDX2 binds to 33 of them. SNAI2 alone binds to 32 of them. Therefore, when siSNAI2 is employed to induce somatic cell reprogramming, the single greatest impairment (in mesodermal cells) to ESC gene expression and the ability to acquire pluripotency is effectively removed.

[0112] These findings suggest that transient inhibition of a single key gatekeeper TF in lineage-constrained cells is sufficient to achieve cell plasticity and reprogramming.

[0113] The dynamic changes in both activating and repressive histone alterations occurring in mesoderm enhancers were observed to occur transiently and simultaneously in the MFSG and ESCG targets, respectively, and to spatially overlap with SNAI2 genome binding. SNAI2 itself continues to present strong gene expression, along with the persistence of the broad-spectrum switching in the histone alteration profiles (in the MFSG and ESCG mesoderm enhancers) observed throughout the progression of mesoderm lineages across 21 different human cell and tissue species (Supplementary Figures 2, 3). Therefore, SNAI2 plays a superficially important role, if not the leading role, in regulating both the sustained expression of mesoderm lineage genes and the sustained repression of pluripotency genes, respectively. However, the precise order and sequence of SNAI2-binding events contributing to the regulation of plasticity program repression and mesoderm lineage maintenance have not yet been established. Similarly, the number and identity of potential co-activators and corepressor factors that may function in conjunction with SNAI2 remain unknown. SNAI2 has been reported to act as a transcriptional repressor by supplementing histone deacetylase 1 / 2 (HDAC1 / 2) and reducing the H3K27 acetylation activation mark (Soleimani et al. 2012). SNAI2 is also known to function in cooperation with Polycomb repression complex 2 (PRC2) to increase the H3K27 trimethylation repression mark and act as a transcriptional repressor (Tien et al. 2015). While the epigenetic regulatory mechanisms employed by SNAI2 have been demonstrated in other cellular environments, it remains unclear whether similar mechanisms are also utilized by SNAI2 in activating mesodermal lineage gatekeeper function, as reported herein.

[0114] In conclusion, these results expand our molecular understanding of lineage constraints, detail, and fate preservation, revealing that certain critical gatekeeper TFs, the master regulators of cell fate, can also be considered to constitute the molecular Achilles heel in specific cellular identity. They act as molecular wedges in the preservation of cell fate and may in some cases be the single most important reason why cellular identity is not immutable. If gatekeeper TFs for specific cell types can be precisely identified, siRNA-mediated cell-specific transient repression of such gatekeeper TFs could be a controversial strategy to help achieve a cellular identity switch of such cells to another transdifferentiated or dedifferentiated plutoppotent / pluripotent cell state, without requiring permanent genetic modification of the targeted cells. In addition, this may potentially enhance the reprogramming efficiency of exogenous TF / small molecule-driven protocols in terms of both cell yield and process time, ultimately helping to achieve the goal of reliably obtaining clinically important somatic or plutoppotent / pluripotent stem cells for therapeutic use. The following are descriptions of the embodiments of the claims originally filed for this application. [1] Somatic fibroblasts comprising an siRNA or shRNA molecule that inhibits the expression of a single lineage-defining transcription factor, and capable of transdifferentiating into another lineage of somatic cells without viral or nonviral delivery of the exogenous transcription factor and / or modulator. [2] The somatic fibroblast according to [1], wherein the transcription factor defining the single lineage is SNAI2, PRRX1, HAND1, CDX2, or a combination thereof. [3] The somatic fibroblast according to any one of [1] to [2], wherein the somatic cell of the other lineage is a somatic cell of the adipocyte lineage, a somatic cell of the osteogenic cell lineage, or a somatic cell of the chondrogenic cell lineage. [4] Somatic fibroblasts comprising an siRNA or shRNA molecule that inhibits the expression of a single lineage-defining transcription factor, which are capable of dedifferentiating into multipotent or pluripotent stem cells without viral or nonviral delivery of exogenous transcription factors and / or modulators. [5] The somatic fibroblast according to [4], wherein the transcription factor defining the single lineage is SNAI2, PRRX1, HAND1, CDX2, or a combination thereof. [6] Somatic fibroblasts according to any one of [4] to [5], wherein the compound or pluripotent stem cell is a dedifferentiated compound mesenchymal stem cell or a dedifferentiated pluripotent stem cell. [7] A population of transdifferentiated somatic cells comprising a population of reprogrammed fibroblasts without permanent genetic alteration, wherein the population of reprogrammed fibroblasts is converted from a population of lineage-constrained fibroblasts to the population of transdifferentiated somatic cells with an efficiency of greater than 1%. [8] The population of somatic cells that has been differentiated, wherein the population of somatic cells that has been differentiated is a population of somatic cells of the adipocyte lineage, a population of somatic cells of the osteogenic cell lineage, or a population of somatic cells of the chondrogenic cell lineage, as described in [7]. [9] A population of transdifferentiated somatic cells as described in [7] or [8], for use in tissue reconstitution procedures.

[10] A population of somatic cells that have been differentiated and transmuted according to any one of [7] to [9], wherein the tissue reconstruction procedure is reconstructive plastic surgery or reconstructive orthopedic surgery.

[11] A population of dedifferentiated stem cells comprising a population of reprogrammed fibroblasts without permanent genetic alteration, wherein the population of reprogrammed fibroblasts is induced to become the population of dedifferentiated stem cells from a population of lineage-constrained fibroblasts with an efficiency of greater than 1%.

[12] The population of dedifferentiated stem cells according to

[11] , wherein the population of dedifferentiated stem cells is a population of dedifferentiated multipotent mesenchymal stem cells or a population of dedifferentiated pluripotent stem cells.

[13] A population of dedifferentiated stem cells as described in

[11] or

[12] for use in tissue reconstitution procedures.

[14] A population of dedifferentiated stem cells as described in

[11] or

[12] , for use in wound treatment.

[15] A population of dedifferentiated stem cells as described in

[11] or

[12] for use in transplant surgery.

[16] A cell culture system for producing a population of differentiated somatic cells, A population of somatic cells that are phylogenetically constrained; siRNA or shRNA molecules that inhibit the expression of a single lineage-defining transcription factor; and Culture medium containing a cell culture medium specific to the aforementioned population of differentiated somatic cells Includes, A cell culture system that does not contain exogenous transcription factors or modulators specific to the differentiated somatic cells.

[17] The cell culture system according to

[16] , wherein the population of lineage-restricted somatic cells is lineage-restricted fibroblasts.

[18] The cell culture system according to

[16] or

[17] , wherein the transcription factor defining the single line is SNAI2, PRRX1, HAND1, CDX2, or a combination thereof.

[19] The cell culture system according to any one of

[16] to

[18] , wherein the population of differentiated somatic cells is an adipocyte lineage, an osteogenic cell lineage, or a chondrogenic cell lineage.

[20] A cell culture system for producing a population of dedifferentiated stem cells, A population of somatic cells that are phylogenetically constrained; siRNA or shRNA molecules that inhibit the expression of a single lineage-defining transcription factor; and Culture medium containing a stem cell medium specific to the aforementioned population of dedifferentiated stem cells Includes, A cell culture system that does not contain exogenous transcription factors or modulators specific to the differentiated somatic cells.

[21] The cell culture system according to

[20] , wherein the population of lineage-restricted somatic cells is lineage-restricted fibroblasts.

[22] The cell culture system according to

[20] or

[21] , wherein the transcription factor defining the single line is SNAI2, PRRX1, HAND1, CDX2, or a combination thereof.

[23] The cell culture system according to any one of

[20] to

[22] , wherein the population of dedifferentiated stem cells is a population of dedifferentiated multipotent mesenchymal stem cells or a population of dedifferentiated pluripotent stem cells.

[24] A method for producing somatic cells that have undergone differentiation, Introducing siRNA or shRNA molecules that temporarily inhibit the expression of a single lineage-defining transcription factor into lineage-constrained somatic cells; and Incubating the lineage-restricted somatic cells in a culture medium specific to the differentiated somatic cells. A method comprising, for executing an exogenous transcription factor and / or modulator without viral or nonviral delivery.

[25] The method according to

[24] , wherein the lineage-restricted somatic cells are lineage-restricted fibroblasts.

[26] The method according to

[24] or

[25] , wherein the transcription factor defining the single line is SNAI2, PRRX1, HAND1, CDX2, or a combination thereof.

[27] The method according to any one of

[24] to

[26] , wherein the differentiated somatic cells are cells of the adipocyte lineage, cells of the osteogenic cell lineage, or cells of the chondrogenic cell lineage.

[28] A method for producing dedifferentiated stem cells, Introducing siRNA or shRNA molecules that temporarily inhibit the expression of a single lineage-defining transcription factor into lineage-constrained somatic cells; and Incubating the lineage-restricted somatic cells in a culture medium specific to the dedifferentiated stem cells. A method comprising, for executing an exogenous transcription factor and / or modulator without viral or nonviral delivery.

[29] The method according to

[28] , wherein the lineage-restricted somatic cells are lineage-restricted fibroblasts.

[30] The method according to

[28] or

[29] , wherein the transcription factor defining the single line is SNAI2, PRRX1, HAND1, CDX2, or a combination thereof.

[31] The method according to any one of

[28] to

[30] , wherein the dedifferentiated stem cells are dedifferentiated compound pluripotent mesenchymal stem cells or dedifferentiated pluripotent stem cells.

[32] An autologous tissue graft containing a population of differentiated somatic cells, (i) The population of differentiated somatic cells is derived from a population of lineage-constrained somatic cells obtained from a subject for use in tissue reconstitution treatment, (ii) The population of transdifferentiated somatic cells includes a population of reprogrammed fibroblasts that have not undergone permanent genetic modification, and the population of reprogrammed fibroblasts is converted from the population of lineage-constrained fibroblasts to the population of transdifferentiated somatic cells with an efficiency of more than 1%. Autologous tissue graft.

[33] The autologous tissue graft according to

[32] , wherein the population of differentiated somatic cells is produced by the method of any one of

[30] to

[35] .

[34] An autologous tissue graft containing a population of dedifferentiated stem cells, (i) The population of dedifferentiated stem cells is derived from a population of lineage-constrained somatic cells obtained from a subject for use in tissue reconstructive treatment, wound treatment, or transplantation surgery. (ii) The population of dedifferentiated stem cells includes a population of reprogrammed fibroblasts that have not undergone permanent genetic modification, and the population of reprogrammed fibroblasts is induced to become the population of dedifferentiated stem cells from a population of lineage-constrained fibroblasts with an efficiency of more than 1%. Autologous tissue graft.

[35] The autologous tissue graft according to

[34] , wherein the population of dedifferentiated stem cells is produced by the method described in any one of

[28] to

[31] .

[36] A method for treating a condition, comprising grafting or transplanting a population of reprogrammed somatic cells, dedifferentiated stem cells, or transdifferentiated somatic cells into or on the surface of the tissue or organ of the condition.

[37] The method according to

[36] , wherein the population of reprogrammed somatic cells, dedifferentiated stem cells, or transdifferentiated somatic cells is part of an autologous tissue graft.

[38] The siRNA or shRNA molecule is an siRNA molecule containing the siRNA sequence and passenger strand sequence shown in Table 1 or Table 2, as described in any one of [1] to [6],

[16] to

[31] , somatic cell fibroblasts, cell culture system or method.

[39] The siRNA or shRNA molecule is an shRNA molecule containing the siRNA sequence, passenger strand sequence and loop sequence shown in Table 1 or Table 2, as described in any one of [1] to [6],

[16] to

[31] .

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Claims

1. Somatic fibroblasts comprising an siRNA or shRNA molecule that inhibits the expression of a single lineage-defining transcription factor, wherein the single lineage-defining transcription factor is SNAI2 or PRRX1, the somatic fibroblasts do not contain exogenous transcription factors and / or small molecule modulators (chemical reprogramming), and are capable of transdifferentiating into another lineage of somatic cells without viral or nonviral delivery of exogenous transcription factors and / or modulators.

2. The somatic fibroblast according to claim 1, wherein the somatic cell of the other lineage is a somatic cell of the adipocyte lineage, a somatic cell of the osteogenic cell lineage, or a somatic cell of the chondrogenic cell lineage.

3. Somatic fibroblasts comprising an siRNA or shRNA molecule that inhibits the expression of a single lineage-defining transcription factor, wherein the single lineage-defining transcription factor is SNAI2 or PRRX1, and the somatic fibroblasts do not contain exogenous transcription factors and / or small molecule modulators (chemical reprogramming), and are capable of dedifferentiating into multipotent or pluripotent stem cells without viral or nonviral delivery of exogenous transcription factors and / or modulators.

4. The somatic fibroblast according to claim 3, wherein the compound or pluripotent stem cell is a dedifferentiated compound mesenchymal stem cell or a dedifferentiated pluripotent stem cell.

5. A cell culture system for producing a population of differentiated somatic cells, A population of somatic cells that are lineage-constrained; A siRNA or shRNA molecule that inhibits the expression of a single lineage-defining transcription factor, wherein the single lineage-defining transcription factor is SNAI2 or PRRX1; and Culture medium containing a cell culture medium specific to the aforementioned population of differentiated somatic cells Includes, A cell culture system that does not contain exogenous transcription factors or modulators specific to the differentiated somatic cells.

6. The cell culture system according to claim 5, wherein the population of lineage-restricted somatic cells is lineage-restricted fibroblasts.

7. The cell culture system according to claim 5 or 6, wherein the population of differentiated somatic cells is an adipocyte lineage, an osteogenic cell lineage, or a chondrogenic cell lineage.

8. A cell culture system for producing a population of dedifferentiated stem cells, A population of somatic cells that are lineage-constrained; A siRNA or shRNA molecule that inhibits the expression of a single lineage-defining transcription factor, wherein the single lineage-defining transcription factor is SNAI2 or PRRX1; and Culture medium containing a stem cell medium specific to the aforementioned population of dedifferentiated stem cells Includes, A cell culture system that does not contain exogenous transcription factors or modulators specific to the dedifferentiated stem cells.

9. The cell culture system according to claim 8, wherein the population of lineage-restricted somatic cells is lineage-restricted fibroblasts.

10. The cell culture system according to claim 8 or 9, wherein the population of dedifferentiated stem cells is a population of dedifferentiated multipotent mesenchymal stem cells or a population of dedifferentiated pluripotent stem cells.

11. A method for producing differentiated somatic cells, Introducing a siRNA or shRNA molecule that temporarily inhibits the expression of a single lineage-defining transcription factor into lineage-constrained somatic cells, where the single lineage-defining transcription factor is SNAI2 or PRRX1; and Incubating the lineage-restricted somatic cells in a culture medium specific to the differentiated somatic cells. A method comprising, for executing an exogenous transcription factor and / or modulator without viral or nonviral delivery.

12. The method according to claim 11, wherein the lineage-restricted somatic cells are lineage-restricted fibroblasts.

13. The method according to claim 11 or 12, wherein the differentiated somatic cells are cells of the adipocyte lineage, cells of the osteogenic cell lineage, or cells of the chondrogenic cell lineage.

14. A method for producing dedifferentiated stem cells, Introducing a siRNA or shRNA molecule that temporarily inhibits the expression of a single lineage-defining transcription factor into lineage-constrained somatic cells, where the single lineage-defining transcription factor is SNAI2 or PRRX1; and Incubating the lineage-restricted somatic cells in a culture medium specific to the dedifferentiated stem cells. A method comprising, for executing an exogenous transcription factor and / or modulator without viral or nonviral delivery.

15. The method according to claim 14, wherein the lineage-restricted somatic cells are lineage-restricted fibroblasts.

16. The method according to claim 14 or 15, wherein the dedifferentiated stem cells are dedifferentiated multipotent mesenchymal stem cells or dedifferentiated pluripotent stem cells.

17. The somatic fibroblast according to any one of claims 1 to 4, wherein the siRNA or shRNA molecule is an siRNA molecule comprising the siRNA sequence and passenger strand sequence shown in Table 1 or Table 2.

18. The somatic fibroblast according to any one of claims 1 to 4, wherein the siRNA or shRNA molecule is an shRNA molecule comprising the siRNA sequence, passenger strand sequence and loop sequence shown in Table 1 or Table 2.

19. The cell culture system according to any one of claims 5 to 10, wherein the siRNA or shRNA molecule is an siRNA molecule containing the siRNA sequence and passenger strand sequence shown in Table 1 or Table 2.

20. The method according to any one of claims 11 to 16, wherein the siRNA or shRNA molecule is an siRNA molecule comprising the siRNA sequence and passenger strand sequence shown in Table 1 or Table 2.

21. The cell culture system according to any one of claims 5 to 10, wherein the siRNA or shRNA molecule is an shRNA molecule comprising the siRNA sequence, passenger strand sequence and loop sequence shown in Table 1 or Table 2.

22. The method according to any one of claims 11 to 16, wherein the siRNA or shRNA molecule is an shRNA molecule comprising the siRNA sequence, passenger strand sequence and loop sequence shown in Table 1 or Table 2.

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