Methods for reprogramming cells

Abstract

This disclosure provides a method for reprogramming cells in the presence of one or more reprogramming factors, reprogrammable cells and their uses, and a kit containing said reprogramming factors.

Description

Invention Field

[0001] This disclosure generally relates to methods for reprogramming cells. Background Technology

[0002] Normally functioning corneal endothelial cells (CECs) maintain corneal transparency and appropriate fluid levels, for example, by balancing fluid “leaking” into the stroma with continuous active pumping from the stroma to move fluid into the anterior chamber of the eye.

[0003] It has been reported that corneal endothelial cells have little or no capacity for proliferation in vivo, and therefore cannot be naturally replaced when damaged or otherwise lost. In the human body, the corneal endothelial cell layer is most densely packed at birth, and thereafter the cell density decreases rapidly with eye growth (allowing the same number of cells to cover a larger area). Subsequently, corneal cell density gradually decreases with age, clearly reflecting the gradual loss of unreplaced cells. As cell density decreases, each cell unfolds and covers a larger area to maintain the barrier and pump functions of the cell layer. However, once the cell density drops too low (below approximately 500 to 1000 cells / mm²), its function is impaired, leading to corneal opacity, stromal edema, loss of visual acuity, and ultimately blindness.

[0004] Although different treatments have been developed, there is a great need for new corneal endothelial reconstruction techniques. Summary of the Invention

[0005] This disclosure provides a method for reprogramming a first type of cell into a second type of cell, comprising culturing the first type of cell in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors includes a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor-β (TGFβ) inhibitor, and a cyclic AMP inducer.

[0006] In some embodiments, the first group of reprogramming factors further comprises basic fibroblast growth factor (bFGF), DNA methyltransferase inhibitors, histone methyltransferase inhibitors (e.g., DOT1L inhibitors), histone deacetylase (HDAC) inhibitors, BMP4, or combinations thereof.

[0007] In some embodiments, the first set of reprogramming factors consists of: (a) a GSK3 inhibitor, a TGFβ inhibitor, and a cyclic AMP inducer; (b) a GSK3 inhibitor, a TGFβ inhibitor, a cyclic AMP inducer, and bFGF; or (c) a GSK3 inhibitor, a TGFβ inhibitor, a cyclic AMP inducer, a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor (e.g., a DOT1L inhibitor), and a histone deacetylase inhibitor.

[0008] In some embodiments, the GSK3 inhibitor is selected from the group consisting of: CHIR99021, LiCl, Li2CO3, and BIO ((2'Z,3'E)-6-bromoindirubin-3'-oxime), TD114-2, Kenpaullone, TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A014418, FRATide, indirubin 3'-oxime, and L803.

[0009] In some embodiments, the TGFβ inhibitor is selected from the group consisting of: SB431542, Repsox, 616452, LDN193189, A8301, GW788388, SD208, SB525334, LY364947, D4476, SB505124, and Tranilast.

[0010] In some embodiments, the cyclic AMP inducers are forskolin, IBMX, Rolipram, 8BrcAMP, prostaglandin E2 (PGE2), NKH477, dibutyryl-cyclic adenosine monophosphate (DBcAMP), and Sp-8-Br-cAMPs.

[0011] In some embodiments, the DNA methyltransferase inhibitor is selected from the group consisting of 5-aza-dC, 5-azacytidine, and RG108.

[0012] In some embodiments, the DOT1L inhibitor is EPZ004777.

[0013] In some embodiments, the histone deacetylase inhibitor is selected from the group consisting of: valproic acid (VPA), trichostocin A (TSA), vorinostat, depsipeptide, Trapoxin, Depudecin, FR901228, and butyrate.

[0014] In some embodiments, the first type of cell is a somatic cell. In some embodiments, the somatic cell is derived from the mesoderm, ectoderm, or endoderm.

[0015] In some embodiments, the somatic cells are fibroblasts. In some embodiments, the fibroblasts are selected from the group consisting of: mouse embryonic fibroblasts (MEF), mouse tail tip fibroblasts (TTF), human embryonic fibroblasts (HEF), human newborn fibroblasts (HNF), adult fibroblasts (HAF), human foreskin fibroblasts (HFF), and mixtures thereof.

[0016] In some embodiments, the somatic cells are exfoliated human renal epithelial cells.

[0017] In some embodiments, the first type of cell is a stem cell. In some embodiments, the stem cells are selected from the group consisting of: human umbilical cord mesenchymal stem cells, human embryonic stem cells, and induced pluripotent stem cells (iPSCs).

[0018] In some embodiments, the second type of cell is a stem cell. In some embodiments, the stem cell is a neural crest cell-like cell (NCC-like cell). In some embodiments, the NCC-like cell is positive for P75, Hnk1, AP2α, and Sox10.

[0019] In some embodiments, the first type of cells are cultured in the presence of the first set of reprogramming factors for (a) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days or (b) no more than 20, 19, 18, 17, 16, 15, 14, 13 or 12 days.

[0020] This disclosure provides a method for reprogramming a second type of cell into a third type of cell, comprising culturing the second type of cell in the presence of a second set of reprogramming factors, wherein the second set of reprogramming factors includes a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0021] In some embodiments, the second set of reprogramming factors further comprises BMP4 and / or a DNA methyltransferase inhibitor. In some embodiments, the casein kinase 1 inhibitor is CKI-7. In some embodiments, the DNA methyltransferase inhibitor is selected from the group consisting of 5-azacytidine, 5-aza-dC, and RG108.

[0022] In some embodiments, the third type of cell is a somatic cell. In some embodiments, the somatic cell is a corneal endothelial cell (CEC)-like cell (CEC-like cell). In some embodiments, the CEC-like cell exhibits ZnO-1 and Na+ content. + / K + -ATPase positive.

[0023] In some embodiments, the second type of cells are cultured in the presence of the second set of reprogramming factors for (a) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days or (b) no more than 20, 19, 18, 17, 16, 15, 14, 13 or 12 days.

[0024] This disclosure provides a method for reprogramming a first type of cell into a third type of cell, comprising step (a) culturing the first type of cell in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors includes a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGFβ) inhibitor, and a cyclic AMP inducer, and step (b) culturing the cell obtained from step (a) in the presence of a second set of reprogramming factors, wherein the second set of reprogramming factors includes a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0025] In some embodiments, the method further includes washing the cells obtained from step (a) before starting step (b). In some embodiments, there is no washing step between step (a) and step (b).

[0026] This disclosure provides a method for reprogramming a first type of cell into a third type of cell, comprising culturing the first type of cell in the presence of a first set of reprogramming factors and a second set of reprogramming factors, wherein the first set of reprogramming factors includes a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGFβ) inhibitor, and a cyclic AMP inducer, and the second set of reprogramming factors includes a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0027] This disclosure provides for NCC-like cell populations generated according to the methods provided herein.

[0028] This disclosure provides a population of corneal endothelial cell-like cells (CEC-like cells) generated according to the methods provided herein.

[0029] This disclosure provides a composition comprising NCC-like cells or CEC-like cells as provided herein.

[0030] This disclosure provides a method for treating diseases or conditions associated with dysfunctional or damaged corneal endothelial cells, comprising administering an effective amount of the CEC-like cells or compositions provided herein to a subject in need. In some embodiments, the subject is a human.

[0031] In some embodiments, the disease or symptom is selected from the group consisting of: Fuch's dystrophy, iridocorneal endothelial syndrome, posterior polymorphic dystrophy, congenital hereditary endothelial dystrophy, age-related macular degeneration (AMD), retinitis pigmentosa, glaucoma, corneal dystrophy, contact lens use, cataract surgery, and late endothelial failure during corneal transplantation.

[0032] This disclosure provides a kit for reprogramming a first type of cell into a second type of cell, wherein the kit contains a first set of reprogramming factors, and the first set of reprogramming factors includes a glycogen synthase kinase 3 (GSK3) inhibitor, a TGFβ inhibitor, and a cyclic AMP inducer.

[0033] This disclosure provides a kit for reprogramming a second type of cell into a third type of cell, wherein the kit contains a second set of reprogramming factors, and the second set of reprogramming factors contains a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0034] This disclosure provides a kit for reprogramming a first type of cell into a third type of cell, wherein the kit comprises a first set of reprogramming factors and a second set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a TGFβ inhibitor, and a cyclic AMP inducer, and the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0035] This disclosure provides a method for identifying drugs that affect the effects of NCC, chemically induced NCC (ciNCC), or chemically induced CEC (ciCEC), comprising administering a drug candidate to the NCC, ciNCC, or ciCEC and detecting the cell response to the drug candidate, thereby identifying the drug. Attached Figure Description

[0036] Figure 1 This section demonstrates the conversion of fibroblasts into ciNCCs under conditions defined by chemical composition. A) A schematic diagram of the induction method starting from mouse fibroblasts. B) Morphological changes at different time points during neural crest cell-like cell induction. C) The number of ciNCC colonies generated under specified conditions. Data are mean ± SD, n = 3 independent experiments. D) In-situ fluorescence microscopy images of SOX10 cells. + E) Displays images of the colony under specified conditions. + Colony number. Data are mean ± SD, n = 3 independent experiments. F) Immunostaining of ciNCC markers for P75, Hnk1, and AP2α on day 12.

[0037] Figure 2 The differentiation potential of ciNCCs derived from fibroblasts is shown. A) Immunohistochemical analysis demonstrates that ciNCCs can differentiate into peripheral neurons (as indicated by immunocytochemistry of Tuj1 and peripheral proteins) and Schwann cells (as indicated by immunocytochemistry of S100β and GFAP). A bright-field image on the right shows melanocytes differentiated from ciNCCs. B) Further differentiation of ciNCCs into chondrocytes, adipocytes, and osteocytes is shown (as indicated by Alcian blue, Oil Red O, and Alizarin Red staining, respectively).

[0038] Figure 3 The study showed that ciNCCs can further differentiate into corneal endothelial cell-like cells (or chemically induced CECs, ciCECs). Figure 3 Image A shows immunofluorescence staining of corneal endothelial markers (including Na+-K+ ATPase, AQP1, Vimentin, N-cadherin, laminin, and AQP1). Cell nuclei were stained with DAPI. Scale bar, 50 μm. Figure 3 B shows a transmission electron microscope image that reveals the tight junctions of corneal endothelial cells. Figure 3 C presents a scheme for a two-step lineage reprogramming strategy to functionally generate corneal endothelial cells from fibroblasts.

[0039] Figure 4 Demonstrates lineage tracing of fibroblast reprogramming towards corneal endothelial-like cells (or ciCEC). A) Demonstrates lineage tracing used to trace from Fsp1-Cre:R26R. tdTomato A schematic diagram of the genetic fate mapping method for the origin of corneal endothelial-like cells reprogrammed by MEF. B) Showing the genetic fate mapping method from Fsp1-Cre:R26R. tdTomato A lineage tracing of the origin of MEF-reprogrammed corneal endothelial-like cells. C) Immunocytochemical analysis showing FSP1-ciNCCs positive for P75, Hnk1, AP2α, and SOX10. D) Fsp1-Cre:R26R cells induced by these MEFs. tdTomato Representative morphological changes of MEF and ciCEC.

[0040] Figure 5This demonstrates the generation of corneal endothelial cell-like cells from different human cell types using small molecules. These include human embryonic skin fibroblasts (HEF), human newly generated fibroblasts (HNF), adult fibroblasts (HAF), human umbilical cord mesenchymal stromal cells (MSC), and urine cells (UC).

[0041] Figure 6 Clinical observations of ciCECs and control groups at different time points are presented. A) Bright-field image of ciCECs at passage 10 (P10). CiCECs at passage 10 express Na+-K+ ATPase and ZO-1. B) Slit-lamp photograph showing significant improvement in corneal transparency in the ciCEC group after injection on day 7 after ciCEC transplantation (Figure 1), while corneal opacity and stromal edema remained severe in the untreated control group analyzed by slit-lamp photographs (Figure 3). C) Significant difference in corneal thickness between the CEC-like cell group and the control group as analyzed by visante OCT. D) Confocal microscopy image confirming complete coverage of polygonal cells on Descemet's membrane in the ciCEC group. E) At low dose (1×10⁻⁶), bright-field images of ciCECs at passage 10 (P10) are presented. 6 Cells / mL ciCEC), high dose (2×10) 6 Changes in corneal thickness during clinical observation in cells / mL ciCEC and in the PBS-treated control group. B)-D) Images from left to right: Image 1: ciCEC-treated group, Image 2: intact contralateral eye, Image 3: PBS-treated control group, Image 4: normal eye group.

[0042] Figure 7 Slit-lamp photographs of rabbits in the ciCEC group and control group at different days are shown. The slit-lamp photographs show a significant improvement in corneal transparency in the ciCEC group after injection, with visible pupil and iris texture (ciCEC group, top image). Only about 14 days later, the cornea became noticeably clear, while corneal opacity and stromal edema remained severe in the control group (control group, top image). The lower images of both the ciCEC group and control group show corneal reflectance detected by slit-lamp examination, with each image in the lower image corresponding to an image in the upper image.

[0043] Figure 8This diagram illustrates the conversion of MEF to ciNCC via small molecule induction. A) Schematic diagram of MEF reprogramming into NCC. B) Effects of various chemicals on ciNCC formation. Data are mean ± SD, n = 3 independent experiments. C) Promoting effects of various chemicals on ciNCC formation (data are mean ± SD, n = 3 independent experiments). D) Schematic diagram of our strategy for converting MEF to ciNCC. E) Generation of Wnt1 from MEF using a small molecule cocktail. + ciNCC. F) Wnt1 in the ciNCC cluster induced by candidate mixtures in combinatorial screening + Cell number quantification (independent experiments, n=3). G) Morphological changes at different days during ciNCC induction treatment (scale bar, 50 μm). H) Wnt1 cells induced by the candidate mixture at different days. - tdTomato + Percentage of cells (independent experiments, n=3).

[0044] Figure 9 Characterization of M6-induced ciNCCs. A) Morphology of M6-induced ciNCCs (scale bar, 400 μm). B) Immunostaining of MEF-derived ciNCCs expressing p75, HNK1, AP2a, and nestin (scale bar, 50 μm). C) Representative images of differentiated ciNCCs stained with peripheral neuronal markers (scale bar, 50 μm). D) Differentiation of ciNCCs into Schwann cells and melanocytes and marker expression (scale bar, 50 μm). E) Differentiation of ciNCCs into mesenchymal lineages and further differentiation into adipocytes, chondrocytes, and osteocytes (scale bar, 100 μm).

[0045] Figure 10 Demonstrates the generation of mouse ciCECs from fibroblasts via small molecule induction. A) Schematic diagram of chemical reprogramming of ciCECs from MEF. B) Bright-field images (scale bar, 400 μm) of initial MEF, reprogrammed ciNCC colonies, and ciCECs. C) From Wnt1 - tdTomato + Morphological changes at different days during ciNCC-induced processing of ciCEC (scale bar, 400 μm). D) ciCEC targets corneal endothelial marker Na. + / K +- Immunofluorescence staining of ATPase, AQP1, vimentin, N-cadherin, laminin, and ZO-1 (scale bar, 50 μm). E) LDL uptake function in ciCECs (scale bar, 50 μm). F) Heatmap of differentially expressed genes in samples at specified time points. The numbers below the heatmap indicate independent biological replicates. Red and blue indicate upregulated and downregulated genes, respectively. G) TEM showing tightly connected ciCECs (scale bar, 5 μm).

[0046] Figure 11 Presents gene expression profiling analysis of ciNCC and ciCEC. A) qRT-PCR analysis of NC gene expression at specified time points. Gene expression (log2) is normalized relative to gene expression in MEF. B) qRT-PCR analysis of gene expression in specified NC cells from ciCEC, MEF, and pCEC at different passage numbers. C) Heatmap of differentially expressed genes in samples at specified time points. The numbers below the heatmap indicate independent biological replicates (n = 2–3). Red and blue indicate upregulated and downregulated genes, respectively. D) Principal component analysis of samples from reprogrammed, ciCEC, and control pCEC at days 0 (D0), 7 (D7), and 12 (D12).

[0047] Figure 12 The diagram demonstrates the lineage tracing evidence for ciCEC induced from fibroblasts. A) shows a schematic diagram of the genetic lineage tracing strategy. This was achieved by sorting cells derived from Fsp1-Cre / ROSA26. tdTomato p75 of MEF from E13.5 mouse embryos in the background - / tdTomato + Cells were used to obtain MEF. B) Displaying cells from cells with Fsp1-Cre / ROSA26 tdTomato p75 of MEF with genetic background - / tdTomato + FACS sorting results of cells. C) Showing p75 - / tdTomato + Immunostaining analysis of cells that showed negative results for Sox10, P75, Olig2, Hnk1, AP2, Sox2, and Pax6 (scale bar, 100 μm). (D)p75 - / tdTomato + Cell differentiation toward ciNCC and ciCEC (scale bar, 400 μm). e) Display of ZO-1, laminin, and Na+ in Fsp1-tdTomato-MEF derived ciCECs. + / K + - ATPase and AQP1 positive immunostaining analysis (scale bar, 50 μm).

[0048] Figure 13 Demonstrates the role of small molecules in promoting long-term amplification of ciCEC. A) ciCEC amplification for 3 days in serum-free control medium (scale bar, 200 μm). B) Serial amplification of ciCEC in serum-free medium supplemented with SB431542 and CKI-7 (scale bar, 200 μm). C) Mean population doubling time of ciCEC cultured in media with or without SB431542 and CKI-7 (mean ± SD, n = 3; ***p < 0.001). D) Bright-field image of ciCEC at P5, amplified for 3 days under culture conditions supplemented with SB431542 and CKI-7 (scale bar, 50 μm). E) These ciCECs at P30 were immobilized and targeted to Na+. + / K + - ATPase, AQP1 and ZO-1 staining (scale bar, 50 μm).

[0049] Figure 14 Demonstrates in vivo transplantation of ciCEC. A) A diagram showing the transplantation of ciCEC and a ROCK inhibitor into a rabbit model. B) After transplantation, corneal transparency in the transplanted eye was significantly improved, while corneal opacity and stromal edema remained severe in the untreated control. C) Slit-lamp micrographs showing the significant improvement in corneal transparency after transplantation, while corneal opacity and stromal edema persisted in the untreated control. D) Demonstrates the survival of tdTomato + Immunohistochemistry of ciCEC attached to the des Smith membrane (scale bar, 100 μm). E) Vistate OCT showing improved corneal edema (reduced corneal thickness) in the transplanted eye. F) Trend of corneal thickness after transplantation. There was a significant difference in corneal thickness between the untreated control and the transplanted control. Results are the mean of biological replicates (n=9) and SEM. G) Real-time confocal imaging of the corneal endothelium, confirming full coverage of polygonal cells on the des Smith membrane in the transplanted eye.

[0050] Figure 15 Showcase Wnt1 - Characterization of MEF. A) Schematic diagram of the genetic lineage tracing strategy. Through sorting from Wnt1... - MEF of E13.5 mouse embryos with Cre / ROSA26tdTomato background and tdTomato - Cells acquire Wnt1 - MEF. B) Showing tdTomato from MEF. - FACS sorting results of cells. C) Displaying Wnt1 -Immunostaining analysis of negative results for P75, HNK1, Sox10, and Ap2 in MEF (scale bar, 100 μm). D) Displaying Wnt1 - RT-PCR analysis of specified neural crest gene expression in MEF and primary NCC. Gapdh served as a control. E) Displaying Wnt1 - Representative image of ciNCC colonies from MEF source (scale bar, 400 μm).

[0051] Figure 16 Demonstrates M6's transformation of tdMEF into ciNCCs. A) Morphology of tdMEF and tdMEF-derived ciNCCs (scale bar, 400 μm). B) Representative images of ciNCCs differentiating into ciCECs (scale bar, 400 μm). C) Immunostaining analysis showing NC cell markers (P75, HNK1, Sox10, and Ap2α) expressed in tdMEF-derived ciNCCs (scale bar, 50 μm). D) FSP1-tdTomato + The differentiation process of ciCEC.

[0052] Figure 17 This demonstrates the generation of ciCECs from fibroblasts via small molecules, bypassing the iPSC stage. A) Morphological changes at different time points during MEF-induced ciCECs (scale bar, 400 μm). B) Immunofluorescence staining of MEF-derived ciNCC colonies against the NC marker Sox10 (scale bar, 400 μm). C) Morphological changes at different time points during OG-MEF-induced ciCECs (scale bar, 400 μm). D) FACS analysis showed the absence of Oct4-GFP-positive cells for ciCEC induction. E) Typical ciCEC karyotype (passage 10).

[0053] Figure 18 Demonstrating the proliferative potential of ciCECs. A) Representative images of ciCECs and pCECs at P3 (scale bar, 400 μm). B) Immunofluorescence images of Ki67 and ZO-1 expression in ciCECs and pCECs at P3. C) EdU of ciCECs and pCECs at different passage numbers analyzed by flow cytometry. D) Distribution of ciCECs and pCECs during the cell cycle (G1, S, and G2 phases) (scale bar, 400 μm). Scratching of ciCECs and pCECs at P3 was performed, and migration was observed and imaged after 8 and 20 hours. E) Scratch wound assay of pCECs at P3 and ciCECs at P3, P15, and P30 (scale bar, 200 μm). Migration was observed after 8 and 20 hours. F) Quantification of scratch closure.

[0054] Figure 19 Presenting observational results of transplanted eyes in a rabbit model. A) Slit-lamp microscopy (center) and confocal microscopy (right) images of the transplanted eye in subject #10 at baseline (before cell injection) and at different days after injection of ciCEC supplemented with a ROCK inhibitor. B) Visante OCT showing corneal thickness in ciCEC-transplanted eyes at different days. C) Survival of tdTomato grafts attached to the desmisz membrane. + Immunostaining analysis of ciCEC. D) Changes in corneal thickness at different number of days. Detailed Implementation

[0055] The following description of this disclosure is intended only to illustrate various embodiments thereof. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that such equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0056] A. General Definition

[0057] Unless the context clearly indicates otherwise, the singular terms “a / an” and “the” include plural referents. For example, reference to “a cell” refers to one or more cells, and reference to “the method” includes reference to equivalent steps and methods disclosed herein and / or known to those skilled in the art, and so on. Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and”. While methods and materials similar to or equivalent to those described herein may be used in practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation “eg” is derived from the Latin *exempli gratia* and is used herein to indicate non-limiting examples. Therefore, the abbreviation “eg” is synonymous with the term “for example.”

[0058] As used herein, the term “comprising / comprises” is used when referring to a composition, a method, and its respective components that are essential to said method or composition, but is open to including unspecified elements, whether necessary or not.

[0059] The term "composed of" refers to compositions, methods and their respective components as described herein, excluding any elements not described in the embodiments.

[0060] The terms “about” or “approximately” mean within 20% of a given value or range, preferably within 10%, and more preferably within 5%.

[0061] As used in this article, the term "cell" refers to a single cell, a cell line, or a culture derived from such cells.

[0062] As used herein, the term "reprogramming" or its equivalents refer to the process of altering or reversing the differentiation state of cells in culture or in vivo compared to the differentiation state that cells would have under the same conditions without reprogramming. In other words, in the context of this disclosure, "reprogramming" includes differentiation, dedifferentiation, and transdifferentiation. As used herein, the term "differentiation" refers to a cellular process by which a less specialized cell becomes a more specialized cell type. In contrast, the term "dedifferentiation" refers to a cellular process in which partially or terminally differentiated cells revert to an earlier developmental stage, such as pluripotent or multipotent cells. Furthermore, the term "transdifferentiation" refers to a cellular process that transforms one differentiated cell type into another. Therefore, as used herein, the terms "lower differentiation state," "lower specialization," or "earlier developmental stage" are relative terms and include both complete dedifferentiation (or complete dedifferentiation) and partial differentiation (or partial differentiation). To distinguish it from the cell development described above, "undifferentiated cells" are cells capable of differentiating in many directions, that is, they can differentiate into two or more types of specialized cells. A typical example of an undifferentiated cell is a stem cell.

[0063] Cell types exhibit various levels of potential during differentiation, such as totipotency, pluripotency, and multipotency. The phrase "totipotent stem cell" refers to a cell capable of differentiating into all the cells that make up an organism, such as the cell resulting from the fusion of an egg and a sperm cell. Cells produced from the first few divisions of a fertilized egg can also be totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells, such as ES cells, can produce any embryonic and adult cell type. However, they cannot develop into an embryo or adult on their own because they lack the potential to develop extraembryonic tissues. Extraembryonic tissues are partially derived from the extraembryonic endoderm and can be further classified into parietal endoderm (Reichert's membrane) and visceral endoderm (the part that forms the yolk sac). Both parietal and visceral endoderm support embryonic development but do not themselves form embryonic structures. Other extraembryonic tissues also exist, including extraembryonic mesoderm and extraembryonic ectoderm. As used herein, “pluripotent stem cells” or cells or equivalents that are “pluripotent” refer to a population of cells capable of differentiating into all three germ layers (e.g., endoderm, mesoderm, and ectoderm). Pluripotent cells express a variety of pluripotent cell-specific markers, exhibit cellular morphological characteristics of undifferentiated cells (i.e., dense colonies, high nucleocytoplasmic ratio, and prominent nucleoli), and form teratomas when introduced into immunocompromised animals, such as SCID mice. Teratomas typically contain cellular or tissue characteristics of all three germ layers. These characteristics can be assessed by those skilled in the art using techniques commonly used in the field. See, for example, Thomson et al., Science 282:1145-1147 (1998). Pluripotent cells are capable of proliferating in cell culture and differentiating into a variety of lineage-limited cell populations exhibiting pluripotent characteristics. Pluripotent stem cells, or cells that are pluripotent or equivalent, are more differentiated than pluripotent stem cells but are not terminally differentiated. Pluripotent stem cells therefore possess greater potential than pluripotent stem cells.

[0064] B. Reprogrammed cells

[0065] In one aspect, this disclosure provides a method for reprogramming a first type of cell into a second type of cell, comprising culturing the first type of cell in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors includes a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor-β (TGFβ) inhibitor, and a cyclic AMP inducer.

[0066] In some embodiments, culturing a first type of cells in the presence of a first set of reprogramming factors causes at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the cells to become a second type of cell.

[0067] In some embodiments, the first type of cell is a mammalian cell derived from the mesoderm, ectoderm, or endoderm, such as a human or mouse cell.

[0068] The ectoderm, mesoderm, and endoderm are the three germ layers formed during embryonic development. The mesoderm serves as the middle layer, the ectoderm as the outer layer, and the endoderm as the inner layer. The mesoderm forms the mesenchyme, mesothelium, non-epithelial blood cells, and coelomic cells, constituting muscles (smooth and striated muscles), bones, cartilage, connective tissue, adipose tissue, the circulatory system, the lymphatic system, the dermis, the urogenital system, serous membranes, and the notochord. The endoderm forms the pharynx, esophagus, stomach, small intestine, colon, liver, pancreas, bladder, the epithelial parts of the trachea and bronchi, lungs, thyroid gland, and parathyroid glands. The ectoderm forms the surface ectoderm, neural crest, and neural tube. The surface ectoderm develops into the epidermis, hair, nails, lens of the eye, sebaceous glands, cornea, tooth enamel, and the epithelium of the mouth and nose. The neural crest of the ectoderm develops into the peripheral nervous system, adrenal medulla, melanocytes, facial cartilage, and dentin. The neural tube of the ectoderm develops into the brain, spinal cord, posterior pituitary gland, motor neurons, and retina.

[0069] In some embodiments, the first type of cell is a somatic cell. As used herein, the term "somatic cell" refers to any cell other than germline cells (e.g., sperm and eggs, and the cells that produce them (gametocytes)) and undifferentiated stem cells. Visceral tissues, skin, bone, blood, and connective tissues are all composed of somatic cells. Somatic cells can be any type of somatic cell of any origin. For example, somatic cells may include, but are not limited to, fibroblasts, epithelial cells, supporting cells, endothelial cells, granular epithelial layers, neurons, pancreatic islet cells, epidermal cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, cardiomyocytes, and other muscle cells.

[0070] In some embodiments, the first type of cell is mesodermal cell. Mesodermal cell markers are known in the art, such as CD56 and APJ. In some embodiments, mesodermal cells include fibroblasts, epithelial cells, leukocytes, adipocytes, and keratinocytes. In some embodiments, the first type of cell is fibroblast, including but not limited to mouse embryonic fibroblasts (MEF), mouse newborn fibroblasts (MNF), mouse tail tip fibroblasts (TTF), human embryonic skin fibroblasts (HEF), human newborn fibroblasts (HNF), adult fibroblasts (HAF), human embryonic lung fibroblasts, human foreskin fibroblasts (HFF), and mixtures thereof.

[0071] Fibroblasts can be obtained from any suitable source, such as from commercial sources or from various organ tissues or skin tissues. Preferred fibroblasts are lung fibroblasts, foreskin fibroblasts, and adult dermal fibroblasts. In some embodiments, fibroblasts are obtained from a patient, for example, through a skin biopsy (e.g., reprogramming of human somatic cells to pluripotency with defined factors; George Q. Daley et al., *Nature*, 2008; a method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts from a single punch biopsy of human skin; Normand et al., *In Vitro Cellular & Developmental Biology-Animal*, 1995).

[0072] In some embodiments, the first type of cells is epithelial cells, such as exfoliated human renal epithelial cells. Epithelial cells can be obtained from urine samples.

[0073] In some embodiments, the first type of cell is a leucocyte. Leucocytes can be obtained from a blood sample. Leucocytes are white blood cells that can generally be classified into their subgroups (lymphocytes, monocytes, neutrophils, eosinophils, and basophils) by the following differences: RF signal intensity (changes in impedance at high frequencies), DC signal intensity (changes in direct current caused by the difference in conductivity between a suspended particle and the liquid medium in which the particle is suspended), fluorescence intensity, scattered light intensity, absorbance, scattered light depolarization, etc. (see US5618733A).

[0074] In some embodiments, the first type of cells is adipocytes or keratinocytes. Adipocytes and keratinocytes can also be readily obtained through skin biopsy or collected hair (isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells, Belmonte et al., Nature Protocols, 2010).

[0075] In other embodiments, the first type of cell is an adult cell. As used herein, the term "adult cell" refers to cells found throughout the body after embryonic development. In some embodiments, the first type of cell may be stem cells, such as embryonic stem cells, induced pluripotent stem cells (iPSCs), and adult stem cells, including but not limited to hematopoietic stem cells, vascular endothelial stem cells, cardiac stem cells, myogenic stem cells, mesenchymal stem cells, epidermal stem cells, adipose-derived stem cells, intestinal stem cells, neural stem cells, renal epithelial stem cells, urethral epithelial stem cells, and liver stem cells.

[0076] In some embodiments, stem cells refer to undifferentiated cells capable of proliferating and producing more progenitor cells, which in turn generate a large number of mother cells, which may then produce differentiated or differentiateable daughter cells. Stem cells can divide asymmetrically, with one daughter cell retaining its stem cell state and the other expressing some unique additional specific functions and phenotypes. Alternatively, some stem cells in a population may divide symmetrically into two stem cells, thus maintaining some stem cells in the population as a whole, while other cells in the population only produce differentiated progeny. The daughter cells themselves can be induced to proliferate and produce progeny, which subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. In other embodiments, the term "stem cell" refers to a subpopulation of progenitor cells with the ability or potential to differentiate into more specialized or differentiated phenotypes under specific conditions, retaining the ability to proliferate under certain circumstances without significantly differentiating. In one embodiment, the term stem cell generally refers to naturally occurring mother cells whose progeny (daughters) typically specialize in different directions through differentiation (e.g., by acquiring complete individual characteristics), as occurs in the progressive diversification of embryonic cells and tissues. Differentiated cells can derive from pluripotent cells, which in turn derive from other pluripotent cells, and so on. While each of these pluripotent cells can be considered a stem cell, the range of cell types each can produce can vary significantly. In many biological cases, stem cells are also "pluripotent" because they can produce progeny of more than one unique cell type, but this is not required for "stem-ness." Self-renewal is another typical part of the stem cell definition. Theoretically, self-renewal can occur through either of two main mechanisms.

[0077] The term "embryonic stem cell" is used to refer to pluripotent stem cells within the internal cell mass of the embryonic blastocyst (see U.S. Patents 5,843,780 and 6,200,806, which are incorporated herein by reference). Distinguishing features of embryonic stem cells define their phenotype. Therefore, a cell possesses one or more of the distinctive features of embryonic stem cells that distinguish it from other cells; such features include, but are not limited to, gene expression profiles, proliferative capacity, differentiation capacity, karyotype, responsiveness to specific culture conditions, and so on.

[0078] The term "adult stem cell" or "ASC" is used to refer to any pluripotent stem cell derived from non-embryonic tissues, including fetal, juvenile, and adult tissues. Adult stem cells have been isolated from a wide variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture. As shown above, stem cells have been found to reside in almost every tissue. Therefore, it is evident from the techniques described herein that stem cell populations can be isolated from virtually any animal tissue.

[0079] In some embodiments, the first type of cell may be mesenchymal stem cells, mesenchymal stromal cells, human embryonic stem cells, or induced pluripotent stem cells (iPSCs).

[0080] As used herein, the terms “iPS cell,” “iPSC,” and “induced pluripotent stem cell” are used interchangeably and refer to pluripotent cells that are artificially derived (e.g., induced by complete or partial reversal) from self-differentiating somatic cells (i.e., derived from non-pluripotent cells). Pluripotent cells can differentiate into all cells of the three developmental germ layers.

[0081] Mesenchymal stem cells (MSCs), or mesenchymal stromal cells, are adult stem cells traditionally found in the bone marrow. However, MSCs can also be isolated from other tissues, including umbilical cord blood, peripheral blood, fallopian tubes, fetal liver, and lungs. MSCs can differentiate into various cell types, including osteoblasts (bone cells), chondrocytes / cartilage cells, myocytes (muscle cells), and adipocytes (fat cells that produce bone marrow adipose tissue).

[0082] In some embodiments, the first type of cell is an ectoderm cell. In some embodiments, the first type of cell is an endoderm cell.

[0083] In some embodiments, the second type of cells transdifferentiates from the first type of cells. For example, mesodermal cells are reprogrammed into ectodermal cells. In some embodiments, the second type of cells are ectodermal cells. In some embodiments, the pluripotent stem cells are neural crest cells (NCCs) or neural crest cell-like cells. In some embodiments, the second type of cells are neural crest cells (NCCs) or neural crest cell-like cells.

[0084] "Neural crest cells" or "NCCs" generally refer to neural progenitor cells with the developmental potential to produce pigment cells that co-express melanosome markers and HMB45. Neural crest cells can be identified by expressing markers identified herein and known in the art. To distinguish the chemically induced NCCs (ciNCCs) presented herein from primary NCCs, the resulting ciNCCs are also named neural crest cell-like cells (NCC-like cells). In some embodiments, the resulting NCC-like cells exhibit one or more biomarkers consistent with the primary NCC phenotype. In some embodiments, the resulting NCC-like cells lack expression of one or more biomarkers consistent with the NCC phenotype or have low expression of such biomarkers (e.g., PAX6 expression). Exemplary NC biomarkers present in a manner consistent with the NCC phenotype may include Nestin, SOX10, SOX9, HNK-1, P75 (NGFR), AP2α, PAX3, PAX7, SNAI2, Snail, Twistl, Krox20, CD271, FoxD3, AN2, and Ki67, and / or at least one pluripotency marker, NANOG, ZNF206, or OCT4. In some embodiments, the NC biomarker is P75, Hnk1, AP2α, and / or SOX10. In the embodiments, the expression of the NC biomarker was increased by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 times or more compared to the first type of cells that produced NCC-like cells.

[0085] In some embodiments, the first type of cells is fibroblasts and the second type of cells is NCC-like cells. In some embodiments, the first type of cells is mouse embryonic fibroblasts (MEF) and the second type of cells is NCC-like cells. In some embodiments, the first type of cells is mouse tail tip fibroblasts (TTF) and the second type of cells is NCC-like cells. In some embodiments, the first type of cells is human embryonic skin fibroblasts (HEF) and the second type of cells is NCC-like cells. In some embodiments, the first type of cells is human embryonic lung fibroblasts or human foreskin fibroblasts (HFF) and the second type of cells is NCC-like cells. In some embodiments, the first type of cells is human neoplastic fibroblasts (HNF) or adult fibroblasts (HAF), and the second type of cells is NCC-like cells.

[0086] In some embodiments, the first type of cell is epithelial cells (e.g., human exfoliated renal epithelial cells) and the second type of cell is NCC-like cells. In some embodiments, the first type of cell is leukocytes (e.g., lymphocytes, monocytes, neutrophils, eosinophils, and basophils) and the second type of cell is NCC-like cells. In some embodiments, the first type of cell is adipocytes and the second type of cell is NCC-like cells. In some embodiments, the first type of cell is keratinocytes and the second type of cell is NCC-like cells. In some embodiments, the first type of cell is MSCs, human embryonic stem (ES) cells, or iPSCs and the second type of cell is NCC-like cells.

[0087] In some embodiments, the second type of cell is a certain type of pluripotent stem cell reprogrammed from another type of stem cell (the first type of cell) (such as mesenchymal stem cells, human embryonic stem cells, or induced pluripotent stem cells (iPSCs)).

[0088] The expression of the marker can be detected by any method known in the art, including but not limited to Western blotting, mRNA amplification-based methods (e.g., PCR, isothermal amplification, which may include reverse transcription and can be applied to detect expression from single or multiple cells), RNA blotting, immunostaining, etc. Additionally, the expression of the marker can be inferred from the expression of a reporter construct (e.g., expressing a visually detectable fluorescent protein, expressing an antibiotic resistance gene detectable by cell survival in the presence of antibiotics), which is under the control of a gene element (such as a promoter of one or a fragment of the aforementioned marker) that confers cell type-specific expression. Exemplary reporter constructs are the pOCT4-GFP and pOCT4-LUC genes, which drive the expression of GFP and luciferase, respectively, in ES cells; the expression of either GFP or luciferase can be readily detected using conventional methods. Other methods for detecting marker expression that can be used are known in the art. See also Ausubel, *Current Protocols in Molecular Biology* (Current Protocols, 1988); Ausubel et al., *Short Protocols in Molecular Biology* (Current Protocols; 5th ed., 2002); Sambrook et al., *Molecular Cloning: A Laboratory Manual* (Cold Spring Harbor Laboratory Press, 3rd ed., 2001); and Sambrook et al., *The Condensed Protocols from Molecular Cloning: A Laboratory Manual* (Cold Spring Harbor Laboratory Press, 2006), each of which is incorporated herein by reference in its entirety.

[0089] Any cell culture system known in the art can be used in this disclosure. In some embodiments, an adherent culture system is used in the methods of this disclosure. The term "adherent culture" refers to a cell culture system in which cells are cultured on a solid surface, which may be coated with a matrix. Cells may or may not adhere tightly to the solid surface or matrix. The matrix used for adherent culture may further comprise any one or a combination of, for example, polystyrene, polyester, polycarbonate, poly(N-isopropylacrylamide), polyornithine, laminin, polylysine, purified collagen, gelatin, cellulose, extracellular matrix, fibronectin, tenacin, vitronectin, polyglycolytic acid (PGA), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), matrix gum, hydroxyapatite, and amnion.

[0090] In some embodiments, suspension culture may be used in the methods of this disclosure. As used herein, the term "suspension culture" refers to a cell culture method in which cells do not adhere to a solid carrier or culture vessel. To transfer cells to a suspension culture, cells are removed from a culture vessel, for example by a cell scraper, and transferred to a sterile, low-adhesion culture plate containing culture medium, which does not allow cells to adhere to the plate surface. Thus, cells can be cultured in suspension without adhering to the substrate or the bottom of the culture dish.

[0091] A culture medium suitable for culturing cells is any culture medium suitable for growing a particular cell type in a culture dish. Such media include, for example, Ham's F10 (Sigma), Ham's F12 medium, Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM) (Sigma), IMDM medium, Medium 199, Eagle's Minimum Essential Medium (EMEM), aMEM medium, Fischer's medium, Neurobasal medium (LifeTechnologies Corporation), and mixtures of these media. Additionally, any of the media described in Ham et al., Enzymatic Methods (Meth.Enz.) 58:44 (1979), Barnes et al., Analytical Biochemistry (Anal.Biochem.) 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent Reissue No. 30,985 may be used as a culture medium. Any of these media may be supplemented as needed with required salts (such as sodium chloride, calcium salts, magnesium salts, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), and antibiotics (such as GENTAMYCIN). TM Drugs, trace elements (defined as inorganic compounds typically present in final concentrations in the micromolar range), glucose or equivalent energy sources, albumin, insulin, transferrin, selenium, fatty acids, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antioxidants, pyruvate, cytokines, etc. May also include any other necessary supplements in appropriate concentrations known to those skilled in the art. Culture conditions (e.g., temperature, pH, etc.) are those previously used for cell culture and are obvious to those generally skilled in the art.

[0092] In the cell reprogramming method described herein, cells are cultured in a basal medium supplemented with one or more of the reprogramming factors described herein. For example, prior to the addition of the first set of reprogramming factors, the basal medium may contain DMEM / F12 / Glutamax (GIBCO), 10% KnockOut Serum Replacement (KSR) (GIBCO), 1% NEAA (GIBCO), 10% FBS (GIBCO), and 0.1 mM 2-mercaptoethanol (GIBCO). In another embodiment, prior to the addition of the first set of reprogramming factors, the basal medium may contain DMEM / F12 / Glutamax (GIBCO), 0.075% bovine serum albumin (BSA) (GIBCO), 1% NEAA (GIBCO), and 0.1 mM 2-mercaptoethanol (GIBCO). In another example, the basal medium may contain DMEM / F12 / Glutamax (Gibco), 10% knockout serum replacement (KSR) (Gibco), 1% NEAA (Gibco), and 0.1 mM 2-mercaptoethanol (Gibco) before the addition of the second set of reprogramming factors. In some embodiments, the basal medium for the second set of reprogramming factors does not contain serum. Those skilled in the art will understand that other necessary supplements (as described) may be added to the medium.

[0093] Regarding culture temperature, it has been demonstrated that culturing at 35.0°C or higher promotes cell reprogramming. The culture temperature is one that does not damage the cells, such as preferably 35.0°C to 42.0°C, or more preferably 36.0°C to 40.0°C, or even more preferably 37.0°C to 39.0°C.

[0094] Reprogramming factors are molecules that, upon contact with cells (e.g., expressed by cells, transformed into cells for expression, or exogenously provided to cells), can induce reprogramming, either alone or in combination with other molecules. Reprogramming factors can be provided from exogenous sources, such as by addition to culture media, and can be introduced into cells by methods known in the art, such as by coupling with cell-entry peptides, protein or nucleic acid transfection agents, liposome transfection, electroporation, bioballistic particle delivery systems (gene guns), microinjection, etc. In some embodiments, the reprogramming factor is added to the culture medium without coupling with any other components.

[0095] In some embodiments, a first set of reprogramming factors for reprogramming a first type of cell into a second type of cell includes a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor-β (TGFβ) inhibitor, and a cyclic AMP inducer. In other embodiments, the first set of reprogramming factors further includes basic fibroblast growth factor (bFGF), a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor (e.g., a DOT1L inhibitor), a histone deacetylase inhibitor, BMP4, or a combination thereof.

[0096] In some embodiments, the first set of reprogramming factors consists of a GSK3 inhibitor, a TGFβ inhibitor, and a cyclic AMP inducer. In some embodiments, the first set of reprogramming factors consists of a GSK3 inhibitor, a TGFβ inhibitor, a cyclic AMP inducer, and bFGF. In some embodiments, the first set of reprogramming factors consists of a GSK3 inhibitor, a TGFβ inhibitor, a cyclic AMP inducer, a DNA methyltransferase inhibitor, a histone deacetylase inhibitor, and BMP4. In some embodiments, the first set of reprogramming factors consists of a GSK3 inhibitor, a TGFβ inhibitor, a cyclic AMP inducer, a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor (e.g., a DOT1L inhibitor), and a histone deacetylase inhibitor.

[0097] In some embodiments, reprogramming factors can sometimes be functionally replaced by paralogs within their respective families.

[0098] As used herein, the term "inhibitor" refers to an agent that reduces the expression and / or activity of a targeted expression product (e.g., mRNA encoding the target or a target peptide) by, for example, at least 10% or more (e.g., 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more). The efficacy of an inhibitor (e.g., its ability to reduce target levels and / or activity) can be determined, for example, by measuring the level of the expression product and / or the activity of the target. Methods for measuring the level of a given mRNA and / or peptide are known to those skilled in the art; for example, RT-PCR can be used to determine RNA levels, and Western blotting using antibodies can be used to determine peptide levels. Target activity can be determined using methods known in the art and described herein, such as transcriptional activity assays. In some embodiments, the inhibitor may be an inhibitory nucleic acid; an aptamer; an antibody or a binding fragment thereof; or a small molecule.

[0099] GSK3 (glycogen synthase kinase 3) is a serine / threonine protein kinase involved in many signaling pathways related to glycogen production, apoptosis, stem cell maintenance, and more. GSK3 includes isoforms (GSK3α and GSK3β) encoded by different genes and sharing high amino acid homology. Examples of GSK3 inhibitors include GSK3α inhibitors and GSK3β inhibitors. Specific examples of GSK3 inhibitors include siRNAs targeting the gene encoding GSK3, anti-GSK3 antibodies, CHIR98014 (Milipore, Bedford), CHIR99021 (Milipore, Bedford), Kenpaullone (Milipore, Bedford), AR-AO144-18 (Santa Cruz Biotechnology, Santa Cruz), TDZD-8 (Abcam, Cambridge, US), SB216763 (Abcam, Cambridge), and BIO ((2'Z,3'E)-6-bromoindorubin-3'-oxime) (Andy Biotech, Minneapolis, R&D). Systems, Minneapolis, US), TWS-119 (Cambridge Abogen), SB415286 (Cambridge Abogen), Ro3303544 (US6479490), LiCl, Li2CO3, etc. All of these are commercially available, or can be prepared by those skilled in the art with reference to known literature.

[0100] In some embodiments, the GSK3 inhibitor is selected from the group consisting of CHIR99021, LiCl, Li2CO3, and BIO. In some embodiments, the GSK3 inhibitor is CHIR99021. In some embodiments, the GSK3 inhibitor is BIO. The concentration of the GSK3 inhibitor in the culture medium is appropriately determined according to the type of inhibitor to be used. In the case of CHIR99021 or BIO, the concentration is generally 0.1-10 μM, preferably 1-5 μM, more preferably about 3 μM. One or more classes of GSK3 inhibitors may be used in combination.

[0101] TGFβ (transforming growth factor β) is a multifunctional cytokine belonging to the transforming growth factor superfamily. TGFβ comprises three distinct mammalian isoforms (TGFβ1 to 3, HGNC symbols TGFBETA1, TGFBETA2, and TGFBETA3). TGFβ can be secreted by many cell types, including macrophages, in a latent form, where it is complexed with two other polypeptides (latent TGFβ-binding protein (LTBP) and latent-related peptide (LAP)). The source of the TGFβ inhibitor used in this invention is not particularly limited, as long as it effectively inhibits TGFβ function. TGFβ inhibitors are commercially available or can be prepared by those skilled in the art based on known literature. Specific examples of TGFβ inhibitors include siRNA targeting the gene encoding GSK3, anti-TGFβ antibodies, and chemical antagonists.

[0102] In some embodiments, the TGFβ inhibitor is selected from the group consisting of: SB431542 (Tocris Bioscience, Bristol, UK), Repsox (Tocris Bioscience, Bristol, UK), LDN193189 (Tocris Bioscience, Bristol, UK), and tranilast (Rizaben). In some embodiments, the TGFβ inhibitor is SB431542. The concentration of the TGFβ inhibitor in the culture medium is appropriately determined according to the type of inhibitor to be used. In the case of SB431542, the concentration is generally 0.1-20 μM, preferably 1-10 μM, more preferably about 5 μM. In the case of Repsox, the concentration is generally 0.1-20 μM, preferably 1-15 μM, more preferably about 10 μM.

[0103] As used herein, the term "cyclic AMP inducer" refers to any compound that increases the intracellular concentration of cAMP in cells by at least 2%, preferably at least 5%, more preferably at least 10%, and most preferably at least 20% at an effective concentration. Methods for measuring intracellular cAMP levels are known to those skilled in the art. Preferred cyclic AMP inducers include isobutylmethylxanthine and trichodin. The concentration of trichodin is generally 1-20 μM, preferably 5-15 μM, more preferably about 10 μM.

[0104] bFGF (basic fibroblast growth factor) is a type of protein inherent in the body, known to control cell growth and differentiation, and possessing functions in various tissues and organs such as angiogenesis, smooth muscle cell proliferation, wound healing, tissue repair, hematopoiesis, and nerve cell differentiation. The origin of the bFGF used in this invention is not particularly limited, as long as it is effective for reprogramming. bFGF is commercially available, or it can be prepared by those skilled in the art with reference to known literature. For example, it can be synthesized based on known base sequences and amino acid sequences, such as amino acid sequences obtained from NCBI accessions AAA52448.1 (human) and AAA37621.1 (mouse). The concentration of bFGF in the culture medium is generally 1-50 ng / ml, preferably about 1-20 ng / ml, more preferably about 10 ng / ml or about 20 ng / ml.

[0105] Histone acetylation is a reversible modification, and deacetylation is catalyzed by a family of enzymes known as histone deacetylases (HDACs). HDAC inhibitors include valproic acid (VPA), trachomycin A (TSA), vorinostat (salicylic acid, SAHA, Merck & Co., Inc.), phenolic peptides (romidepsin, FK-228, Gloucester Pharmaceutical Inc.), Trapoxin, Depudecin, FR901228 (Fujisawa Pharmaceuticals), and butyrate. In some embodiments, the HDAC inhibitor is VPA. The concentration of the HDAC inhibitor in the culture medium is appropriately determined based on the type of inhibitor to be used. In the case of VPA, the concentration is generally 1-2000 μM, preferably 10-1000 μM, and more preferably about 500 μM.

[0106] The basic culture medium supplemented with the first group of reprogramming factors should be refreshed every 1, 2 or 3 days.

[0107] In some embodiments, the first type of cells are cultured in the presence of the first set of reprogramming factors for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 days, 1.5 months, or 2 months. In some embodiments, the first type of cells are cultured in the presence of the first set of reprogramming factors for no more than 2 months, 1.5 months, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, the first type of cells are cultured in the presence of the first set of reprogramming factors for 1 day to 2 months, 1 day to 1 month, 1 day to 25 days, 1 day to 20 days, 1 day to 19 days, 1 day to 18 days, 1 day to 17 days, 1 day to 16 days, 5 days to 16 days, 7 days to 16 days, 8 days to 16 days, 9 days to 16 days, 10 days to 16 days, 11 days to 16 days, 12 days to 16 days, 1 day to 15 days, 1 day to 14 days, 1 day to 13 days, 1 day to 12 days, 2 days to 12 days, 3 days to 12 days, 4 days to 12 days, 5 days to 12 days, 6 days to 12 days, and 7 days to 12 days.

[0108] In one aspect, this disclosure provides a method for reprogramming a second type of cell into a third type of cell, comprising culturing the second type of cell in the presence of a second set of reprogramming factors, wherein the second set of reprogramming factors includes a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0109] Casein kinase 1 (CK1) is a serine / threonine-selective enzyme that acts as a regulator of signal transduction pathways in most eukaryotic cell types. CK1 isoforms are involved in Wnt signaling, circadian rhythms, nucleocytoplasmic shuttle of transcription factors, DNA repair, and DNA transcription. The origin of the CK1 inhibitor used in this invention is not particularly limited, as long as it is effective for reprogramming. CK1 inhibitors are commercially available or can be prepared by those skilled in the art with reference to known literature. Specific examples of CK1 inhibitors include siRNA targeting the gene encoding CK1, anti-CK1 antibodies, and chemical antagonists. Preferably, the CK1 inhibitor is CKI-7. The concentration of the CK1 inhibitor in the culture medium is appropriately determined according to the type of inhibitor to be used. In the case of CKI-7, the concentration is generally 0.1-20 μM, preferably 1-10 μM, and more preferably about 5 μM. Other inhibitors of casein kinase 1 include PF 670462 (Andy Biotech, Minneapolis, USA), D4476 (Andy Biotech, Minneapolis, USA), (R)-CR8 (Andy Biotech, Minneapolis, USA), (R)-DRF053 dihydrochloride (Andy Biotech, Minneapolis, USA), TAK 715 (Andy Biotech, Minneapolis, USA), PF 4800567 hydrochloride (Andy Biotech, Minneapolis, USA), LH 846, CKI 7 dihydrochloride (Andy Biotech, Minneapolis, USA), SR3029 (Andy Biotech, Minneapolis, USA), Epiblastin A (Andy Biotech, Minneapolis, USA), and PF 5006739 (Andy Biotech, Minneapolis, USA).

[0110] In some embodiments, the second type of cell is an ectoderm cell. In some embodiments, the ectoderm cell is a neural crest cell (NC) or an NCC-like cell. In some embodiments, the second type of cell is an NC or an NCC-like cell. NC or NCC-like cells can differentiate from human embryonic stem cells (hES cells), for example, using a dual SMAD inhibitor as described herein or as described in WO / 2010 / 096496. NC or NCC-like cells can differentiate from hES cells using a combination of a Wnt agonist (e.g., Wnt3a and / or (2'Z,3'E)-6-bromoindorubin-3'-oxime (BIO)) and a SMAD inhibitor (e.g., SB431542 and / or head protein (Noggin)); see Menendez et al., Proceedings of the National Academy of Sciences (PNAS), November 29, 2011, Vol. 108, No. 48, pp. 19240-19245. For example, efficient induction of NC or NCC-like cells has been reported by exposing hES cells to SB431542 and (2'Z,3'E)-6-bromoindorubin-3'-oxime (BIO) (with or without the head protein) or Wnt3a and SB431542. NCs can also be obtained from neural rosette culture, for example by culturing hES cells on MS5 matrix feeder cells (see Lee et al., Stem Cells 25(8), 1931-1939 (2007), which is incorporated herein by reference in its entirety). NCs can also be obtained from a wide range of tissues, including in developing embryos, neural tubes, sciatic nerves, intestines, and dorsal root ganglia; and in larvae and adults, dorsal root ganglia, bone marrow, skin, heart, cornea, teeth, and carotid bodies. See Nagoshi et al., Journal of Cellular Biochemistry 107:1046-1052 (2009); Crane and Trainor, Annu. Rev. CellDev. Biol. 2006. 22:267-86; and Blum, Brain Research Bulletin 83 (2010):189-193, each of which is incorporated herein by reference in its entirety. In some embodiments, NCC-like cells are obtained by reprogramming first-type cells according to this application.

[0111] In some embodiments, culturing a second type of cell in the presence of a second set of reprogramming factors causes at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the cells to transform into a third type of cell.

[0112] In some embodiments, the third type of cell is an ectodermal cell. In some embodiments, the third type of cell is a neural somatic cell. In some embodiments, the third type of cell is a corneal endothelial cell-like cell (CEC-like cell).

[0113] "Corneal endothelial cells" or "CECs" generally refer to mitochondrial-rich cells arranged on the posterior surface of the cornea and facing the anterior chamber of the eye in living organisms. To distinguish the chemically induced CECs (ciCECs) presented herein from primary CECs, the resulting CECs are also named corneal endothelial cell-like cells (CEC-like cells). CEC-like cells obtained by the reprogramming method disclosed herein can be identified or recognized by exhibiting one or more of the following endogenous CEC properties: expression of CEC markers, the ability to form a uniformly sized, predominantly hexagonal cell monolayer, and the ability to form a "leak pump" (allowing solutes and nutrients to leak from the aqueous humor to a more superficial layer of the cornea while simultaneously actively pumping water from the stroma to the aqueous humor in the opposite direction). Exemplary CEC markers include, but are not limited to: Na + / K + ATPase, tight junction protein 1 (TJP1 / ZO-1), KLF13, AQP1, collagen VIII, SLC 16A3, CFTR, NBC1, CA2, AE2 / SCL4A2, SCL16A1, CA12, CA4, FoxCl. For example, CECs typically express collagen VIII, Na+, and other proteins. + K +The ATPase pump and ZO-1 are present, but vWF and CD31 (the latter being present in vascular endothelial cells) are not expressed. Additionally, CECs may express one or more corneal endothelial pump markers (including AQP1, CA2, CA4, CA12, SCL14A2, SLC 16A1, SLC 16A3, SLC 16A7, CFTR, NHE1, ADC Y10, voltage-dependent anion channels VDAC2 and VDAC3, chloride channel proteins CLCN2 and CLC), periocular neural crest markers (including PITX2 and FOXCl), and / or cell adhesion and matrix proteins (including occlin, connexin 43, 9.3E antigen, collagen III, collagen IV, N-cadherin, VE-cadherin, E-cadherin, β-catenin, laminin α4, Nidogen-2, and Netrin 4). For example, CEC can express at least one corneal endothelial pump marker, at least one periocular neural crest marker, and at least one cell adhesion and matrix protein.

[0114] In some embodiments, the resulting CEC-like cells exhibit one or more biomarkers consistent with the primary CEC phenotype. In some embodiments, the resulting CEC-like cells express tight junction protein 1 (TJP1 / ZO-1), N-cadherin, and Na+ / K+ ATPase. In some embodiments, the expression of the CEC biomarker is increased by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 times or more compared to NCCs or NCC-like cells that produce CEC-like cells.

[0115] The markers can be detected or measured using methods known in the field. For example, adhesion junctions formed by N-cadherin can be confirmed by examining expression at the protein level (e.g., using antigen-antibody reactions) or the gene level (e.g., using RT-PCR). Cellular Na+ + / K +-ATPase pumping function can be measured, for example, according to the methods described in Investigative Ophthalmology & Visual Science, 2010, Vol. 51, No. 8, 3935-3942, and Current Eye Research, 2009, Vol. 34, 347-354, using a Ussing chamber.

[0116] In some embodiments, the second set of reprogramming factors further includes BMP4 and / or DNA methyltransferase inhibitors.

[0117] Bone morphogenetic proteins (BMPs) are a group of growth factors that promote bone and cartilage formation. BMPs interact with specific receptors on the cell surface called bone morphogenetic protein receptors (BMPRs). Signal transduction via BMPRs leads to the mobilization of members of the SMAD family of proteins. Signaling pathways involving BMPs, BMPRs, and SMADs are important in cardiac, central nervous system, and cartilage development, as well as postnatal bone development. They play a crucial role in embryonic patterning and early skeletal formation during embryonic development. Therefore, interference with BMP signaling can affect the body plan of the developing embryo. Examples of BMP inhibitors include, but are not limited to, DMH2 (Bedford Millipore, USA), Dorsomorphin (Cambridge Biocare, USA), LDN193189 (Bristol-Tocresse Life Sciences, UK), DMH-1 (Bristol-Tocresse Life Sciences, UK), K 02288 (Bristol-Tocresse Life Sciences, UK), and ML 347 (Bristol-Tocresse Life Sciences, UK). The concentration of BMP inhibitor in the culture medium is appropriately determined according to the type of inhibitor to be used, such as 0.1nM-10μM, 0.1nM-5μM, 0.1nM-2.5μM, 0.1nM-2μM, 0.5nM-2μM, 1nM-2μM, 1nM-1.5μM, 1nM-1000nM, 5nM-1000nM, 10nM-1000nM, 50nM-1000nM, 50nM-500nM, 50nM-200nM, 100nM-200nM, 100nM-150nM, or 100nM.

[0118] BMP4 (bone morphogenetic protein 4) is a member of the bone morphogenetic protein family, which is part of the transforming growth factor-β superfamily. BMP4 is found in the ventral region and in the early embryonic development of the eye, heart blood, and auditory bullae. The origin of the BMP4 inhibitor used in this invention is not particularly limited, as long as it is effective for reprogramming. BMP4 inhibitors are commercially available or can be prepared by those skilled in the art with reference to known literature. Specific examples of BMP4 inhibitors include siRNA targeting the gene encoding BMP4, chordin, head protein, and anti-BMP4 antibodies. The concentration of BMP4 inhibitor in the culture medium should be appropriately determined according to the type of inhibitor to be used, such as 0.01-100 μg / ml, 0.01-50 μg / ml, 0.01-25 μg / ml, 0.01-10 μg / ml, 0.01-5 μg / ml, 0.01-1 μg / ml, 0.01-0.5 μg / ml, 0.01-0.1 μg / ml, 0.01-0.05 μg / ml, 0.05-10 μg / ml, 0.1-10 μg / ml, 0.1-5 μg / ml, 0.1-4 μg / ml, 0.1-3 μg / ml, or 0.1-2 μg / ml or 10 ng / ml.

[0119] DNMT (DNA methyltransferase) family enzymes catalyze the transfer of methyl groups into DNA. DNA methylation provides a wide range of biological functions. The DNMT inhibitors used in this invention are not particularly limited in origin, as long as they are effective for reprogramming. DNMT inhibitors are commercially available or can be prepared by those skilled in the art with reference to known literature. Specific examples of DNMT inhibitors include siRNAs targeting genes encoding DNMT, anti-DNMT antibodies, and chemical antagonists.

[0120] In some embodiments, the DNMT inhibitor is selected from the group consisting of: decitabine (Bristol-Torres Life Sciences, UK), 5-azacytidine (Bristol-Torres Life Sciences, UK), 5-aza-dC (Bristol-Torres Life Sciences, UK), and RG108 (Bristol-Torres Life Sciences, UK). The concentration of the DNMT inhibitor in the culture medium is appropriately determined according to the type of inhibitor to be used, such as 0.1-100 μM, 0.1-50 μM, 0.1-25 μM, 0.1-10 μM, 0.5-10 μM, 1-10 μM, and 5 μM.

[0121] DOT1L (telomere silencing interferon 1-like protein) is a class of histone methyltransferases (HMTs) that catalyze the methylation of histone 37 lysine residues within chromatin structures (e.g., telomere chromatin). The DOT1L inhibitors used in this invention are not particularly limited, provided they are effective for reprogramming. DOT1L inhibitors are commercially available or can be prepared by those skilled in the art with reference to known literature. Specific examples of DOT1L inhibitors include siRNAs targeting genes encoding DOT1L, anti-DOT1L antibodies, and chemical antagonists. In some embodiments, the DOT1L inhibitor is a small molecule DOT1L inhibitor. Without wishing to be bound by theory, it is known that DOT1L can catalyze H3K27 methylation via the AdoMet binding site on DOT1L using adenosylmethionine (AdoMet) as a cofactor. Therefore, any chemical mimicking the AdoMet molecular structure, which removes AdoMet from its binding site on DOT1L, is contemplated as a DOT1L inhibitor in this disclosure. In some embodiments, the DOT1L inhibitors are selected from the group consisting of: EPZ004777, EPZ5676 (also known as pinometostat), SGC 0946, and SYC-522.

[0122] In some embodiments, the second type of cells are cultured in the presence of the second set of reprogramming factors for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 days, 1.5 months, or 2 months. In some embodiments, the second type of cells are cultured in the presence of the second set of reprogramming factors for no more than 2 months, 1.5 months, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, the second type of cells are cultured in the presence of the second set of reprogramming factors for 1 day to 2 months, 1 day to 1 month, 1 day to 25 days, 1 day to 20 days, 1 day to 19 days, 1 day to 18 days, 1 day to 17 days, 1 day to 16 days, 5 days to 16 days, 7 days to 16 days, 8 days to 16 days, 9 days to 16 days, 10 days to 16 days, 11 days to 16 days, 12 days to 16 days, 1 day to 15 days, 1 day to 14 days, 1 day to 13 days, 1 day to 12 days, 2 days to 12 days, 3 days to 12 days, 4 days to 12 days, 5 days to 12 days, 6 days to 12 days, and 7 days to 12 days.

[0123] In one aspect, this disclosure provides a method for reprogramming a first type of cell into a third type of cell, comprising step (a) culturing the first type of cell in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors includes a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGFβ) inhibitor, and a cyclic AMP inducer, and step (b) culturing the cell obtained from step (a) in the presence of a second set of reprogramming factors, wherein the second set of reprogramming factors includes a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0124] In one aspect, this disclosure provides a method for reprogramming a first type of cell into a third type of cell, comprising culturing the first type of cell in the presence of a first set of reprogramming factors and a second set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGFβ) inhibitor, and a cyclic AMP inducer, and the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0125] In some embodiments, the first set of reprogramming factors further comprises bFGF. In some embodiments, the second set of reprogramming factors further comprises BMP4 and / or a DNA methyltransferase inhibitor.

[0126] In some embodiments, culturing first-type cells in the presence of a first group and a second group of reprogramming factors causes at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the cells to become third-type cells.

[0127] In some embodiments, the method further includes washing the cells obtained from step (a) before starting step (b). In some embodiments, there is no washing step between step (a) and step (b).

[0128] C. Pharmaceutical compositions and treatment methods

[0129] In one aspect, this disclosure provides a population of NCC-like cells generated according to the methods provided herein. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the population are NCC-like cells.

[0130] In one aspect, this disclosure provides a method for generating patient-specific neural crest cell-like cells. In one embodiment, the first type of cells is obtained from a subject suffering from a neurological disease.

[0131] In one aspect, this disclosure provides a population of corneal endothelial cell-like cells (CEC-like cells) generated according to the methods provided herein. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the population of this disclosure are CEC-like cells.

[0132] In some embodiments, NCC-like cells or CEC-like cells generated according to the methods provided herein are purified in vitro. In some embodiments, the reprogramming methods produce high-purity NCC-like cells or CEC-like cells and do not require purification. For example, in some embodiments, the reprogramming methods provided herein can yield cell compositions containing at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more NCC-like cells or CEC-like cells. In some embodiments, the reprogramming methods provided herein produce NCC-like cells or CEC-like cells of low purity or below the desired purity and require purification. In some embodiments, NCC-like cells or CEC-like cells are purified by substantially separating the NCC-like cells or CEC-like cells from other cells in the composition. In the case of CEC-like cell purification, the other cells in the composition may include undifferentiated NCC-like cells and / or NCC-like cells differentiated into a non-desired cell lineage or phenotype.

[0133] In one aspect, this disclosure provides a composition comprising NCC-like cells or CEC-like cells generated according to the methods provided herein. The composition may include one or more pharmaceutically acceptable carriers and diluents.

[0134] The term "pharmaceutically acceptable" indicates that the specified carrier, mediator, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other components constituting the formulation and physiologically compatible with its recipient. Pharmaceutically acceptable carriers used in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous mediators, non-aqueous mediators, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering / chelating agents, diluents, adjuvants, excipients or non-toxic excipients, other components known in the art, or various combinations thereof.

[0135] The compositions described herein may also include components that facilitate transplantation. The compositions described herein may be pyrogen-free or substantially pyrogen-free and pathogen-free, wherein pathogens include bacterial contaminants, mycoplasma contaminants, and viruses.

[0136] Compositions containing NCC-like cells or CEC-like cells as described herein may further contain immunosuppressants or immune tolerance agents.

[0137] On the other hand, this disclosure relates to the therapeutic use of the CEC-like cells provided herein. For example, the CEC-like cells of the present invention can be used in cell therapy as grafts for treating diseases requiring corneal endothelial transplantation, such as bullous keratosis, corneal edema, corneal leukoma, etc.

[0138] In one aspect, this disclosure provides a method for treating a disease or condition associated with dysfunctional or damaged corneal endothelial cells, comprising administering to a subject in need an effective amount of the CEC-like cells provided herein or a composition containing CEC-like cells.

[0139] In some embodiments, the application of corneal endothelial cells induces an ocular healing process. In some embodiments, the application of corneal endothelial cells replenishes diseased tissue. In some embodiments, the application of corneal endothelial cells has a regenerative effect on damaged or diseased ocular tissue.

[0140] The route of administration may include any suitable means, including but not limited to topical application to the ocular site, injection into the ocular site, transplantation at the ocular site, etc. In some embodiments, the specific administration mode selected will depend on the specific treatment, the patient's disease state or symptom, the nature or route of administration of other drugs or therapeutic agents administered to the subject, etc. In some embodiments, corneal endothelial cells may be administered to the subject in a single dose or in multiple doses at selected time intervals, such as in titrated doses. When multiple doses are administered, the doses may be spaced apart from each other, for example, one week, one month, one year, or ten years. One or more growth factors, hormones, interleukins, cytokines, small molecules, or other cells may also be administered before, during, or after cell administration to further bias the cells toward a specific cell type.

[0141] As used herein, the term "effective amount" refers broadly to the amount of a compound or cell sufficient to achieve such treatment against a disease when administered to a patient. An effective amount can be a prophylaxis effective amount and / or a preventative effective amount. An effective amount can be an amount that effectively reduces signs / symptoms, effectively prevents the occurrence of signs / symptoms, reduces the severity of signs / symptoms, eliminates the occurrence of signs / symptoms, slows the development of signs / symptoms, prevents the development of signs / symptoms, and / or achieves prevention and control of signs / symptoms. "Effective amount" can vary depending on the disease and its severity, as well as the patient's age, weight, medical history, susceptibility, and pre-existing conditions. For the purposes of this invention, the term "effective amount" is synonymous with "therapeutic effective amount."

[0142] As used herein, “treating / treatment” encompasses the treatment of a subject, such as a human, animal, or mammal, of a disease or medical condition as described herein, and includes: (i) suppressing the disease or condition, i.e., halting its development; (ii) alleviating the disease or condition, i.e., causing the condition to subside; (iii) slowing the progression of the condition; and / or (iv) suppressing, alleviating, or slowing the progression of one or more symptoms of the disease or medical condition.

[0143] As used herein, the term "subject" is not limited to a specific species or sample type. For example, the term "subject" can refer to a patient, and often to a human patient. However, the term is not limited to humans and therefore encompasses a wide range of mammal species, such as non-human veterinary mammals like dogs, cats, rabbits, pigs, rodents, horses, or monkeys.

[0144] The CEC-like cells described herein are cell clusters, such as clumps obtained through concentration and filtration, and analogues thereof, which are used as pharmaceutical agents of the present invention. Furthermore, it is possible to add protective agents, such as glycerol, DMSO (dimethyl sulfoxide), propylene glycol, acetamide, etc., to the pharmaceutical agent, and to cryopreserve the mixture. For safer use of the pharmaceutical agent, it may undergo treatment under conditions that cause denaturation of pathological proteins, such as heat treatment, radiation treatment, etc., while preserving the function of corneal endothelial cells.

[0145] In some embodiments, CEC-like cells may be administered in combination with surgery. In some embodiments, the surgery may be Descemet's stripping with endothelial keratoplasty (DSEK), which involves the removal of Descemet's membrane and corneal endothelium, followed by transplantation of donor tissue. Alternatively, the surgery may be penetrating keratoplasty (PKP), in which the entire cornea is removed and replaced. Other surgeries may include lamellar keratoplasty, Descemet's Membrane Endothelial Keratoplasty (DMEK), DSAEK, and DLEK.

[0146] "Diseases or conditions associated with dysfunctional or damaged corneal endothelial cells" include any disease or condition suitable for treatment with CEC-like cells, including those in which the subject's CEC number is reduced or dead, density is decreased, or they otherwise become dysfunctional. Primary diseases affecting the corneal endothelium include Fuchs' dystrophy, iridocorneal endothelial syndrome, posterior polymorphic dystrophy, and congenital hereditary endothelial dystrophy. Effective treatments may include replacement of corneal endothelium with secondary diseases or conditions including age-related macular degeneration (AMD), retinitis pigmentosa, glaucoma, corneal dystrophy, contact lens use, cataract surgery, and late endothelial failure during corneal transplantation. Corneal endothelial cell diseases also include any damage to the cornea, such as damage caused by chemical irritants, resulting from: contact lens use, reactions or sensitivities (e.g., to contact lens care solutions, cosmetics, eye drops, medications, fumes, etc.), scratches, abrasions, bruises, contusions, foreign bodies in the eye (e.g., sand or dust), or exposure to ultraviolet light (from, for example, sunlight, fluorescent lights, snow reflection, water reflection, or arc welding or other exposures). In some embodiments, a disease or condition associated with dysfunctional or damaged corneal endothelial cells results in vision loss in the subject. In some embodiments, the vision loss in the subject is permanent or irreversible.

[0147] In some embodiments, the corneal endothelial cells are immune-compatible with the subject (e.g., allogeneic or autologous).

[0148] In some embodiments, the treatment method may further include administering an immunosuppressant or an immune tolerance agent to the subject. The immunosuppressant or immune tolerance agent may be administered in an amount sufficient to reduce the risk of rejection of the CEC-like cells.

[0149] Immunosuppressants or immune tolerance agents may contain one or more of the following: anti-lymphocyte globulin (ALG) polyclonal antibody, anti-thymocyte globulin (ATG) polyclonal antibody, or azathioprine. (Anti-IL-2Ra receptor antibody), cyclosporin (cyclosporin A) (Anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid (Anti-CD20 antibody), sirolimus, tacrolimus, mycophenolate mofetil, and corticosteroids.

[0150] Immunosuppressants can be administered at least about 1, 2, 4, 5, 6, 7, 8, 9, or 10 mg / kg. When used, immunosuppressants can be administered systemically or locally, and can be administered before, concurrently with, or after CEC-like cell administration. Immunosuppressive therapy can continue for weeks, months, years, or indefinitely after cell administration. For example, a patient may receive 5 mg / kg cyclosporine for 6 weeks after CEC-like cell administration.

[0151] In one aspect, this disclosure provides for the use of the CEC-like cells or compositions provided herein in the manufacture of medicaments for treating diseases or conditions associated with dysfunctional or damaged corneal endothelial cells.

[0152] In one aspect, this disclosure provides a method for treating a disease or condition associated with dysfunctional or damaged corneal endothelial cells, comprising administering to a subject in need an effective amount of the NCC-like cells provided herein or a composition containing NCC-like cells.

[0153] In one aspect, this disclosure provides for the use of the NCC-like cells or compositions provided herein for the manufacture of medicaments for the treatment of diseases or conditions associated with dysfunctional or damaged corneal endothelial cells.

[0154] The NCC-like cells described in this article can be used to treat neurological diseases.

[0155] As used herein, “neurological disease” is defined as a disorder of the nervous system and includes disorders involving the central nervous system (cerebrum, brainstem, and cerebellum), the peripheral nervous system (including cranial nerves), and the autonomic nervous system (parts of which are located in the central and peripheral nervous systems). In particular, neurological disease includes any disease in which the neural crest cells or Schwann cells are impaired, altered, or destroyed. Examples of neurological diseases related to Schwann cells include demyelinating diseases, multiple sclerosis, myelopathy, experimental allergic encephalomyelitis (EAE), acute disseminated encephalomyelitis (ADEM), post-infectious or post-vaccination encephalomyelitis, peripheral neuropathy, Schwannomatosis, and other related conditions. Figure 3Charcot-Marie-Tooth disease, Guillain-Barré syndrome, and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).

[0156] This disclosure also provides methods for drug discovery and / or drug screening. Analyses for drug discovery and / or drug screening are also provided. In some embodiments, these methods include administering a drug candidate to NCC-like cells or CEC-like cells and detecting the cell response to the drug candidate. Detecting the response can identify whether the drug candidate has suitable properties (e.g., toxicity or therapeutic efficacy). In some embodiments, the methods can be used to determine cell health and viability in the presence of the drug candidate. In some embodiments, the methods can be used to test the toxicity of the drug candidate. In some embodiments, the methods can be used to assess changes in cell population phenotype in the presence of the drug candidate.

[0157] In one aspect, this disclosure provides a method for using NCC-like cells to screen for drugs that reverse, inhibit, or prevent neurological diseases or neurological side effects of medications (e.g., diabetes medications).

[0158] D. Reagent kit

[0159] In one aspect, this disclosure provides a kit for reprogramming a first type of cell into a second type of cell, wherein the kit comprises a first set of reprogramming factors, and the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGFβ) inhibitor, and a cyclic AMP inducer.

[0160] In one aspect, this disclosure provides a kit for reprogramming a second type of cell into a third type of cell, wherein the kit comprises a second set of reprogramming factors, and the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0161] In one aspect, this disclosure provides a kit for reprogramming a first type of cell into a third type of cell, wherein the kit comprises a first set of reprogramming factors and a second set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a TGFβ inhibitor, and a cyclic AMP inducer, and the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0162] The kit may further include instructions for use and separate packaging for each component in the kit.

[0163] All publications and patents cited in this specification are incorporated herein by reference in their entirety.

[0164] Example

[0165] Example 1: Materials and Methods

[0166] 1. Animals

[0167] All animal experiments were approved by the Animal Ethics Committee of Wenzhou Medical University, Wenzhou, China. Oct4-GFP transgenic allele-carrying mice (CBA / CaJ×C57BL / 6J) were from the Jackson Laboratory; Wnt1-cre and Fsp1-Cre mice were from the Jackson Laboratory (BALB / c-Tg(S100a4-cre)1Egn / YunkJ); ROSA26-tdTomato mice were from the Jackson Laboratory (Gt(ROSA)26Sortm14(CAG-tdTomato)Hze). 129Sv / Jae and C57BL / 6 mice were from Beijing Vital River Laboratory. Wnt1-Cre / ROSA26 tdTomato and Fsp1-Cre / ROSA26 tdTomato Mice were obtained by crossing Wnt1-Cre and Fsp1-Cre mice with ROSA26-tdTomato mice, respectively (see [link to relevant documentation]). Figure 4 A). New Zealand white rabbits were sourced from JOINN Laboratories (Suzhou) Inc., Suzhou, China. All animals were handled in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. All animals were housed under stable conditions (21℃±2℃) with a 12-hour dark / light cycle.

[0168] 2. Cell Culture

[0169] As previously described, primary mouse embryonic fibroblasts (MEFs) were isolated from mouse embryos at day 13.5 (E13.5) (Liu, C., Hu, X., Li, Y., Lu, W., Li, W., Cao, N., Zhu, S., Cheng, J., Ding, S. and Zhang, M. (2019). "Conversion of mouse fibroblasts into oligodendrocyte progenitor-like cells through a chemical approach." Journal of Molecular Cell Biology. "Small molecules enable cardiac reprogramming of mouse fibroblasts with a single factor, Oct4". Cell Reports 6, 951-960. (2014). Wang, H., Cao, N., Spencer, CI, Nie, B., Ma, T., Xu, T., Zhang, Y., Wang, X., Srivastava, D. and Ding, S. (2014). In simple terms, the head, tail, limbs, and internal organs of the embryo were carefully removed and discarded. The remaining tissue was sliced ​​into small pieces, trypsinized with 0.25% trypsin / EDTA (Gibberellic acid), and seeded onto 10-cm culture dishes. Fibroblasts were cultured in DMEM (Gibico) supplemented with 10% fetal bovine serum (FBS, Gibbec), 2 mM GlutaMAX (Gibico), 0.1 mM non-essential amino acids (Sigma), 100 IU / mL penicillin, and 100 mg / mL streptomycin (Gibico). All fibroblasts were expanded two generations and subsequently used for further experiments. This was to prepare Wnt1. - MEF will be derived from Wnt1-Cre / ROSA26 tdTomato Mouse embryonic fibroblasts targeting tdTomato - Cells are sorted using FACS.

[0170] Primary mouse corneal endothelial cells (CECs) (CP-M179) and culture medium (CM-M179) were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). Primary mouse CECs were cultured in DMEM containing 10% FBS (Gibberellic Acid), 0.1 mM non-essential amino acids (Sigma), 2 mM GlutaMAX (Gibberellic Acid), and 1% penicillin-streptomycin (Gibberellic Acid). mESCs were maintained in ESC medium composed of 10% FBS (Gibberellic Acid), LIF, 0.1 mM non-essential amino acids (Sigma), 2 mM GlutaMAX (Gibberellic Acid), 1% penicillin-streptomycin (Gibberellic Acid), 0.1 mM 2-mercaptoethanol (Gibberellic Acid), CHIR99021 (3 mM), and PD0325901 (1 mM).

[0171] Human embryonic fibroblasts (HEF), human newly formed fibroblasts (HNF), and adult fibroblasts (HAF) were purchased from ScienCell Research Laboratories. Human umbilical cord mesenchymal stem cells (MSCs) were provided by Nuwacell Ltd. (Hefei, China). The cells were maintained in fibroblast culture medium containing DMEM supplemented with 10% FBS, 1% GlutaMAX, and 1% NEAA. Human exfoliated renal epithelial cells (UCs) were derived from 100–300 mL urine samples from normal individuals, as previously reported by Zhou, T. et al. (2012). Generation of human induced pluripotent stem cells from urine samples. Nature Experiment Handbook 7, 2080–2089.

[0172] 3. FACS sorting used to generate tdMEF

[0173] To prepare tdMEF, the resulting fibroblasts were targeted with tdTomato as previously described. + / p75 － Cells were sorted using FACS. Primary MEFs were sorted from those with Fsp1-Cre / Rosa26. tdTomato (Fsp1-Cre mice × Rosa26) tdTomatoE13.5 mouse embryos with a genetic background of [insert genetic background here]. Second-generation MEFs were dissociated with 0.25% trypsin at 37°C for 5 minutes and neutralized with MEF medium. These MEFs were stained with a specific antibody against p75 and subjected to [insert genetic background here]. + / p75 － FACS sorting of cells. During FACS sorting, 24-well culture plates coated with Matrigel were pre-warmed at 37°C for at least 30 minutes, followed by seeding with tdMEF. Immediately after FACS sorting, tdMEF cells were seeded at 15,000 cells / well into pre-warmed Matrigel-coated 24-well culture plates in MEF medium supplemented with 1 μM thiazovivin (Tzv) at 37°C in 5% CO2 and 20% O2 for 5 hours to allow MEF to ligate into the plate. After 5 hours, the medium was changed to Tzv-free MEF medium, and tdMEFs were cultured overnight at 37°C in 5% CO2.

[0174] 4. Small molecule compounds and libraries

[0175] Small molecules were obtained from Sigma, including the GSK3b inhibitor CHIR99021 (SML1046), the TGFb inhibitor SB431542 (S4317), the DNA methylation inhibitor 5-aza-dC (A3656), the cyclic AMP inducer trichodin (F6886), and CKI-7 (C0742). bFGF was obtained from Peprotech. The DOT1L inhibitor EPZ004777 (S7353) and the ROCK inhibitor Y-27632 (S1049) were obtained from Selleck.

[0176] 5. Immunocytochemistry

[0177] To further investigate the expression of typical neural crest (NC) cell markers, reprogrammed MEFs were fixed, immunostained, and analyzed. In short, cells were washed once with 1×PBS and fixed with 4% paraformaldehyde at room temperature for 10 minutes, followed by permeabilization with 0.2% Triton X-100 in 1×PBS for 10 minutes, and then blocked with 7.5% BSA for at least 1 hour. All primary antibodies were diluted in 7.5% BSA and incubated overnight at 4°C. Cells were washed five times at room temperature with 1×PBS for 10 minutes each. Secondary antibodies Alexa-488, Alexa-555, and Alexa-647 were purchased from Invitrogen, diluted in 7.5% BSA, and incubated at room temperature for 1 hour, followed by five 10-minute washes with 1×PBS. Cell nuclei were stained with DAPI. The antibodies used in this study are listed in Table 2.

[0178] Table 1: Antibodies used in this study

[0179]

[0180]

[0181] 6. Statistical Analysis

[0182] All experiments were conducted independently at least three times. Results are expressed as mean ± SD. Data were analyzed by unpaired two-tailed Student's t-test to compare two groups, and by one-way ANOVA and Tukey's test or Dunnett's multiple comparisons test to compare multiple groups. All analyses were performed using SPSS Statistics 19.0 software. A p-value < 0.05 was considered significant.

[0183] 7. Cell cycle analysis

[0184] Cells were carefully dissociated into single-cell suspensions using acutase solution, washed twice with PBS, and then fixed overnight with cold 70% ethanol. The fixed cells were washed twice with PBS, followed by ribonuclease (100 μg / mL, Sigma) treatment and propidium iodide (50 μg / mL, Sigma) staining at 37°C for 30 min. Cells approximately 1 × 10⁻⁶ cells were analyzed using FACSCantoII (Becton Dickinson).6 Cells were analyzed to determine cell cycle distribution patterns. ModFit 4.1 (Verity Software House) was used to analyze the percentage of cells in the G1, S, and G2 / M phases of the cell cycle.

[0185] 8. Transplantation

[0186] All rabbits weighing 2.0–2.5 kg were anesthetized intramuscularly with ketamine hydrochloride (60 mg / kg) and toluenethiazide (10 mg / kg, Bayer, Munich, Germany). The rabbits were divided into two groups (n = 10 per group), with their right eye used for this experiment. After disinfection and aseptic covering of the surgical site, a 6 mm corneal incision was made at the 12 o'clock position using a suture cutter, and a viscoelastic agent (Healon; Amersham Pharmacia Biotech AB) was infused into the anterior chamber. After marking the corneal surface with a marker (Devon Industries, Madrid, Spain), a 6.0 mm diameter circular opening for descemetorhexis removal was created in the center of the cornea using a 30-gauge needle (Terumo, Tokyo, Japan), and the descemetorhexis membrane was removed from the anterior chamber of the eye. As previously described, the corneal endothelium was mechanically scraped from the Desmith membrane using a lacrimal duct irrigator (Shandong Weigao). Fsp-ciCECs were dissociated using 0.25% trypsin-EDTA at a concentration of 1 × 10⁻⁶. 7 Cells were resuspended at a density of 100 cells / mL in basal medium and kept on ice. The anterior chamber was washed three times with PBS. Following this procedure, 1 × 10⁶ cells were resuspended in 100 μL of basal DMEM containing 100 μM ROCK inhibitor Y-27632 (Celec) using a 26-gauge needle. 6 One cultured ciCEC was injected into the anterior chamber of the right eye. Subsequently, rabbits in the cryo-induced (cryo-induced injury alone), CE-induced (cryo-induced injury and CE injection) and spherical-induced (cryo-induced injury and spherical injection) groups maintained an eye-down position for 24 hours to allow cells to connect via gravity, while rabbits in the spherical-induced (eye-up) group maintained an eye-up position for 24 hours under deep anesthesia. Each operated eye was externally examined two or three times a week, and photographs were taken on days 3, 7, 14, and 28 post-injection. Central corneal thickness was measured using an ultrasonic pachymeter, and intraocular pressure was measured using a pneumatic tonometer on days 0.5, 1, 3, 7, 14, 21, and 28 post-surgery. The average of the three readings was taken.

[0187] 9. Cell proliferation analysis

[0188] via Click-iT TM The ethynyldeoxyuridine (EdU) Alexa Fluor 488 Imaging Kit (Ingenieur) was used to determine the proliferation rate of ciCECs cultured in differentiation medium alone or in M5 medium alone, according to the manufacturer's instructions. In short, CECs were passaged at 5 × 10⁻⁶... 3 The cells were seeded at a low density of 10 cells / cm² onto a glass slide and incubated for 24 hours.

[0189] 10. Transmission electron microscopy (TEM) analysis

[0190] For TEM analysis, cells were fixed in 2.5% EM-grade glutaraldehyde (Servicebio) at 4°C for 2–4 hours, washed with 0.1M phosphate-buffered saline (pH 7.4), and post-fixed in 1% osmium tetroxide at 4°C for 2–4 hours. After washing and a final rinse in 100% acetone via continuous ethanol solution (50–100%), the cells were incubated in 1:1 acetone / Pon 812 (SPI) for 2 hours, followed by overnight incubation in 1:2 acetone / Pon 812. Samples were embedded in Pon 812 and polymerized at 60°C for 48 hours, then sectioned (60–80 nm) using a Daitom microscope. Sections were stained with 2% uranium acetate, followed by lead citrate staining, and visualized using an HT7700 transmission electron microscope (Hitachi).

[0191] 11. Karyotype analysis

[0192] Cells were treated with 0.1 μg / mL colchicine (Gibco) at 37°C for 2 hours, triedpsinized, resuspended, and incubated in 0.075 M potassium chloride at 37°C for 15 minutes. They were then fixed with a 3:1 methanol:acetic acid solution and subsequently dropped onto a slide to disperse the chromosomes. Chromosomes were visualized using Giemsa (Sevier Biosciences) staining.

[0193] 12. RNA sequencing and analysis

[0194] Total RNA from each sample was isolated using TRIzol reagent and purified using the RNeasy 23 Mini Kit (Kaigen) according to the manufacturer's instructions. RNA quality and quantity were assessed using a NanoDrop 2000, an Agilent 2100 Bioanalyzer, and an Agilent RNA 6000 Nano Kit. RNA library construction and sequencing were performed using Annoroad GeneTechnology. Sequencing libraries were generated using the NEBNext Ultra RNA Library Preparation Kit (NEB) for Illumina 24, following the manufacturer's recommendations, and library clustering was performed using the HiSeq PE Cluster Kit v4-cBot-HS (Tsinghua Biosciences). After clustering, the libraries were sequenced on the Tsinghua Biosciences platform, generating 150 bp paired-end reads. Initial data analysis was performed on BMKCloud (http: / / www.biocloud.net / ).

[0195] 13. FACS cytometrics

[0196] For MEF preparation, fibroblasts with the desired genotype are cultured in MEF medium until they reach confluence of more than 80%. Cells are washed twice with 1×PBS and treated with 0.25% trypsin at 37°C for 5 minutes. After harvesting, cells are passed through a 70-μm filter, washed twice with pre-cooled buffer (1×PBS, 1.5% FBS, 0.5% BSA), and resuspended in the buffer. At the recommended concentration, cells are incubated on ice for 30 minutes with FITC-conjugated P75 antibody (Ebola) or isotype control (BD), or at room temperature for 45 minutes, followed by six washes with FACS buffer. Cells are then resuspended in FACS buffer and sorted using BD FACSAria II.

[0197] Example 2: Generation of ciNCC and ciCEC from fibroblasts

[0198] 1. Culture medium preparation

[0199] Stage I culture medium preparation

[0200] The basal medium contained DMEM / F12 / Glutamax (Gibco), 10% KSR (Gibco), 10% FBS (Gibco), 1% NEAA (Gibco), and 0.1 mM 2-mercaptoethanol (Gibco), supplemented with small molecules Repsox (10 μM), Chir99021 (10 μM), laryngin (10 μM), and bFGF (10 ng / ml).

[0201] Before the addition of the first set of reprogramming factor molecules, the basal medium may also contain DMEM / F12 / Glutamax (Gibco), 0.075% bovine serum albumin (BSA) (Gibco), 1% NEAA (Gibco), and 0.1 mM 2-mercaptoethanol (Gibco), and produces comparable or better results in cell reprogramming (data not shown).

[0202] In addition, a combination of small molecules Repsox (10 μM), Chir99021 (10 μM), and salivain (10 μM) can achieve a similar induction effect (data not shown). Shake the culture medium for 30 minutes to ensure complete dissolution.

[0203] Stage II culture medium preparation

[0204] DMEM / F12 / Glutamax (Gibco), 10% KSR (Gibco), 1% NEAA (Gibco), 0.1mM 2-mercaptoethanol (Gibco), supplemented with 5μM SB431542 and 5μM CKI-7.

[0205] 2. Chemical induction of ciCEC from fibroblasts

[0206] To investigate whether small molecules can chemically reprogram fibroblasts into corneal endothelial cells, we designed a two-step method to directly reprogram mouse fibroblasts into neural crest cells and differentiate them into corneal endothelial cells. Figure 1 A). In Wnt1-Cre-Rosa Tomato In mice, tdTomato is faithfully expressed in the neural crest (NC) under the control of the Wnt1 gene. Therefore, from the Wnt1-Cre / ROSA26 genotype at embryonic stage E13.5... tdTomato MEFs were isolated from mice, and neural crest populations were labeled with tdTomato expression. We performed fluorescence-activated cell sorting (FACS) to collect tdTomato. -The populations were selected to exclude any neural crest or progenitor cells. These MEFs were negative for typical NC markers (including Sox10, P75, Hnk1, and AP2α) (data not shown). Additionally, these MEFs were also negative for typical neural stem cell (NSC) markers (including Sox2, Pax6, and Olig2) (data not shown). No methods have been reported for inducing ciNCCs from fibroblasts using small molecules. To investigate whether small molecules can chemically reprogram fibroblasts into ciNCCs, we selected over 20 small molecules as our potential candidate molecules for neural crest lineage reprogramming based on targeted epigenetic modifications and regulation of neural crest developmental signaling. The candidate small molecules focused on three main classes of reprogramming factors comprising three small molecules: (1) TGF-β signaling inhibitors, such as Repsox (R), which inhibits mesoderm and endoderm specialization; (2) GSK3 inhibitors, such as CHIR99021 (C), which promotes neural development; and (3) cyclic AMP inducers, such as trichomoniasis (F). These three compounds were combined with basic fibroblast growth factor (bFGF) as a chemically defined culture medium (hereinafter referred to as RCF) to reprogram mouse embryonic fibroblasts (MEF) into ciNCCs.

[0207] In short, MEFs were seeded at 50,000 cells / well in 6-well plates. After overnight culture, the MEFs were treated in reprogrammed medium with a defined RCF chemistry. In RCF medium, numerous small, dense clusters of cells with well-defined edges rapidly emerged within twelve days. Figure 1 B). To identify combinations of RCFs sufficient to reprogram mouse fibroblasts into chemically induced neural crest cells (ciNCCs), Wnt1 was used. － MEF was used to observe tdTomato expression. Results showed that the RCF compound could reprogram MEF cells into tdTomato-positive neural crest-like cells. Figure 1 D). To further investigate the expression of typical NC markers, RCF-treated MEF cells were fixed, immunostained, and analyzed. These cells expressed HNK1, p75, and AP2α. Figure 1 F). Those highly proliferative and self-regenerating Sox10-positive cells of fibroblast origin were subsequently called ciNCCs.

[0208] During the reprogramming process, numerous small, dense clusters of cells with well-defined edges rapidly appear within 7 days. Figure 1 B and Figure 1 C). After treatment with Repsox, Chir99021, trichomoniasis-1, and bFGF, we observed tdTomato after 10 days of treatment. + cell( Figure 1 D and Figure 1 E). On day 12, markers of neural crest cells (NC) (HNK1, p75, and AP2α) were observed (see [link to article]). Figure 1 F). However, in the absence of bFGF, a combination of small molecules Repsox (10 μM), Chir99021 (10 μM), and salivacin (10 μM) can achieve a similar inductive effect (data not shown).

[0209] After 14 days of induction of ciNCCs with SB431542 and CKI-7 differentiation medium, ciNCCs differentiated into corneal endothelial cell-like cells (ciCECs) (see [link]). Figure 3 C). Figure 3 A shows the markers (Na2+) of stage II-induced ciCECs detected by immunofluorescence staining on day 14. + -K + ATPase, AQP1, vimentin, N-cadherin, laminin, and AQP1 were also observed. Tight junctions of corneal endothelial cells were also observed under transmission electron microscopy.

[0210] To avoid potential contamination of the initial MEF with neural crest cells (ciNCCs), we performed lineage tracing experiments to trace the origin of ciNCCs and ciCECs. Figure 4 B). In Fsp1-Cre-RosaTomato mice, tdTomato is faithfully expressed in ciNCCs and ciCECs under the control of the Fsp1 gene. In Fsp1-ciNCCs, the expression of ciNCC markers P75, Hnk1, AP2α, and SOX10 was observed by immunocytochemistry (see [link to Fsp1-ciNCC]). Figure 4 C). Figure 4 D shows representative morphological changes in Fsp1-Cre:R26RtdTomato MEFs and ciCECs induced from these MEFs.

[0211] Human cells (human embryonic skin fibroblasts (HEF), human neoplastic fibroblasts (HNF), adult fibroblasts (HAF), human umbilical cord mesenchymal stromal cells (MSC), and urine cells (UC)) were induced using the same procedure described above. Preferably, the chemical factors added in stage I were Repsox (10 μM), Chir99021 (10 μM), trichodin (10 μM), 5-azacytidine (5 μM), VPA (500 μM), and BMP4 (10 ng / ml). The chemical factors added in stage II were 5 μM SB431542 and 5 μM CKI-7, consistent with those mentioned above. Induced ciNCCs and ciCECs derived from the above cells are shown in [the table / image / image]. Figure 5middle.

[0212] Example 3: Characterization of ciNCC and ciCEC obtained in Example 2

[0213] 1.1 RNA preparation and RT-PCR

[0214] To confirm the expression of NC genes (including Sox10, P75, Pax3, and Msx1), total RNA was extracted using the RNeasy Plus mini kit (Kaigen). In short, 1 μg of total RNA was used for reverse transcription with the iScript cDNA Synthesis Kit (Bio-Rad), and the resulting cDNA was diluted five times in H2O for PCR use. For semi-quantitative PCR, 1 μl of 1 / 5 diluted cDNA was used as the template for the PCR program: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, then 72°C for 10 min. Quantitative PCR was performed following the FAST SYBR Green master mixture (ABI) protocol. All PCRs were repeated three times, and the expression of individual genes was normalized relative to Gapdh expression. Primer sequences are listed in Table 2.

[0215] Table 2: Primers for qRT-PCR

[0216]

[0217] 1.2 RNA sequencing and analysis pipeline

[0218] To validate the transcriptome analysis of ciNCC, RNA sequencing was performed. RNA sequencing libraries were prepared using the Ovation RNA Sequencing System v2 Kit (NuGEN). Total RNA (50 ng) was reverse transcribed to synthesize first-strand cDNA using a combination of random hexamer and poly-T chimeric primers. The RNA template was then partially degraded by heating, and second-strand cDNA was synthesized using DNA polymerase. Double-stranded DNA was then amplified using single-primer isothermal amplification (SPIA). SPIA is a linear cDNA amplification process in which ribonuclease H degrades the RNA in the DNA / RNA heteroduplex at the 5′ end of the double-stranded DNA, after which the SPIA primer binds to the cDNA, and polymerase initiates replication at the 3′ end of the primer by replacing the existing forward strand. Random hexamers were then used to linearly amplify the second-stranded cDNA. Finally, libraries derived from SPIA-amplified cDNA were prepared using the Ultralow V2 Library Kit (NuGEN). The RNA sequencing libraries were analyzed using a bioanalyzer and quantified by qPCR (KAPA). On a HiSeq 2500 instrument (Qide), three RNA sequencing libraries were pooled into each pathway for paired-end 100bp sequencing. Known adapters and low-quality regions of the reads were adjusted using Fastq-mcf. Sample QC was assessed using FastQC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Reads were aligned to the mouse reference assembly mm9 using TopHat 2.0.13 (Kim, D. et al. 2011. TopHat 2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biology 14.). Using SubreadfeatureCounts (Liao et al. 2014. "FeatureCounts: an efficient general-purpose program for assigning sequence reads to genomic features." Bioinformatics 30, 923-930.), we combined gene-level expression with Ensembl gene annotations for mm9.Before processing differential expression, we used RUVSeq to adjust for batch effects (David Risso et al., 2014, "Normalization of RNA-seq data using factor analysis of control genes or samples," *Nature Biotechnology*, 32, 896-902). Genes from at least two samples that did not have a CPM (counts per million) value between .5 and 5000 were filtered out. Differential expression p-values ​​were calculated using edgeR (Robinson, MD et al., 2010, "edgeR: a Bioconductor package for differential expression analysis of digital gene expression data," *Bioinformatics*, 26, 139-140). The built-in R function "p.adjust" was used to calculate the false discovery rate (FDR) using the Benjamini-Hochberg method (Benjamini and Hochberg, (1995). "Controlling the false discovery rate: a practical and powerful approach to multiple testing." Journal of the Royal Statistical Society Series B, 57, 289-300). Gene ontology analysis was performed using DAVID Bioinformatics Resources 6.7 or ToppGene. Heatmaps were generated using Cluster 3.0 and viewed via Java Treeview. Transcriptome analysis revealed that these cells were very similar to mouse NC cells, but different from MEF cells.

[0219] 1.3 Western Blotting

[0220] Western blotting was performed to confirm the expression of NC-specific and CEC-specific markers. Briefly, cells were collected in the presence of a phosphatase inhibitor (Cell Signaling Technology) and mixed with an equal volume of 2×SDS-PAGE sample buffer containing DTT. The sample was boiled and clarified by centrifugation. After electrophoresis, the proteins were transferred to a PVDF membrane. The efficiency of the transfer was determined by briefly staining the membrane in Ponceau S. The membrane was blocked with 5% BSA at room temperature for at least 1 hour and incubated overnight at 4°C with the desired antibody diluted in TBST solution of 5% BSA. The membrane was washed five times with TBST (10 minutes each time) and then incubated for 1 hour at room temperature with HRP-conjugated secondary antibody diluted in TBSA. After five washes with TBST, the blot was developed using an ECL exogenous detection kit. These results collectively demonstrate the robust and universal effectiveness of the chemical mixture for reprogramming fibroblasts into ciNCC and ciCEC (data not shown).

[0221] 2. Maintenance and differentiation potential of ciNCC

[0222] For maintaining ciNCC after FACS sorting and purification, cells were cultured on poly-D-lysine / laminin-coated culture plates in neural crest medium consisting of neural matrix medium (Gibco) supplemented with 1×N2, 1×B27, 10 μg / ml bFGF, 10 μg / ml EGF and 10 ng / ml BMP4.

[0223] To characterize the differentiation potential of expanded ciNCC, it was first cultured under differentiation conditions. Neuronal differentiation was induced by FGF2 / EGF withdrawal and exposure to BDNF, GDNF, NGF, and dibutyryl cyclic AMP (dbcAMP), generating peripheral neurons. Immunostaining showed that ciNCC produced Tuj1+ and peripheral protein+ neurons at passage 5, and this differentiation potential was well maintained during long-term culture. Figure 2 A). Schwann cell differentiation, as assessed by S100b and GFAP+ expression, was induced in the presence of CNTF, neuregulin 1b, and dbcAMP.

[0224] For melanocyte differentiation, after 7 days of induction treatment, ciNCCs were cultured in melanocyte differentiation medium (EBM2 basal medium, 5% (v / v) FBS, 100 ng / ml SCF (LifeTech), 200 nM endothelin 3 (EDN3, Sigma), 50 ng / ml WNT1, 10 ng / ml FGF2, 5 μg / ml insulin, 1 pM cholera toxin, 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma), and 10 μM SB431542 (Sigma)). Melanocytes were observed after 3-4 weeks of treatment. Figure 2 A).

[0225] To examine the differentiation potential toward the mesenchymal lineage, we cultured ciNCCs under MSC culture conditions. Under these conditions, cells exhibiting mesenchymal morphology and marker expression (CD105+) emerged (data not shown). After another month of culture, most cells expressed CD105 and a set of surface markers specific to mesenchymal stem cells. We used an established mesenchymal stem cell differentiation protocol and demonstrated that mesenchymal precursor cells generated from ciNCCs can differentiate into adipocytes, chondrocytes, and osteoblasts. Figure 2 B) Differentiation.

[0226] 3. Functional characterization of neurons from the ciNCC source

[0227] To determine the function of ciNCC-derived neurons, we examined their electrophysiological properties. Whole-cell patch-clamp recordings were obtained from ciNCC-derived neurons after co-culturing with rat cortical neurons for 10–20 days under differentiation conditions. The cultured neurons were transferred to a perfusion stage on an Olympus BX51WI upright microscope and perfused at 2.5 mL / min at room temperature with artificial cerebrospinal fluid (aCSF) containing the following components (in mM): NaCl 11 g, KCl 2.5 g, NaH₂PO₄ 1 g, NaHCO₃ 26.2 g, glucose 1 g, CaCl₂ 2.5 g, and MgSO₄ 1.3 g, with the osmolarity adjusted to 300 osm L⁻¹. The aCSF was bubbled with 95% O₂ and 5% CO₂ throughout the recording process. Data were collected, filtered at 2 kHz, and digitized at 10 kHz using a MultiClamp 700B amplifier (Axon Instruments). Offline analysis was performed in Igor Pro (Wavemetrics). Action potentials were recorded in a whole-cell current-clamp configuration. The electrode solution used for the current-clamp experiments contained the following substances (in mM): potassium gluconate 123, KCl 10, MgCl2 1, HEPES 10, EGTA 1, CaCl2 0.1, K2ATP 1, Na4GTP 0.2, and glucose 4, with the pH adjusted to 7.2 using KOH. The membrane potential was maintained at approximately -70 mV, and step currents from -20 to 50 pA were injected at 10-pA intervals. Whole-cell currents were recorded at a restraint potential of -70 mV, with voltage steps from -70 mV to +30 mV delivered in 20-mV increments. Spontaneous postsynaptic currents were recorded in whole-cell voltage-clamp mode. The whole-cell electrode solution used for synaptic current recording contained the following substances (in mM): CsCl 135, HEPES 10, EGTA 1, Mg-ATP 4, Na4GTP 0.4, and QX-314 10, pH 7.4. To sample excitatory and inhibitory currents, 1 mM glutamate and 100 μM GABA were ejected at 10 p.si for 100 ms, with voltages maintained at -70 mV and 0 mV, respectively. As expected, it generated repetitive action potential sequences induced by depolarization of the membrane in current-clamp mode, indicating that the ciNCC-derived neurons possess normal neuronal activity (data not shown).

[0228] 4. Cell proliferation analysis

[0229] To measure the proliferation capacity of ciCECs, cell proliferation analysis was performed. This was conducted using Click-iT... TMThe proliferation rate of ciCECs cultured in Stage II medium was determined using the Ethynyldeoxyuridine (EdU) Alexa Fluor 488 Imaging Kit (Ingenie Biotech / LifeScience) according to the manufacturer's instructions. In short, CECs were passaged at 5 × 10⁻⁶... 3 Cells were seeded at a low density of [number] cells / cm² onto FNC-coated slides and cultured for 24 hours. The results showed that these cells had high proliferative capacity (data not shown).

[0230] 5. Safety of ciCEC verified by teratoma formation test

[0231] To assess the potential risk of tumor development, 1×10 6 Subcutaneous injection of ciCEC into NOD-SCID mice resulted in teratoma formation at 4 to 8 weeks. To generate chimeras, ciCEC was injected into ICR blastocysts and transplanted into pseudopregnant ICR females. Germline transmission in the resulting chimeric mice was determined by mating F2 mice with ICR mice. All animal experiments were approved by and conducted according to the guidelines of the Animal Care and Use Committee of the Guangzhou Institutes of Biomedicine and Health. No tumors formed within 6 months after ciCEC transplantation, while large teratomas appeared after 4–8 weeks in recipients of mESC transplantation (data not shown). This indicates that ciCEC has minimal (if present) tumorigenic potential. Furthermore, ciCEC maintained a normal karyotype during 30 consecutive passages in vitro (data not shown). To better understand in vivo differentiation, ciCEC was transplanted into the eyes of NOD / SCID mice. Four to eight weeks after ciCEC transplantation, no tumors formed within six months following the transplantation (data not shown).

[0232] 6. Transplantation of ciCEC into damaged rabbit eyes

[0233] To assess the in vivo transplantation and expansion capabilities of ciCEC, we transplanted it into a rabbit model of bullous keratopathy induced by mechanical scraping of corneal endothelium from Desmith's membrane. Rabbits were divided into two groups (n=10 per group), and the right eye was used for this experiment. ciCEC was injected into the anterior chamber ( Figure 6 Image B (Figure 1). Each recipient receives 1×10⁻⁶. 6 One (low dose) ciCEC or 2×10 6 One (high-dose) ciCEC. Untreated normal rabbits ( Figure 6 Image B, number 4) and PBS injection only ( Figure 6The rabbit in image B (3rd image) was used as a control.

[0234] After disinfection and aseptic covering of the surgical site, a 6mm sclerokeratotomy was performed at the 12 o'clock position using a suture cutter (Alcon Surgical, Shanghai, China), and viscoelastic agent (Healon; Amersham Pharmacia Biotech AB) was infused into the anterior chamber. After marking the corneal surface with a marker (Devon Industries, Madrid, Spain), a 6.0mm diameter circular opening for descemetorhexis was created in the center of the cornea using a 30-gauge needle (Termall, Tokyo, Japan), and the descemetorhexis membrane was removed from the anterior chamber of the eye. The corneal endothelium was mechanically scraped from the descemetorhexis membrane using a lacrimal duct irrigator (Weigao, Shandong). Fsp-ciCEC was dissociated using 0.25% trypsin-EDTA and treated with 1×10⁻⁶... 7 Cells / mL were resuspended in PBS and kept on ice. The anterior chamber was washed three times with PBS.

[0235] Following this procedure, a 26-gauge needle was used to transfer 1×10⁶ cells suspended in 100 μl of basic DMEM containing 100 μM ROCK inhibitor Y-27632 (which promotes cell adhesion to the implantation site) (ROCK inhibitor, Silex). 6 One ciCEC was injected into the anterior chamber of the right eye. Figure 6 B, Image 1).

[0236] Following the procedure, the rabbits were placed in a prone position for 2–3 hours. Each operated eye was examined externally two or three times a week, and photographs were taken on days 1, 3, 5, 7, 14, 21, 35, and 42 post-surgery. Slit-lamp photographs showed a significant improvement in corneal transparency in the CEC-like cell group (ciCEC group) after injection, and pupillary and iris textures were visible. Only about 7 days later, the cornea became noticeably clear, while corneal opacity and stromal edema remained severe in the control group. Figure 6 Image B, from left to right, is the first and third image respectively. Visante OCT also shows a rapid decrease in corneal thickness after CEC-like cell injection. Figure 6 C, each image corresponds to the one above. Figure 6 Image B). Confocal microscopy images confirmed complete coverage of polygonal cells on the des Smith membrane in the ciCEC group ( Figure 6 D, Image 1. Figure 6 Each image in D corresponds to the image above. Figure 6 B and Figure 6Image C). It was found that the mean corneal thickness in the ciCEC group at days 1, 3, 5, 7, 14, 21, 35, and 42 was significantly smaller than that in the untreated control group. Figure 6 E). Slit-lamp images show significant improvement in corneal transparency in the ciCEC group at days 1, 3, 7, 14, 21, and 28. Figure 7 ).

[0237] Example 4: Generation of ciNCC and ciCEC from fibroblasts according to another embodiment

[0238] Preparation of M6 reprogramming medium

[0239] The basal medium contained knockout DMEM (Gibico), 10% KSR (Gibico), 10% FBS (Gibico), 1% NEAA (Gibico), and 0.1 mM 2-mercaptoethanol (Gibico), supplemented with small molecules Chir99021 (3 μM), SB431542 (5 μM), laryngin (10 μM), VPA (500 mM), EPZ004777 (5 μM), and 5-aza-dC (0.5 μM). The medium was shaken for 30 minutes to ensure complete dissolution of all components.

[0240] Preparation of differentiation culture medium

[0241] DMEM / F12 / GlutaMAX (Gibco), 10% KSR (Gibco), 1% NEAA (Gibco) and 0.1mM 2-mercaptoethanol (Gibco) supplemented with SB431542 (5μM) and CKI-7 (5μM).

[0242] From fibroblast chemical transformation NCC

[0243] MEF at 5×10 4 10 cells / well were seeded into fibroblast medium in 6-well tissue culture plates. The culture plates were pre-spread with fibronectin or laminin for more than two hours. After overnight culture, the medium was replaced with M6 chemical medium, which was refreshed every 2 days. NCC-like cells appeared and increased on days 3–5. After induction for 7–10 days, FACS sorting was performed to collect Wnt1 cells. + cell.

[0244] CEC-like cells induced by mouse ciNCC

[0245] From days 12 to 16, the M6 ​​chemo-medium medium was replaced with SB431542 and CKI-7 medium, which were refreshed every 2 days. Endothelial-like cell clusters appeared and increased on day 8, and Oct4-GFP positive clusters appeared on day 12. CEC-like cells appeared as early as day 20. Between days 30 and 35, CEC-like cell colonies were counted or further analyzed.

[0246] ciNCC differentiation

[0247] Approximately 5×10 3 One ciNCC was seeded on a laminine-coated glass coverslip in a 24-well plate containing NCSC medium and incubated for the first 24 hours. After 24 hours, the cells were subjected to differentiation conditions. For neuronal differentiation, the medium was switched to neuronal differentiation medium (NCC medium without bFGF and EGF, supplemented with 200 μM ascorbic acid, 2 μM db-cAMP, 25 ng / ml BDNF, 25 ng / ml NT3, and 50 ng / ml GDNF). Half of the medium was replaced every 2–3 days. Specific neuronal markers were analyzed from day 10 to day 20 post-differentiation. To differentiate into oligodendrocytes, cells were cultured for 1 day in the presence of 5 μM retinoic acid and 200 ng / ml Shh, and then cultured for 3–5 days in the presence of 20 ng / ml PDGF-AA, 20 ng / ml bFGF, and 200 ng / ml SHH. Subsequently, they were cultured for 8–12 days in differentiation medium containing 40 ng / ml T3, 200 ng / ml Shh, 1 nM LDN193189, 5 mM db-cAMP, and 10 ng / ml NT3. The medium was changed every other day. For astrocyte differentiation, 50 ng / ml BMP4 was added to the differentiation medium for 8–12 days, and the medium was changed every other day.

[0248] result

[0249] Given that corneal endothelium originates from NCCs, we designed a two-step approach to reprogram mouse fibroblasts into CEC-like cells using small molecules. The first step involves guiding the chemical reprogramming of mouse embryonic fibroblasts (MEFs) into ciNCCs. To screen for small molecules with the potential to convert fibroblasts into ciNCCs, we performed lineage tracing experiments to track the conversion process and exclude any NCCs or progenitor cells from the starting MEF. Figure 8 A, Figure 15A). Wnt1-Cre transgenic mice have been confirmed as a lineage-tracing report model for NC development. In Wnt1-Cre / ROSA26tdTomato mice, the tdTomato protein is faithfully expressed in the NCC. Therefore, MEFs were isolated from Wnt1-Cre / ROSA26tdTomato mice at E13.5. Because the NCC population was labeled with tdTomato, we performed fluorescence-activated cell sorting (FACS) to collect tdTomato. - The population was designed to exclude any NCC or progenitor cells (purified cells are referred to below as Wnt1-tdTomato). - MEF; Figure 15 B). We confirmed that Wnt1-tdTomato - MEF was also negative for other NCC markers (including Sox10, P75, Pax3, Hnk1, and AP2α). Figure 15 C, D). Additionally, these Wnt1-tdTomato - MEF was negative for typical NSC markers (including Sox2, Pax6, and nestin) (data not shown).

[0250] It has been reported that some small molecules used to enhance reprogramming can promote lineage reprogramming. To generate ciNCCs from MEFs, based on (1) epigenetic and signal transduction regulation of NCC development and (2) enhanced neural lineage reprogramming, we selected a group of 16 small molecules as candidates. Initially, the small molecule candidates for NC lineage reprogramming successfully focused on three classes of reprogramming factors, including Chir99021 (a GSK3 inhibitor), SB431542 (a TGF-β inhibitor), and trichomoniasis (a cAMP agonist). Figure 8 B). For subsequent screening and optimization, we found that VPA (HDAC inhibitor), EPZ004777 (DOT1L inhibitor), and 5-aza-dC (DNA methylation inhibitor) further enhanced the inhibition of Wnt1-tdTomato + Cell induction ( Figure 8 C). In this study, we used a chemically defined culture medium combined with a mixture of the aforementioned six small molecules (hereinafter referred to as M6) to reprogram MEF into ciNCC ( Figure 8 D). When treated with M6 medium, Wnt1-tdTomato expression was observed in each cell as early as day 3. Figure 8 E). M6 reprogrammed medium effectively induced Wnt1-tdTomato at 3.97%. + cell( Figure 8F). Induced Wnt1-tdTomato were observed in M6 reprogrammed medium on days 5–7. + colony ( Figure 8 G, Figure 15 E). These Wnt1-tdTomato + Cells and colonies exhibited a morphology similar to that of primary NCCs (pNCCs). Around day 12, Wnt1-tdTomato in small colonies... + The number of cells increased significantly. Figure 8 H). Although Wnt1-tdTomato + The efficiency of NCC generation was only comparable to that of human fibroblasts transformed with TFs31, but in this study only 2-5% of the cells were Wnt1-tdTomato positive. These results were reproducible in different batches of MEF (n=8), and MEFs with different genetic backgrounds (C57BL / 6, 129×C57BL / 6, and 129) could also be transformed into ciNCCs under the M6 ​​condition. Taken together, these results indicate that M6 can reprogram MEFs into ciNCCs.

[0251] Example 5: Characterization of ciNCC and ciCEC obtained in Example 4 and additional studies

[0252] Characterization of transformed ciNCC

[0253] The reprogramming process of ciNCCs has two phases: an initial phase (days 0-7) and an amplification phase (days 7-12). The first phase involves culturing MEFs in reprogramming medium to initiate epigenetic activation. A small number of NCC-like clusters with well-defined margins appear. The second phase involves culturing the epigenetically activated cells in a specific small molecule medium. Most clusters amplify and gradually grow during this second phase.

[0254] To obtain ciNCC, we perform FACS to collect Wnt1-tdTomato + Cells. Established ciNCCs were continuously propagated in standard NCC amplification medium containing N2, B27, bFGF, and EGF. Morphologically, M6-induced cells at P3 maintained typical NCC characteristics in monolayer culture. Figure 9 A). After passage, ciNCCs became morphologically uniform. Further characterization of M6-induced Wnt1-tdTomato + In our cells, we attempted to examine gene expression. Our results showed that ciNCCs express multiple NCC markers, including P75, HNK1, AP2α, and nestin (…). Figure 9B). Furthermore, we tested whether ciNCCs possessed the potential to differentiate into peripheral neurons, Schwann cells, and others. For ciNCC differentiation, these cells were cultured in different lineage differentiation media. After 2–4 weeks of culture, the expression of markers was assessed by immunostaining to examine the differentiated cells. Notably, ciNCCs also produced cells expressing neuron-specific markers (including Tuj1 and peripheral proteins). Figure 9 C). Regarding melanocyte differentiation, we observed melanocytes ( ) 2-3 weeks after induction. Figure 9 C). Our immunostaining results showed that ciNCCs can differentiate into Schwann cells. The induced Schwann cells were GFAP cells. + and S100β + cell( Figure 9 D). Further in vitro differentiation of these ciNCCs yields mesenchymal lineages, resulting in typical mesenchymal cell morphology. Our results demonstrate that these ciNCC-derived mesenchymal cells can generate osteoblasts, adipocytes, and chondrocytes (D). Figure 9 E). Together, these data suggest that our ciNCCs can be induced to differentiate into the peripheral nervous system lineage and the mesenchymal lineage.

[0255] Transplantation into animal models to demonstrate ciCEC function

[0256] To assess the in vivo corneal endothelial regeneration capacity of ciCEC, we transplanted it into a well-established rabbit model of bullous keratopathy induced by mechanical scraping of the corneal endothelium from Desmith's membrane. Previous studies have shown that injection of human pCEC supplemented with a ROCK inhibitor restored endothelial function. Based on this study, ciCEC was injected in combination with a ROCK inhibitor (Y27636) into the anterior chamber of the eye. Figure 14 A). Each recipient receives 1×10 6 One ciCEC was used. The contralateral eye (normal) and the untreated eye (PBS injection) served as experimental controls. Corneal edema decreased much earlier after ciCEC transplantation compared to the untreated eye. We observed a gradual increase in corneal transparency in the transplanted eye after transplantation, compared to the untreated eye which showed no change in corneal transparency. Figure 14 B. Figure 19 A). Seven days later, the cornea of ​​the transplanted eye became clear, while the corneal opacity and stromal edema remained poor in the untreated eye. Slit-lamp examination showed that the corneal transparency of the transplanted eye was also significantly improved after injection, and the pupillary and iris textures were clearly visible. Figure 14 C Figure 19 A).

[0257] Next, we investigated the survival of ciCECs in the transplanted eye. The eyeball was enucleated on day 28 post-surgery to assess the transplanted ciCEC cells. Fluorescence microscopy confirmed the presence of tdTomato-labeled transplanted cells. Immunohistochemistry showed ZO-1 expression, indicating pump function of the transplanted ciCEC cells. Figure 14 D、 Figure 19 C). Visante optical coherence tomography (OCT) of the anterior segment also showed a reduction in corneal thickness after ciCEC injection. Figure 14 E). Confocal microscopy confirmed complete coverage of polygonal endothelial cells on the des Smith membrane in the ciCEC transplantation disease model, which was undetectable in the untreated model due to its severe corneal opacity. Figure 14 G). Under magnification, on day 28, the transplanted ciCEC was tightly adhered to the posterior corneal surface in a monolayer, while the des Smith membrane in the untreated model was exposed and had no detectable CEC.

[0258] There was a rapid decrease in corneal thickness within 4 weeks after ciCEC injection, followed by a more gradual decrease over the next 2 weeks. Figure 14 F, Figure 19 (D) In ​​the untreated group, the mean corneal thickness was approximately 1200 μm throughout the 42-day observation period. In contrast, it decreased rapidly in the transplanted group, significantly less than the mean corneal thickness in the untreated group. We observed that at days 14 (P<0.01), 21, 28, 35, and 42 postoperatively (P<0.001), the mean corneal thickness in the ciCEC transplanted group was significantly less than that in the control group, indicating a significant reversal of corneal edema. These results strongly suggest that ciCEC transplantation refills and self-organizes the posterior corneal surface and has the ability to regenerate corneal endothelium.

[0259] Small molecules promote the induction of ciNCC into ciCEC.

[0260] To generate mouse CEC-like cells from ciNCCs, we also sought a group of small molecules as candidates based on the importance of small molecules in the in vitro CEC organogenesis and maintenance. In the initial screening, we found that SB431542 and CKI-7 could induce CEC-like cells from ciNCCs. These two compounds were subsequently included in the differentiation conditions (…). Figure 10 A). To determine whether ciNCCs could further differentiate into mature CECs, we treated ciNCCs with a differentiation medium containing both small molecules. After culturing in this differentiation medium for 7–15 days, small populations outside or within clusters exhibited typical compact aggregates with a uniform and polygonal morphology. Figure 10 B). We also observed that these colonies rapidly expanded, and by days 12–15, small clusters merged into larger clusters. Figure 10 B). These CEC-like cells grow rapidly and have strong proliferative capacity. ciNCC-induced CEC-like cells form monolayers of hexagonal and pentagonal cells. To confirm that the CEC-like cells are derived from ciNCC, we differentiated tdTomato cells using CEC differentiation medium containing 5 μM SB431542 and 5 μM MKI-7. + ciNCC. These tdTomato + ciNCCs can also be subsequently induced to differentiate into CEC-like cells. Figure 10 C). Na + / K + The expression of ATPase, AQP1, vimentin, ZO-1, and N-cadherin was further verified by immunofluorescence staining. Figure 10 D). In this study, we identified the function of CEC-like cells by the uptake of Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL). Figure 10 E). Global gene expression analysis via RNA sequencing revealed that CEC-like cells share a similar gene expression profile with primary CECs (pCECs), but this profile differs from that of the initial MEF. Figure 10 F). Tight junctions were observed in CEC-like cells via TEM. Figure 10 To further monitor the reprogramming process, we used qRT-PCR to further confirm the expression of a set of NCC and CEC markers at different time points. By day 12, robust expression of NCC genes, including Hnk1, P75, Sox10, Sox9, Pax3, and Ap2, was detected in cells. Figure 11 A). Additionally, CEC genes (such as Slc4a1c, Col8a1, Na) were detected by qRT-PCR. + / K + Similar dynamics to the activation of genes for ATPase, Aqp1, and N-cadherin (ATPase, Aqp1, and N-cadherin). Figure 11 B). Genes known to be enriched in pCECs are highly upregulated in ciCECs. To verify the transformation process from fibroblasts to CECs, we analyzed the transcriptome using RNA sequencing (B). Figure 11 C). The expression of 16 CEC-specific genes was significantly upregulated in ciCEC, consistent with pCEC. However, fibroblast-specific genes were significantly downregulated. Notably, a group of NCC marker genes were initially upregulated and subsequently downregulated during the induction process. Principal component analysis revealed that M6-treated cells differed from the initial MEF, indicating that chemical reprogramming induced significant transcriptional changes. Figure 11D). These results indicate that CEC-like cells acquire CEC properties. In general, these data demonstrate that the combination of SB431542 and CKI-7 effectively promotes the generation of CECs within 10–15 days in ciNCC culture. Those fibroblast-derived CEC-like cells are subsequently referred to as chemically induced CEC-like cells (ciCECs).

[0261] Used to confirm the lineage tracing of ciCEC induced from fibroblasts.

[0262] To confirm the origin of the initial fibroblasts used for small molecule-based reprogramming, we sought a genetic lineage tracing strategy for purifying fibroblast-specific protein 1 (Fsp1)-tdTomato-positive fibroblasts. Figure 12 A). Fsp1-Cre has been identified as a specific fibroblast marker for lineage tracing; therefore, Fsp1-Cre mice were crossed with ROSA26tdTomato mice. MEF cells were isolated from transgenic mice at E13.5 (Fsp1-Cre / ROSA26tdTomato) and fibroblasts specifically expressed tdTomato; these cells are named tdMEF (tdTomato) below. Figure 16 A). To avoid potential contamination of MEF by NCC progenitor cells, we performed FACS to collect tdTomato + / p75 - group( Figure 12 B). These tdMEFs were negative for all NCC markers (including P75, HNK1, Sox10, and AP2α). Figure 12 C).

[0263] Subsequently, these tdMEFs were induced using the M6 ​​medium described above. Epithelial clusters expressing tdTomato were observed during ciNCC induction. Figure 12 D、 Figure 16 B). We made Fsp1-tdTomato + ciNCCs were passaged and cultured in NCC amplification medium for further experiments (after 2 weeks of induction). Immunofluorescence analysis confirmed these Fsp1-tdTomato... + ciNCC was positive for NC markers P75, HNK1, SOX10, and AP2α, indicating that Fsp1-tdTomato + The ciNCC colonies have differentiated toward CEC-like cells. Figure 16 C). In addition, Fsp1-tdTomato + ciNCC can differentiate into tdTomato + ciCEC( Figure 16D). Through immunostaining, we found that differentiated CEC-like cells co-expressed Na. + / K + -ATPase, AQP1, laminin, ZO-1, Na + / K + -ATPase and tdTomato( Figure 12 E). Notably, all of these ciCECs also express tdTomato, demonstrating a transformation from fibroblasts. These results clearly confirm that ciNCCs and ciCECs transform from fibroblasts through a two-step lineage reprogramming.

[0264] CiCECs generated through chemicals bypass the induced pluripotent stem cell (iPSC) stage.

[0265] Because our lineage reprogramming mechanism may be similar to that of chemically reprogrammed iPSCs, we sought to assess whether ciCECs undergo an iPSC phase. We compared chemical iPSC reprogramming from MEFs derived from those carrying Oct4 promoter-driven GFP (OG2) reporter genes. We observed that these M6-treated MEFs underwent a characteristic mesenchymal-to-epithelial transition (MET), with small cell colonies gradually appearing near day 6. Figure 17 A). These cell colonies express the NCC marker Sox10 (A). Figure 17 B). In contrast, based on our method, we did not observe any Oct4-GFP positive cells throughout the entire process from MEF to ciCEC. Figure 17 C, 3D). Furthermore, ciCEC maintained a normal karyotype during ten consecutive passages in vitro. Figure 17 E). To assess the potential risk of tumor development, a total of 5 × 10 6 one CICEC and 2×10 6 Mouse embryonic stem cells (ESCs) were subcutaneously transplanted into NOD / SCID mice. Notably, no tumors formed within 6 months after transplantation with ciCECs, while large teratomas (data not shown) appeared after 4–8 weeks in mice transplanted with ESCs. This result indicates that ciCECs do not possess tumorigenic potential. To better understand their in vivo differentiation, we transplanted ciCECs into the anterior chamber of the eyes of NOD / SCID mice. No tumors formed from the transplanted ciCECs within 4–8 weeks, and within 6 months post-transplantation. These results demonstrate that our method can directly reprogram MEFs into ciNCCs and ultimately into ciCECs, bypassing the iPSC stage.

[0266] long-term in vitro amplification capacity of ciCEC

[0267] Maintaining the morphology and normal physiological function of cultured CECs in vitro has proven challenging. We aimed to test whether large quantities of functional ciCECs could be generated from fibroblasts to enable large-scale application of ciCECs. Based on our observations, ciCECs cultured in medium containing SB431542 (5 μM) and CKI-7 (5 μM) were small hexagonal cells (without epithelial-to-mesenchymal transition-like cells). Figure 18 A) We hypothesize that SB431542 and CKI-7 will promote the growth of ciCECs in vitro. We evaluated the long-term in vitro expansion capacity of ciCECs by sequential passage at a 1:6 ratio and found that the phenotype was similar between P3 and P30. Figure 13 A, B). This result demonstrates that SB431542 and CKI-7 strongly promote ciCEC expansion. In small molecule-based media, these ciCECs maintained themselves as a homogeneous cell population with a hexagonal morphology for at least 30 passages (P30). Furthermore, we successfully cultured these ciCECs clone-derived to 10 passages and exhibited a consistent morphology. Our immunostaining results showed that the proportion of Ki67-positive cells in P3 ciCECs was higher than that in P3 pCECs (…). Figure 18 B). ciCECs exhibit high proliferative capacity because 24.6%, 37.8%, and 48.1% of these cells in P1, P3, and P6, respectively, showed EdU incorporation ( Figure 18 C). FACS analysis using propidium iodide (PI) staining showed that the cell cycle distribution (G0 / G1, S, and G2 / M phases) was 46.30%, 45.11%, and 8.59% for ciCECs in P3, and 67.30%, 21.50%, and 11.20% for pCECs in P3, respectively. Figure 18 D). It rapidly expands into large homogeneous colonies, with a population doubling time of 22.3 ± 3.7 hours. Figure 13 C). Of particular interest are the large “vacuole-like” structures found on the surface of ciCECs at P2 to P10. Figure 13 D). These vacuolar structures disappear at P20 as cells proliferate continuously. Notably, ciCECs at P30 also express typical CEC markers, including Na+ / K+-ATPase, AQP1, and ZO-1 (…). Figure 13 E). When the ability to migrate into the gaps created by scratches was analyzed by imaging, ciCECs cultured in media containing SB431542 and CKI-7 at different passage numbers showed stronger proliferation and migration abilities compared to pCECs. Figure 18 E, F). Together, these results demonstrate that SB431542 and CKI-7 have robust and universal effects on long-term in vitro amplification of ciCECs.

Claims

1. A method for reprogramming a first type of cell into a third type of cell, comprising step (a) culturing the first type of cell in the presence of a first set of reprogramming factors, and step (b) culturing neural crest cell-like cells obtained from step (a) in the presence of a second set of reprogramming factors; When the first type of cell is mouse embryonic fibroblasts, the first group of reprogramming factors consists of 10 μM Chir99021, 10 μM Repsox, 10 μM trichomoniasis, and 10 ng / ml bFGF, or 10 μM Repsox, 10 μM Chir99021, and 10 μM trichomoniasis, or 3 μM Chir99021, 5 μM SB431542, 10 μM trichomoniasis, 500 mM VPA, 5 μM EPZ004777, and 0.5 μM 5-aza-dC; the second group of reprogramming factors consists of 5 μM SB431542 and 5 μM CKI-7. When the first type of cells are human embryonic skin fibroblasts, human newly generated fibroblasts, adult fibroblasts, human umbilical cord mesenchymal matrix cells, or urine cells, the first group of reprogramming factors consists of 10 μM Repsox, 10 μM Chir99021, 10 μM trichomoniasis, 5 μM 5-azacytidine, 500 μM VPA, and 10 ng / ml BMP4, and the second group of reprogramming factors consists of 5 μM SB431542 and 5 μM CKI-7; The third type of cell is corneal endothelial cell-like cells.

2. The method according to claim 1, wherein the neural crest-like cells are positive for P75, Hnk1, AP2α, and Sox10.

3. The method of claim 1, wherein the first type of cells are cultured in the presence of the first set of reprogramming factors for (a) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days or (b) no more than 20, 19, 18, 17, 16, 15, 14, 13 or 12 days.

4. The method according to claim 1, the method further comprising washing the cells obtained from step (a) prior to starting step (b).

5. The method according to claim 1, wherein there is no washing step between step (a) and step (b).

6. The method according to claim 1, wherein the corneal endothelial cell-like cells are ZO-1 and Na-1. + / K + -ATPase positive.

7. The method of claim 1, wherein the cells obtained from step (a) are cultured in the presence of the second set of reprogramming factors for (a) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days or (b) no more than 20, 19, 18, 17, 16, 15, 14, 13 or 12 days.

8. A kit for reprogramming a first type of cell into a third type of cell, wherein the kit comprises a first set of reprogramming factors and a second set of reprogramming factors; When the first type of cell is mouse embryonic fibroblasts, the first group of reprogramming factors consists of 10 μM Chir99021, 10 μM Repsox, 10 μM trichomoniasis, and 10 ng / ml bFGF, or 10 μM Repsox, 10 μM Chir99021, and 10 μM trichomoniasis, or 3 μM Chir99021, 5 μM SB431542, 10 μM trichomoniasis, 500 mM VPA, 5 μM EPZ004777, and 0.5 μM 5-aza-dC; the second group of reprogramming factors consists of 5 μM SB431542 and 5 μM CKI-7. When the first type of cells are human embryonic skin fibroblasts, human newly generated fibroblasts, adult fibroblasts, human umbilical cord mesenchymal matrix cells, or urine cells, the first group of reprogramming factors consists of 10 μM Repsox, 10 μM Chir99021, 10 μM trichomoniasis, 5 μM 5-azacytidine, 500 μM VPA, and 10 ng / ml BMP4, and the second group of reprogramming factors consists of 5 μM SB431542 and 5 μM CKI-7; The third type of cell is corneal endothelial cell-like cells.

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