WO2022110494A1 - 用于重编程细胞的方法 - Google Patents

用于重编程细胞的方法 Download PDF

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WO2022110494A1
WO2022110494A1 PCT/CN2020/141524 CN2020141524W WO2022110494A1 WO 2022110494 A1 WO2022110494 A1 WO 2022110494A1 CN 2020141524 W CN2020141524 W CN 2020141524W WO 2022110494 A1 WO2022110494 A1 WO 2022110494A1
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cells
inhibitor
type
reprogramming factors
inhibitors
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PCT/CN2020/141524
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English (en)
French (fr)
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林伟锋
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北京赛尔湃腾科技咨询合伙企业(有限合伙)
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Priority to US18/254,585 priority Critical patent/US20230416676A1/en
Priority to JP2023532738A priority patent/JP2023554248A/ja
Priority to EP20963382.5A priority patent/EP4289941A1/en
Publication of WO2022110494A1 publication Critical patent/WO2022110494A1/zh

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Definitions

  • the present disclosure generally relates to methods for reprogramming cells.
  • CECs corneal endothelial cells
  • corneal endothelial cells have little or no ability to proliferate in vivo and thus cannot be naturally replaced when damaged or otherwise lost.
  • the corneal endothelial cell layer is most densely aggregated at birth, and thereafter the cell density decreases rapidly as the eye grows (so that the same number of cells cover a larger area). Thereafter, corneal cell density gradually decreased with age, clearly reflecting the progressive loss of cells that were not replaced.
  • the cell density decreases, each cell spreads out and covers a larger area to maintain the barrier and pump functions of the cell layer.
  • the cell density falls too low (below about 500 to 1000 cells/mm2), its function is compromised, causing corneal opacity, stromal edema, loss of visual acuity, and eventual blindness.
  • the present disclosure provides a method for reprogramming a first type of cells into a second type of cells, comprising culturing the first type of cells in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors Contains glycogen synthase kinase 3 (GSK3) inhibitors, transforming growth factor-beta (TGFbeta) inhibitors, and cyclic AMP inducers.
  • GSK3 glycogen synthase kinase 3
  • TGFbeta transforming growth factor-beta
  • cyclic AMP inducers cyclic AMP inducers.
  • the first set of reprogramming factors further comprises basic fibroblast growth factor (bFGF), DNA methyltransferase inhibitors, histone methyltransferase inhibitors (eg, DOT1L inhibitors), A histone deacetylase (HDAC) inhibitor, BMP4, or a combination thereof.
  • bFGF basic fibroblast growth factor
  • DNA methyltransferase inhibitors eg, DNA methyltransferase inhibitors
  • histone methyltransferase inhibitors eg, DOT1L inhibitors
  • a histone deacetylase (HDAC) inhibitor eg, BMP4, or a combination thereof.
  • the first set of reprogramming factors consists of (a) a GSK3 inhibitor, a TGF[beta] inhibitor, and a cyclic AMP inducer, (b) a GSK3 inhibitor, a TGF[beta] inhibitor, a cyclic AMP inducer and bFGF or (c) GSK3 inhibitors, TGF ⁇ inhibitors, cyclic AMP inducers, DNA methyltransferase inhibitors, histone methyltransferase inhibitors (eg, DOT1L inhibitors), and histone deacetylases inhibitor.
  • 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, campalone (Kenpaullone), TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A014418, FRATide, indirubin 3'-oxime and L803.
  • the TGF ⁇ inhibitor is selected from the group consisting of SB431542, Repsox, 616452, LDN193189, A8301, GW788388, SD208, SB525334, LY364947, D4476, SB505124, and Tranilast.
  • the cyclic AMP inducer is forskolin, IBMX, Rolipram, 8BrcAMP, prostaglandin E2 (PGE2), NKH477, dibutyryl monocyclic adenylate (DBcAMP) ), Sp-8-Br-cAMPs.
  • the DNA methyltransferase inhibitor is selected from the group consisting of 5-aza-dC, 5-Azacytidine, and RG108.
  • the DOT1L inhibitor is EPZ004777.
  • the histone deacetylase inhibitor is selected from the group consisting of: valproic acid (VPA), trichostatin A (TSA), vorinostat, Depsipeptide, Trapoxin, Depudecin, FR901228 and butyrate.
  • the first type of cells are somatic cells.
  • the somatic cells are derived from mesoderm, ectoderm, or endoderm.
  • the somatic cells are fibroblasts.
  • the fibroblasts are selected from the group consisting of mouse embryonic fibroblasts (MEF), mouse tail tip fibroblasts (TTF), human embryonic fibroblasts (HEF), human neoplastic cells Fibroblasts (HNF), adult fibroblasts (HAF), human foreskin fibroblasts (HFF), and mixtures thereof.
  • the somatic cells are human exfoliated renal epithelial cells.
  • the first type of cells are stem cells.
  • 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).
  • the second type of cells are stem cells.
  • the stem cells are neural crest cell-like cells (NCC-like cells).
  • the NCC-like cells are positive for P75, Hnk1, AP2 ⁇ , and Sox10.
  • the cells of the first type are cultured (a) in the presence of the first set of reprogramming factors for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days or (b) not more than 20, 19, 18, 17, 16, 15, 14, 13 or 12 days.
  • the present disclosure provides a method of reprogramming cells of a second type into cells of a third type, comprising culturing the cells of the second type in the presence of a second set of reprogramming factors, wherein the second set of reprogramming factors comprises TGF ⁇ inhibitors and casein kinase 1 inhibitors.
  • the second set of reprogramming factors further comprises BMP4 and/or DNA methyltransferase inhibitors.
  • the casein kinase 1 inhibitor is CKI-7.
  • the DNA methyltransferase inhibitor is selected from the group consisting of 5-azacytidine, 5-aza-dC, and RG108.
  • the third type of cells are somatic cells.
  • the somatic cells are corneal endothelial cell (CEC)-like cells (CEC-like cells).
  • CEC-like cells are positive for ZO-1 and Na + /K + -ATPase.
  • the second type of cells is cultured (a) in the presence of the second set of reprogramming factors for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days or (b) not more than 20, 19, 18, 17, 16, 15, 14, 13 or 12 days.
  • the present disclosure provides a method of reprogramming cells of a first type into cells of a third type, comprising step (a) culturing the cells of the first type in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors
  • the factors comprise a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGF ⁇ ) inhibitor, and a cyclic AMP inducer, and step (b) culturing in the presence of a second set of reprogramming factors obtained from step (a) cells in which the second group of reprogramming factors includes TGF ⁇ inhibitors and casein kinase 1 inhibitors.
  • the method further comprises washing the cells obtained from step (a) before starting step (b). In certain embodiments, there is no washing step between steps (a) and (b).
  • the present disclosure provides a method of reprogramming cells of a first type into cells of a third type, comprising culturing the cells of the first type in the presence of a first set of reprogramming factors and a second set of reprogramming factors, wherein the first set of reprogramming factors
  • One group of reprogramming factors includes glycogen synthase kinase 3 (GSK3) inhibitors, transforming growth factor (TGF ⁇ ) inhibitors, and cyclic AMP inducers
  • a second group of reprogramming factors includes TGF ⁇ inhibitors and casein kinase 1 inhibitors agent.
  • the present disclosure provides populations of NCC-like cells produced according to the methods provided herein.
  • the present disclosure provides corneal endothelial cell-like cell (CEC-like cell) populations produced according to the methods provided herein.
  • the present disclosure provides a composition comprising the NCC-like cells or CEC-like cells provided herein.
  • the present disclosure provides a method of treating a disease or condition associated with dysfunctional or damaged corneal endothelial cells comprising administering to a subject in need thereof an effective amount of a CEC-like cell provided herein or a combination provided herein thing.
  • the subject is a human.
  • the disease or condition is selected from the group consisting of: Fuch's dystrophy, iridocorneal endothelial syndrome, posterior polymorphic dystrophy, congenital hereditary endothelial dystrophy, age Associated macular degeneration (AMD), retinitis pigmentosa, glaucoma, corneal dystrophy, contact lens use, cataract surgery, and advanced endothelial failure in corneal transplantation.
  • Fuch's dystrophy iridocorneal endothelial syndrome
  • posterior polymorphic dystrophy congenital hereditary endothelial dystrophy
  • age Associated macular degeneration (AMD) age Associated macular degeneration
  • retinitis pigmentosa glaucoma
  • corneal dystrophy contact lens use
  • cataract surgery cataract surgery
  • advanced endothelial failure in corneal transplantation advanced endothelial failure in corneal transplantation.
  • the present disclosure provides a kit for reprogramming cells of a first type into cells of a second type, wherein the kit comprises a first set of reprogramming factors, and the first set of reprogramming factors comprises glycogen synthase kinase 3 (GSK3) inhibitors, TGF ⁇ inhibitors, and cyclic AMP inducers.
  • GSK3 glycogen synthase kinase 3
  • the present disclosure provides a kit for reprogramming cells of a second type into cells of a third type, wherein the kit comprises a second set of reprogramming factors, and the second set of reprogramming factors comprises a TGF ⁇ inhibitor and a protein protein kinase 1 inhibitor.
  • the present disclosure provides a kit for reprogramming cells of a first type into cells of a third type, wherein the kit comprises a first set of reprogramming factors and a second set of reprogramming factors, wherein the first set of reprogramming factors Factors include glycogen synthase kinase 3 (GSK3) inhibitors, TGF ⁇ inhibitors, and cyclic AMP inducers, and a second group of reprogramming factors includes TGF ⁇ inhibitors and casein kinase 1 inhibitors.
  • GSK3 glycogen synthase kinase 3
  • the present disclosure provides a method of identifying a drug that affects 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 cellular response to the drug candidate responses to identify drugs.
  • ciNCC chemically-induced NCC
  • ciCEC chemically-induced CEC
  • Figure 1 shows the transformation of fibroblasts into ciNCCs by chemically defined conditions.
  • C) Shows the number of ciNCC colonies generated under the indicated conditions. Data are mean ⁇ SD, n 3 independent experiments.
  • Figure 2 shows the differentiation potential of ciNCC transformed from fibroblasts.
  • Right brightfield image shows melanocytes differentiated from ciNCC.
  • Figure 3 shows that ciNCCs can be further differentiated into corneal endothelial cell-like cells (or chemically induced CECs, ciCECs).
  • Figure 3A shows immunofluorescence staining for corneal endothelial markers including Na+-K+ ATPase, AQP1, Vimentin, N-cadherin, laminin, and AQP1. Nuclei were stained with DAPI. Scale bar, 50 ⁇ m.
  • Figure 3B shows a transmission electron microscope image showing tight junctions of corneal endothelial cells.
  • Figure 3C shows a scheme of a two-step lineage reprogramming strategy for the functional generation of corneal endothelial cells from fibroblasts.
  • Figure 4 shows Lineage tracing of fibroblast reprogramming towards corneal endothelial cell-like cells (or ciCECs).
  • FIG. 5 shows the generation of corneal endothelial-like cells from different human cell types by small molecules.
  • Human embryonic skin fibroblasts HEF
  • human neoplastic fibroblasts HNF
  • adult fibroblasts HAF
  • human umbilical cord mesenchymal stromal cells MSC
  • urine cells UC
  • Figure 6 shows the clinical observations at different days for the ciCEC group and the control group in rabbits.
  • A) Brightfield images of ciCECs at passage 10 (P10) are shown. ciCECs at passage 10 expressed Na+-K+ ATPase and ZO-1.
  • B) Shows a slit lamp photograph showing a significant improvement in the transparency of the cornea in the ciCEC group following day 7 injection of ciCEC transplantation (panel 1), while corneal opacity and stromal edema in the untreated control group analyzed by slit lamp photographs Still serious (Figure 3).
  • C) Demonstrates significant corneal thickness differences in the CEC-like cell group and the control group analyzed by visante OCT.
  • Figure 7 shows slit lamp photographs at different days for ciCEC and control groups in rabbits. Slit-lamp photographs demonstrate significantly improved corneal transparency in the ciCEC group after injection, and pupil and iris texture can be seen (ciCEC group, upper panel). After only about 14 days, the cornea became visibly clear, while corneal opacity and stromal edema remained severe in the control group (control group, upper panel).
  • the lower panels for both the ciCEC and control groups show corneal reflections detected by slit lamp, and each image in the lower panel corresponds to the image in the upper panel.
  • Figure 8 shows the conversion of MEFs to ciNCCs by small molecule induction.
  • B) Effects of individual chemicals on ciNCC generation. Data are mean ⁇ SD, n 3 independent experiments.
  • C) Promotion effect of each chemical on ciNCC generation (data are mean ⁇ SD, n 3 independent experiments).
  • F) Quantification of Wnt1 + cell numbers in ciNCC clusters induced by candidate mixtures in combinatorial screening (independent experiments, n 3).
  • G Morphological changes at different days during ciNCC induction treatment (scale bar, 50 ⁇ m).
  • Figure 9 shows the characterization of M6-induced ciNCC.
  • Figure 10 shows the generation of mouse ciCECs from fibroblasts by small molecule induction.
  • Figure 11 shows gene expression profiling of ciNCC and ciCEC.
  • C) Heatmap of differentially expressed genes in samples at indicated time points. Numbers below the heatmap indicate independent biological replicates (n 2-3). Red and blue indicate up- and down-regulated genes, respectively.
  • Figure 12 shows lineage tracing demonstrating induction of ciCECs from fibroblasts.
  • Figure 13 shows that small molecules promote long-term expansion of ciCECs.
  • C) Mean population doubling time of ciCECs cultured in medium with or without SB431542 and CK1-7 (mean ⁇ SD, n 3; ***p ⁇ 0.001).
  • Figure 14 shows ciCEC engraftment in vivo.
  • A Graph depicting transplantation of ciCEC and ROCK inhibitor into model rabbits.
  • B After transplantation, corneal transparency was significantly improved in transplanted eyes, while corneal opacity and stromal edema remained severe in untreated controls.
  • C Slit lamp micrograph showing that after transplantation, the transparency of the transplanted cornea was significantly improved, while corneal opacity and stromal edema remained in the untreated control.
  • D Immunohistochemistry showing attachment of viable tdTomato + ciCECs to De Smith's membrane (scale bar, 100 ⁇ m).
  • E Visante OCT demonstrating improved corneal edema (reduced corneal thickness) in transplanted eyes.
  • G Real-time confocal imaging of the corneal endothelium demonstrating complete coverage of polygonal cells on Desmi's membrane in the transplanted eye.
  • FIG. 15 shows the characterization of Wnt1 - MEFs.
  • Figure 16 shows that M6 converts tdMEFs to ciNCCs.
  • Figure 17 shows the generation of ciCECs from fibroblasts by small molecules that do not pass through the iPSC stage.
  • D) Oct4-GFP positive cells were absent for ciCEC induction as determined by FACS analysis.
  • Figure 18 shows the proliferative potential of ciCECs.
  • D Distribution of ciCECs and pCECs in the cell cycle (G1, S and G2 phases) (scale bar, 400 ⁇ m). ciCECs at P3 and pCECs at P3 were scratched and migration was observed after 8 and 20 hours and images were taken.
  • Figure 19 shows observations on the transplanted eyes of the rabbit model.
  • compositions comprising/comprises
  • methods comprising/comprises
  • components essential to the methods or compositions, but includes unspecified elements, whether or not Whether necessary, is still open.
  • cell refers to a single cell, cell line or culture derived from such cells.
  • reprogramming/reprogramming/reprogrammed refers to an altered state of differentiation in culture or in vivo compared to the differentiated state a cell would have under the same conditions without reprogramming or The process of reversing the differentiated state of a cell.
  • reprogramming includes differentiation, dedifferentiation, and transdifferentiation.
  • differentiation refers to a cellular process by which less specialized cells become more specialized cell types.
  • the term “dedifferentiation” refers to a cellular process in which partially or terminally differentiated cells revert to an earlier developmental stage, such as cells with pluripotency or pluripotency.
  • the term “transdifferentiation” refers to the cellular process of converting one differentiated cell type into another differentiated cell type.
  • the terms “lower differentiated state” or “less specialized” or “early developmental stage” are relative terms and include fully dedifferentiated states (or fully dedifferentiated) and partially differentiated states (or partially differentiated states) ).
  • an "undifferentiated cell” is a cell that is capable of differentiating in many directions, ie, it is capable of differentiating into two or more types of specialized cells.
  • Typical examples of undifferentiated cells are stem cells.
  • Totipotent stem cells refers to cells that are capable of differentiating into all the cells that make up an organism, such as cells resulting from the fusion of egg cells and sperm cells. Cells produced from the first few divisions of a fertilized egg may also be totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells, such as ES cells, can give rise to any embryonic and adult cell type. However, it cannot develop into embryonic or adult animals alone because it lacks the potential to develop extraembryonic tissues.
  • Extraembryonic tissue is derived in part from extraembryonic endoderm and can be further classified into parietal endoderm (Reichert's membrane) and visceral endoderm (the part that forms the yolk sac) . Both the luminal endoderm and the visceral endoderm support embryonic development but do not themselves form embryonic structures. Other extraembryonic tissues also exist, including extraembryonic mesoderm and extraembryonic ectoderm.
  • pluripotency refer to cell populations that can differentiate into all three germ layers (eg, endoderm, mesoderm, and ectoderm).
  • Pluripotent cells express a variety of pluripotent cell-specific markers, have the cellular morphological characteristics of undifferentiated cells (ie, dense colonies, high nucleocytoplasmic ratio, and prominent nucleoli), and when introduced into immunocompromised animals such as SCID mice Teratoma is formed in the middle. Teratomas typically contain cellular or tissue features of all three germ layers. Those of ordinary skill in the art can assess these characteristics by using techniques commonly used in the art. See, eg, Thomson et al., Science 282:1145-1147 (1998). Pluripotent cells are capable of proliferating in cell culture and differentiating toward a variety of lineage-restricted cell populations that exhibit pluripotent properties. 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 have a higher potential than pluripotent stem cells.
  • the present disclosure provides a method for reprogramming cells of a first type into cells of a second type, comprising culturing the cells of the first type in the presence of a first set of reprogramming factors, wherein the first
  • the panel of reprogramming factors includes glycogen synthase kinase 3 (GSK3) inhibitors, transforming growth factor-beta (TGFbeta) inhibitors, and cyclic AMP inducers.
  • culturing the first type of cells in the presence of the first set of reprogramming factors results in 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 become cells of the second type.
  • the cells of the first type are mammalian cells derived from mesoderm, ectoderm or endoderm, such as human or mouse.
  • Ectoderm, mesoderm, and endoderm are the three germ layers formed during embryonic development, with mesoderm as the middle layer, ectoderm as the outer layer and endoderm as the inner layer.
  • the mesoderm forms the mesenchyme, mesothelial, non-epithelial blood cells, and coelomocytes, which make up muscles (smooth and striated), bone, cartilage, connective tissue, adipose tissue, circulatory system, lymphatic system, dermis, urogenital system, serosa and notochord.
  • the endoderm forms the pharynx, esophagus, stomach, small intestine, colon, liver, pancreas, bladder, trachea and bronchial epithelial parts, lungs, thyroid and parathyroid glands.
  • the ectoderm forms the superficial ectoderm, the neural crest, and the neural tube, where the superficial ectoderm develops into the epidermis, hair, nails, lens of the eye, sebaceous glands, cornea, enamel, and epithelium of the mouth and nose; the neural crest of the ectoderm develops into the surrounding Nervous system, adrenal medulla, melanocytes, facial cartilage, and dentin; and the neural tube of the ectoderm develops into the brain, spinal cord, posterior pituitary gland, motor neurons, and retina.
  • the first type of cells are somatic cells.
  • somatic cell refers to any cell other than germline cells (eg, sperm and eggs, and the cells that make them (gametocytes)) and undifferentiated stem cells. Internal organs, skin, bone, blood, and connective tissue are all composed of somatic cells. Somatic cells can be any type of somatic cell of any origin.
  • somatic cells can include, but are not limited to, fibroblasts, epithelial cells, Sertoli cells, endothelial cells, epithelial granulosa, neurons, pancreatic islet cells, epidermal cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, Melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), red blood cells, macrophages, monocytes, mononuclear cells, cardiomyocytes, and other muscle cells.
  • the cells of the first type are mesoderm cells. Markers for mesodermal cells are known in the art, such as CD56 and APJ.
  • mesodermal cells include fibroblasts, epithelial cells, leukocytes, adipocytes, and keratinocytes.
  • the first type of cells are fibroblasts, including but not limited to mouse embryonic fibroblasts (MEFs), mouse neoplastic fibroblasts (MNFs), mouse tail tip fibroblasts (TTFs) ), human embryonic skin fibroblasts (HEF), human neoplastic fibroblasts (HNF), adult fibroblasts (HAF), human embryonic lung fibroblasts, human foreskin fibroblasts (HFF), and mixtures thereof.
  • MNFs mouse embryonic fibroblasts
  • MNFs mouse neoplastic fibroblasts
  • TTFs mouse tail tip fibroblasts
  • HEF human embryonic skin fibroblasts
  • HNF human neoplastic fibroblasts
  • HNF human neoplastic fibroblasts
  • HFF human embryonic lung fibroblasts
  • HFF human foreskin fibroblasts
  • 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.
  • fibroblasts are obtained from a patient, for example, by a skin biopsy (e.g., reprogramming of human somatic cells to pluripotency with defined factors).
  • a skin biopsy e.g., reprogramming of human somatic cells to pluripotency with defined factors.
  • the cells of the first type are epithelial cells, such as human exfoliated kidney epithelial cells.
  • Epithelial cells can be obtained from urine samples.
  • the first type of cells are leucocytes.
  • Leukocytes can be obtained from blood samples.
  • Leukocytes are white blood cells that can generally be classified into their subpopulations (lymphocytes, monocytes, neutrophils, eosinophils, and basophils) by the following differences: RF signal intensity (high frequency change in electrical impedance), DC signal intensity (the change in DC current caused by the difference in conductivity between the suspended particles and the liquid medium in which the particles are suspended), fluorescence intensity, scattered light intensity, absorbance, scattered light depolarization, etc. etc. (see US5618733A).
  • the first type of cells are adipocytes or keratinocytes.
  • Adipocytes and keratinocytes are also readily obtained from skin biopsy or harvested hair (isolation and cultivation of human keratinocytes from skin or plucked hair) and culture of human keratinocytes for the generation of induced pluripotent stem cells), Belmonte et al. Nature Protocols 2010).
  • the first type of cells are adult cells.
  • the term "adult cell” refers to cells found throughout the body after embryonic development.
  • the first type of cells 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 Mesenchymal stem cells, epidermal stem cells, adipose-derived stem cells, intestinal stem cells, neural stem cells, renal epithelial stem cells, urothelial stem cells and liver stem cells.
  • 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 Mesenchymal stem cells, epidermal stem cells, adipose-derived stem
  • stem cells refer to undifferentiated cells that can proliferate and give rise to more progenitor cells, which can give rise to numerous mother cells, which can in turn give rise to differentiated or differentiated daughter cells.
  • Stem cells can divide asymmetrically, with one daughter cell retaining the stem cell state and the other daughter cell expressing some unique other specific function and phenotype.
  • some of the stem cells in the population can 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.
  • Daughter cells themselves can be induced to proliferate and produce progeny, which then differentiate into one or more mature cell types, while also retaining one or more cells with the developmental potential of the parent.
  • stem cells refers to a subpopulation of progenitor cells with the ability or potential to differentiate into a more specialized or differentiated phenotype under specific circumstances, which retains the ability to proliferate without being substantially differentiated under certain circumstances ability.
  • stem cell generally refers to a naturally occurring parent cell, the progeny (progeny) of which are typically specialized in different directions through differentiation (eg, by acquiring fully individual characteristics), as in the progressive diversification of embryonic cells and tissues occur in the process. Differentiated cells can be derived from pluripotent cells, which themselves are derived from pluripotent cells, and so on.
  • stem cells Although each of these pluripotent cells can be considered a stem cell, the range of cell types that each can produce can vary significantly. In many biological instances, stem cells are also "pluripotent" in that 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 definition of stem cells. In theory, self-renewal can occur through either of two mechanisms.
  • embryonic stem cells is used to refer to the pluripotent stem cells of the inner cell mass of embryonic blastocysts (see US Pat. Nos. 5,843,780, 6,200,806, which are incorporated herein by reference). Distinguishing characteristics of embryonic stem cells define the embryonic stem cell phenotype. Thus, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell that allow the cell to be distinguished from other cells. Exemplary embryonic stem cell distinguishing characteristics include, but are not limited to, gene expression profiles, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.
  • adult stem cell or "ASC” is used to refer to any pluripotent stem cell derived from non-embryonic tissues, including fetal, larval, and adult tissues.
  • ASC adult stem cells
  • 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 nearly every tissue. Thus, stem cell populations can be isolated from virtually any animal tissue as can be seen from the techniques described herein.
  • the first type of cells may be mesenchymal stem cells, mesenchymal stromal cells, human embryonic stem cells, or induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • iPS cells As used herein, the terms “iPS cells,” “iPSCs,” and “induced pluripotent stem cells” are used interchangeably and refer to artificially derived (eg, induced by complete or partial reversal) from differentiated somatic cells (ie, derived from non-differentiated somatic cells). pluripotent cells). Pluripotent cells can differentiate into all cells of the three developing germ layers.
  • MSCs Mesenchymal stem cells
  • mesenchymal stem cells are adult stem cells traditionally found in the bone marrow.
  • mesenchymal stem cells can also be isolated from other tissues including umbilical cord blood, peripheral blood, fallopian tubes and fetal liver and lung.
  • MSCs can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), and adipocytes (fat cells that produce adipose tissue in the bone marrow). )).
  • the first type of cells are ectodermal cells. In certain embodiments, the first type of cells are endoderm cells.
  • the cells of the second type are transdifferentiated from the cells of the first type.
  • mesoderm cells are reprogrammed into ectoderm cells.
  • the second type of cells are ectodermal cells.
  • the pluripotent stem cells are neural crest cells (NCCs) or neural crest cell-like cells.
  • the second type of cells are neural crest cells (NCCs) or neural crest cell-like cells.
  • Neural crest cells generally refer to neural progenitor cells that have the developmental potential to generate 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) provided herein from primary NCCs, the resulting ciNCCs are also named neural crest cell-like cells (NCC-like cells). In some embodiments, the NCC-like cells of the resulting NCC exhibit one or more biomarkers consistent with the phenotype of the primary NCC.
  • the resulting NCC-like cells lack or have lower expression of one or more biomarkers consistent with the NCC phenotype (eg, expression of PAX6).
  • exemplary NC biomarkers whose presence is consistent with the NCC phenotype can include Nestin, SOX10, SOX9, HNK-1, P75 (NGFR), AP2 ⁇ , PAX3, PAX7, SNAI2, Snail, Twist1, Krox20, CD271, FoxD3, AN2 and Ki67, and/or at least one of the pluripotency markers NANOG, ZNF206 or OCT4.
  • the NC biomarker is P75, Hnk1, AP2 ⁇ and/or SOX10.
  • the expression of the NC 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.
  • the first type of cells are fibroblasts and the second type of cells are NCC-like cells.
  • the first type of cells are mouse embryonic fibroblasts (MEFs) and the second type of cells are NCC-like cells.
  • the first type of cells are mouse tail tip fibroblasts (TTFs) and the second type of cells are NCC-like cells.
  • the first type of cells are human embryonic skin fibroblasts (HEFs) and the second type of cells are NCC-like cells.
  • the cells of the first type are human embryonic lung fibroblasts, human foreskin fibroblasts (HFF) and the cells of the second type are NCC-like cells.
  • the cells of the first type are human neoplastic fibroblasts (HNF) or adult fibroblasts (HAF), and the cells of the second type are NCC-like cells.
  • the cells of the first type are epithelial cells (eg, human exfoliated kidney epithelial cells) and the cells of the second type are NCC-like cells.
  • the cells of the first type are leukocytes (eg, lymphocytes, monocytes, neutrophils, eosinophils, and basophils) and the cells of the second type are NCC-like cells.
  • the first type of cells are adipocytes and the second type of cells are NCC-like cells.
  • the cells of the first type are keratinocytes and the cells of the second type are NCC-like cells.
  • the first type of cells are MSCs, human embryonic stem (ES) cells or iPSCs and the second type of cells are NCC-like cells.
  • the second type of cells is certain cells reprogrammed from another type of stem cells (the first type of cells), such as mesenchymal stem cells, human embryonic stem cells, or induced pluripotent stem cells (iPSCs) types of pluripotent stem cells.
  • the first type of cells such as mesenchymal stem cells, human embryonic stem cells, or induced pluripotent stem cells (iPSCs) types of pluripotent stem cells.
  • 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 (eg, PCR, isothermal amplification, etc., which can include reverse transcription and can be applied to detect the single cell or multiple cell expression), Northern blotting, immunostaining, etc.
  • the expression of the marker can be achieved by the expression of a reporter construct (eg, the expression of a fluorescent protein that can be detected visually, the expression of an antibiotic resistance gene that can be detected by cell survival in the presence of antibiotics, etc.) It is inferred that the reporter construct is under the control of a genetic element conferring cell-type specific expression, such as a promoter of one of the aforementioned markers or a fragment thereof.
  • 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 of which can be readily detected using routine methods. Other methods of detecting marker expression that can be used are known in the art.
  • adherent culture systems are used in the methods of the present disclosure.
  • adherent culture refers to a cell culture system whereby cells are cultured on a solid surface, which in turn can be coated with a matrix. Cells may or may not adhere tightly to a solid surface or matrix.
  • the matrix for adherent culture may further comprise, for example, any one or a combination of the following: 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-glycolic acid (PLGA), matrigel, hydroxyapatite, and amniotic membrane.
  • 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-glycolic acid (PLGA), matrigel, hydroxyapatit
  • suspension culture can be used in the methods of the present disclosure.
  • the term "suspension culture” as used herein refers to a mode of cell culture in which cells do not adhere to a solid support or culture vessel. To transfer cells into suspension cultures, cells are removed from the culture vessel, such as by a cell scraper, and transferred to sterile low-adherence plates containing culture medium that do 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 dish.
  • a medium suitable for growing cells is any medium suitable for growing a cell type in a petri dish.
  • Such media include, for example, Ham's F10 (Sigma), Ham's F12 medium, Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Dulbecco's Modified Eagle's Medium (DMEM) (Sigma), IMDM medium, Medium 199, Eagle's Minimum Essential Medium (EMEM), aMEM medium, Fisher's culture Fischer's medium, Neurobasal medium (Life Technologies Corporation), and mixtures of these media.
  • Ham's F10 Ham's F12 medium, Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma) and Dulbecco's Modified Dulbecco's Modified Eagle's Medium (DMEM) (Sigma), IMDM medium, Medium 199, Eagle's Minimum Essential Medium (EMEM), aMEM medium, Fisher's culture Fischer's medium, Neurobasal medium
  • any of these media may be supplemented as needed with required salts (eg, sodium chloride, calcium, magnesium, and phosphate), buffers (eg, HEPES), nucleotides (eg, adenosine and thymidine) , antibiotics (such as GENTAMYCIN TM drugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), glucose or equivalent energy sources, albumin, insulin, transferrin, selenium, fatty acids, 2-Mercaptoethanol, thiol glycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antioxidants, pyruvate, cytokines, and more. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions (eg, temperature, pH, etc.) are those previously used for cell culture and will be apparent to the ordinarily skilled artisan.
  • salts eg, sodium chlor
  • the minimal medium may contain DMEM/F12/Glutamax (GIBCO), 10% KnockOut Serum Replacement (KSR) (GIBCO) prior to the addition of the first set of reprogramming factors ), 1% NEAA (Gebeco), 10% FBS (Gebeco), and 0.1 mM 2-mercaptoethanol (Gebeco).
  • the minimal medium may contain DMEM/F12/Glutamax (Gebeco), 0.075% bovine serum albumin (BSA) (Gebeco), 1% prior to addition of the first set of reprogramming factors NEAA (Gebeco) and 0.1 mM 2-mercaptoethanol (Gebeco).
  • the minimal medium may contain DMEM/F12/Glutamax (Gebeco), 10% Knockout Serum Replacement (KSR) (Gebeco), 1% NEAA ( Gibeco) and 0.1 mM 2-mercaptoethanol (Gibeco).
  • the minimal medium for the second set of reprogramming factors is serum-free. Those skilled in the art will appreciate that other necessary supplements (as described) can be added to the medium.
  • the culture temperature is a temperature 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 more preferably 37.0°C to 39.0°C.
  • a reprogramming factor is a molecule that, alone or in combination with other molecules, can cause reprogramming when contacted with a cell (eg, expressed by a cell, transformed into a cell for expression, provided exogenously to a cell, etc.).
  • Reprogramming factors can be provided from exogenous sources, for example, by addition to the culture medium, and can be introduced into cells by methods known in the art, such as by coupling to cell entry peptides, protein or nucleic acid transfectants, lipids In vivo transfection, electroporation, biolistic particle delivery system (gene gun), microinjection, etc.
  • the reprogramming factor is added to the medium without being coupled to any other components.
  • the first set of reprogramming factors that can be used to reprogram cells of a first type to cells of a second type comprises glycogen synthase kinase 3 (GSK3) inhibitors, transforming growth factor-beta (TGF[beta]) ) inhibitors and cyclic AMP inducers.
  • the first set of reprogramming factors further comprises basic fibroblast growth factor (bFGF), DNA methyltransferase inhibitors, histone methyltransferase inhibitors (eg, DOT1L inhibitors), a set of A protein deacetylase inhibitor, BMP4, or a combination thereof.
  • the first set of reprogramming factors consists of GSK3 inhibitors, TGF[beta] inhibitors, and cyclic AMP inducers. In certain embodiments, the first set of reprogramming factors consists of a GSK3 inhibitor, a TGF[beta] inhibitor, a cyclic AMP inducer, and bFGF. In certain embodiments, the first set of reprogramming factors consists of GSK3 inhibitors, TGF[beta] inhibitors, cyclic AMP inducers, DNA methyltransferase inhibitors, histone deacetylase inhibitors, and BMP4.
  • the first set of reprogramming factors is comprised of GSK3 inhibitors, TGF ⁇ inhibitors, cyclic AMP inducers, DNA methyltransferase inhibitors, histone methyltransferase inhibitors (eg, DOT1L inhibitors) ) and histone deacetylase inhibitors.
  • reprogramming factors can sometimes be functionally substituted by paralogs within their respective families.
  • the term "inhibitor” refers to a reduction in the expression and/or activity of a targeted expression product (eg, mRNA or target polypeptide encoding a target), eg, by at least 10% or more (eg, 10% or more). , 50% or higher, 70% or higher, 80% or higher, 90% or higher, 95% or higher, or 98% or higher).
  • a targeted expression product eg, mRNA or target polypeptide encoding a target
  • the efficacy of an inhibitor eg, its ability to reduce the level and/or activity of the target
  • the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody or binding fragment thereof; or a small molecule.
  • 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 (GSK3 ⁇ and GSK3 ⁇ ) that are encoded by different genes and have high homology at the amino acid level.
  • Examples of GSK3 inhibitors include GSK3 ⁇ inhibitors and GSK3 ⁇ inhibitors.
  • GSK3 inhibitors include siRNA against the gene encoding GSK3, anti-GSK3 antibodies, CHIR98014 (Milipore, Bedford), CHIR99021 (Bedford Millipore), Kenpaullone ( Bedford Millipore), AR-AO144-18 (Santa Cruz Biotechnology, Santa Cruz), TDZD-8 (Abcam, Cambridge, US), SB216763 ( Cambridge, USA), BIO ((2'Z,3'E)-6-bromo indirubin-3'-oxime) (R&D Systems, Minneapolis, US) ), TWS-119 (Cambridge, USA), SB415286 (Cambridge, USA), Ro3303544 (US6479490) LiCl, Li 2 CO 3 and the like. All of these are commercially available or can also be prepared by one of ordinary skill in the art with reference to known literature.
  • the GSK3 inhibitor is selected from the group consisting of CHIR99021 , LiCl, Li2CO3, and BIO. In certain embodiments, the GSK3 inhibitor is CHIR99021. In certain embodiments, the GSK3 inhibitor is BIO.
  • the concentration of the GSK3 inhibitor in the medium is appropriately determined according to the kind of inhibitor to be used. In the case of CHIR99021 or BIO, the concentration is generally 0.1-10 [mu]M, preferably 1-5 [mu]M, more preferably about 3 [mu]M.
  • One or more classes of GSK3 inhibitors can be used in combination.
  • TGF[beta] (transforming growth factor beta) is a multifunctional cytokine belonging to the transforming growth factor superfamily.
  • TGF ⁇ includes three different mammalian isoforms (TGF ⁇ 1 to 3, HGNC symbols TGFBETA1, TGFBETA2, TGFBETA3).
  • TGF[beta] can be secreted by many cell types, including macrophages, in a latent form, in which TGF[beta] is complexed with two other polypeptides, latent TGF[beta] binding protein (LTBP) and latency-associated peptide (LAP).
  • the source of the TGF ⁇ inhibitor to be used in the present invention is not particularly limited as long as it can effectively inhibit the TGF ⁇ function.
  • TGF[beta] inhibitors are commercially available or can also be prepared by one of ordinary skill in the art with reference to known literature. Specific examples of TGF ⁇ inhibitors include siRNA against the gene encoding GSK3, anti-TGF ⁇ antibodies, and chemical antagonists.
  • the TGF ⁇ inhibitor is selected from the group consisting of: SB431542 (Tocris Bioscience, Bristol, UK), Repsox (Tocris Bioscience, Bristol, UK) Science), LDN193189 (Tocris Life Sciences, Bristol, UK) and Tranilast (Rizaben).
  • the TGF ⁇ inhibitor is SB431542.
  • the concentration of the TGF ⁇ inhibitor in the medium is appropriately determined according to the kind of inhibitor to be used. In the case of SB431542, the concentration is generally 0.1-20 [mu]M, preferably 1-10 [mu]M, more preferably about 5 [mu]M. In the case of Repsox, the concentration is generally 0.1-20 [mu]M, preferably 1-15 [mu]M, more preferably about 10 [mu]M.
  • cyclic AMP inducer refers to any compound that at an effective concentration increases the intracellular concentration of cAMP in a cell by at least 2%, preferably by at least 5%, more preferably by at least 10%, most preferably by at least 20% .
  • Methods for measuring intracellular cAMP levels are known to those skilled in the art.
  • Preferred cyclic AMP inducers include isobutylmethylxanthine and forskolin.
  • the concentration of forskolin is generally 1-20 [mu]M, preferably 5-15 [mu]M, more preferably about 10 [mu]M.
  • bFGF Basic Fibroblast Growth Factor
  • Basic Fibroblast Growth Factor is a type of protein inherent in the body and is known to control cell growth and differentiation, and has functions in various tissues and organs such as angiogenesis, smooth muscle cell proliferation, wound Functions of therapy, tissue repair, hematopoiesis, neural cell differentiation, and more.
  • the origin of bFGF to be used in the present invention is not particularly limited as long as it is effective for reprogramming.
  • bFGF is commercially available or can also be prepared by one of ordinary skill in the art with reference to known literature. For example, it can be synthesized based on a known base sequence and amino acid sequence, for example, the amino acid sequence can be obtained under NCBI Accession Nos. AAA52448.1 (human) and AAA37621.1 (mouse).
  • the concentration of bFGF in the medium is generally 1-50 ng/ml, preferably about 1-20 ng/ml, more preferably about 10 ng/ml or about 20 ng/m
  • Histone acetylation is a reversible modification, and deacetylation is catalyzed by a family of enzymes called histone deacetylases (HDACs).
  • HDAC inhibitors include valproic acid (VPA), tricomycin A (TSA), vorinostat (suberoylanilide hydroxamic acid, SAHA, Merck & Co., Inc.), Depsipeptides (Romidepsin, FK-228, Gloucester Pharmaceutical Inc.), Trapoxin, Depudecin, FR901228 (Fujisawa Pharmaceuticals) and butyrate.
  • the HDAC inhibitor is VPA.
  • the concentration of the HDAC inhibitor in the medium is appropriately determined according to the kind of inhibitor to be used. In the case of VPA, the concentration is generally 1-2000 [mu]M, preferably 10-1000 [mu]M, more preferably about 500 [mu]M.
  • Minimal medium supplemented with the first set of reprogramming factors was refreshed every 1, 2 or 3 days.
  • the cells of the first type 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 certain embodiments, the cells of the first type 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.
  • the cells of the first type 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 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, 7 days to 12 days.
  • 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
  • the present disclosure provides a method for reprogramming a second type of cells into a third type of cells, comprising culturing the second type of cells in the presence of a second set of reprogramming factors, wherein the second Groups of reprogramming factors include TGF ⁇ inhibitors and casein kinase 1 inhibitors.
  • Casein kinase 1 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, transcription factor nucleocytoplasmic shuttling, DNA repair, and DNA transcription.
  • the origin of the CK1 inhibitor to be used in the present invention is not particularly limited as long as it is effective for reprogramming.
  • CK1 inhibitors are commercially available, or can also be prepared by one of ordinary skill in the art with reference to known literature. Specific examples of CK1 inhibitors include siRNA against the gene encoding CK1, anti-CK1 antibodies, and chemical antagonists.
  • the CK1 inhibitor is CKI-7.
  • the concentration of the CK1 inhibitor in the medium is appropriately determined according to the kind of inhibitor to be used. In the case of CKI-7, the concentration is generally 0.1-20 [mu]M, preferably 1-10 [mu]M, more preferably about 5 [mu]M.
  • casein kinase 1 Other inhibitors of casein kinase 1 include PF 670462 (Andy Bio, Minneapolis, USA), D4476 (Andy Bio, Minneapolis, USA), (R)-CR8 (Minneapolis, USA) Andy Biological Company), (R)-DRF053 Dihydrochloride (Minneapolis Andy Biological Company, United States), TAK 715 (Minneapolis Andy Biological Company, United States), PF 4800567 Hydrochloride (United States Andy Biological Company) Andy Bio, Minneapolis), LH 846, CKI 7 dihydrochloride (Andy Bio, Minneapolis, USA), SR 3029 (Andy Bio, Minneapolis, USA), Epiblastin A (Andy Bio, Minneapolis, USA), PF 5006739 (Andy Bio, Minneapolis, USA).
  • the second type of cells are ectodermal cells.
  • the ectodermal cells are neural crest cells (NCs) or NCC-like cells.
  • the second type of cells are NC or NCC-like cells.
  • NC or NCC-like cells can be differentiated from human embryonic stem cells (hES cells), eg, using dual SMAD inhibitors as described herein or as described in WO/2010/096496.
  • NC or NCC-like cells can be treated with Wnt agonists (e.g. Wnt3a and/or (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO)) with SMAD inhibitors (e.g.
  • SB431542 and/or or Noggin were differentiated from hES cells; see Menendez et al., Proceedings of the National Academy of Sciences (PNAS) Nov. 29, 2011, Vol. 108, No. 48, 19240-19245.
  • hES cells were treated with SB431542 and (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO) (in the presence or absence of noggin) or Efficient induction of NC or NCC-like cells following Wnt3a and SB431542 contact.
  • BIO (2'Z,3'E)-6-bromoindirubin-3'-oxime
  • NCs can also be obtained from neural rosette cultures, for example, by culturing hES cells on MS5 stromal feeder cells (see Lee et al., Stem Cells 25(8), 1931-1939 (2007), which have is incorporated herein by reference in its entirety). NCs can also be obtained from a number of tissues, including in developing embryos, neural tube, sciatic nerve, gut, and dorsal root ganglia; and in larvae and adults, dorsal root ganglia, bone marrow, skin, heart, cornea, teeth, and neck in the arterial body. See Nagoshi et al., Journal of Cellular Biochemistry 107:1046-1052 (2009); Crane and Trainor, Annu. Rev. Cell Dev. Biol.
  • NCC-like cells are obtained by reprogramming cells of a first type according to the present application.
  • culturing the second type of cells in the presence of the second set of reprogramming factors results in 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 cells become the third type of cells .
  • the third type of cells are ectodermal cells. In certain embodiments, the third type of cells are neural somatic cells. In certain embodiments, the third type of cells are corneal endothelial cell-like cells (CEC-like cells).
  • CECs Corneal endothelial cells
  • CECs generally refer to mitochondria-rich cells that line the posterior surface of the cornea and face the anterior chamber of the eye in living organisms.
  • ciCECs chemically induced CECs
  • CEC-like cells corneal endothelial cell-like cells
  • CEC-like cells obtained by the reprogramming methods disclosed herein can be identified or recognized by their exhibiting one or more of the following endogenous CEC properties: cells of uniform size that express CEC markers, form predominantly hexagons The ability of a monolayer, the ability to form a "leak pump” (allowing solutes and nutrients to leak from the aqueous humor to the more superficial layers of the cornea while actively pumping water in the opposite direction from the stroma to the aqueous humor).
  • Exemplary CEC markers include, but are not limited to: Na + /K + ATPase, Claudin 1 (TJP1/ZO-1), KLF13, AQP1, Collagen VIII, SLC 16A3, CFTR, NBC1, CA2, AE2/SCL4A2, SCL16A1, CA12, CA4, FoxCl.
  • CECs typically express collagen VIII, the Na + K + ATPase pump, and ZO-1, and do not express vWF and CD31 (the latter present in vascular endothelial cells).
  • CECs may express one or more corneal endothelial pump markers (which include AQP1, CA2, CA4, CA12, SCL14A2, SLC 16A1, SLC 16A3, SLC 16A7, CFTR, NHE1, ADC Y10, the voltage-dependent anion channel VDAC2 and VDAC3, chloride channel proteins CLCN2 and CLC), periocular neural crest markers (which include PITX2 and FOXCl) and/or cell adhesion and matrix proteins (which include Occludin, Connexin43, 9.3E Antigen, Collagen III, Collagen IV, N-Cadherin, VE-Cadherin, E-Cadherin, ⁇ -catenin, Laminin ⁇ 4, Nidogen-2, and Axon-Guiding Factor (Netrin) 4 ).
  • CECs 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
  • the resulting CEC-like cells exhibit one or more biomarkers consistent with the primary CEC phenotype.
  • the resulting CEC-like cells express Claudin 1 (TJP1/ZO-1), N-cadherin, and Na+/K+ ATPases.
  • 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.
  • Labels can be detected or measured by methods known in the art. For example, adherent junctions by N-cadherin can be confirmed by examining expression at the protein level (eg, using an antigen-antibody reaction) or gene level (eg, using RT-PCR).
  • the Na + /K + -ATPase pumping function of cells can be described, for example, according to "Investigative Ophthalmology & Visual Science", 2010 Vol. 51, No. 8, 3935-3942, and “Current Eye Research ( Current Eye Research), 2009 Vol. 34, 347-354 and measured using a Ussing chamber.
  • the second set of reprogramming factors further comprises BMP4 and/or DNA methyltransferase inhibitors.
  • Bone morphogenetic proteins 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 through the BMPR results in the mobilization of members of the SMAD family of proteins. Signaling pathways involving BMP, BMPR, and Smad are important in heart, central nervous system, and cartilage development, as well as postnatal bone development. It plays an important role in embryonic patterning and early bone formation during embryonic development. Thus, interfering with BMP signaling affects the body plan of the developing embryo.
  • BMPRs bone morphogenetic protein receptors
  • BMP inhibitors include but are not limited to DMH2 (Bedford Millipore, USA), Dorsomorphin (Cambridge, USA), LDN193189 (Tocris Life Sciences, Bristol, UK), DMH- 1 (Tocris Life Sciences, Bristol, UK), K 02288 (Tocris Life Sciences, Bristol, UK) and ML 347 (Tocris Life Sciences, Bristol, UK).
  • the concentration of the BMP inhibitor in the medium is appropriately determined according to the kind of inhibitor to be used, such as 0.1 nM-10 ⁇ M, 0.1 nM-5 ⁇ M, 0.1 nM-2.5 ⁇ M, 0.1 nM-2 ⁇ M, 0.5 nM-2 ⁇ M, 1 nM-2 ⁇ M , 1nM-1.5 ⁇ M, 1nM-1000nM, 5nM-1000nM, 10nM-1000nM, 50nM-1000nM, 50nM-500nM, 50nM-200nM, 100nM-200nM, 100nM-150nM or 100nM.
  • BMP4 bone morphogenetic protein 4
  • BMP4 is a member of the bone morphogenetic protein family that is part of the transforming growth factor-beta superfamily. BMP4 is found in the ventral margin region and in early embryonic development in the eye, heart, and auditory vesicles.
  • the origin of the BMP4 inhibitor to be used in the present invention is not particularly limited as long as it is effective for reprogramming.
  • BMP4 inhibitors are commercially available or can also be prepared by one of ordinary skill in the art with reference to known literature. Specific examples of BMP4 inhibitors include siRNA against the gene encoding BMP4, Chordin, Noggin, and anti-BMP4 antibodies.
  • the concentration of the BMP4 inhibitor in the medium is appropriately determined according to the kind of inhibitor to be used, such as 0.01-100 ⁇ g/ml, 0.01-50 ⁇ g/ml, 0.01-25 ⁇ g/ml, 0.01-10 ⁇ g/ml, 0.01-5 ⁇ g/ml , 0.01-1 ⁇ g/ml, 0.01-0.5 ⁇ g/ml, 0.01-0.1 ⁇ g/ml, 0.01-0.05 ⁇ g/ml, 0.05-10 ⁇ g/ml, 0.1-10 ⁇ g/ml, 0.1-5 ⁇ g/ml, 0.1-4 ⁇ g/ ml, 0.1-3 ⁇ g/ml or 0.1-2 ⁇ g/ml or 10 ng/ml.
  • the DNMT (DNA methyltransferase) family of enzymes catalyzes the transfer of methyl groups to DNA. DNA methylation provides a wide variety of biological functions.
  • the origin of the DNMT inhibitor to be used in the present invention is not particularly limited as long as it is effective for reprogramming.
  • DNMT inhibitors are commercially available or can also be prepared by one of ordinary skill in the art with reference to known literature. Specific examples of DNMT inhibitors include siRNA against the gene encoding DNMT, anti-DNMT antibodies, and chemical antagonists.
  • the DNMT inhibitor is selected from the group consisting of: decitabine (Bristol, UK), 5-azacytidine (Bristol, UK) Chris Life Sciences), 5-aza-dC (ToChris Life Sciences, Bristol, UK) and RG108 (ToChris Life Sciences, Bristol, UK).
  • concentration of the DNMT inhibitor in the medium is appropriately determined according to the kind of inhibitor to be used, such as 0.1-100 ⁇ M, 0.1-50 ⁇ M, 0.1-25 ⁇ M, 0.1-10 ⁇ M, 0.5-10 ⁇ M, 1-10 ⁇ M, 5 ⁇ M.
  • DOT1L telomere silencing disruptor 1-like protein
  • HMTs histone methyltransferases
  • the DOT1L inhibitor to be used in the present invention is not particularly limited as long as it is effective for reprogramming.
  • DOT1L inhibitors are commercially available or can also be prepared by one of ordinary skill in the art with reference to known literature.
  • Specific examples of DOT1L inhibitors include siRNA against the gene encoding DOT1L, anti-DOT1L antibodies, and chemical antagonists.
  • the DOT1L inhibitor is a small molecule DOT1L inhibitor.
  • DOT1L is known to catalyze H3K27 methylation through the AdoMet binding site on DOT1L using adenosylmethionine (AdoMet) as a cofactor. Therefore, any chemical that mimics the molecular structure of AdoMet that can displace AdoMet from its binding site on DOT1L is within the contemplation of the present disclosure as a DOT1L inhibitor.
  • the DOT1L inhibitor is selected from the group consisting of EPZ004777, EPZ5676 (also known as pinometostat), SGC 0946, and SYC-522.
  • the second type of cells is 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.
  • the cells of the second type 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.
  • 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 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, 7 days to 12 days.
  • the present disclosure provides a method of reprogramming cells of a first type into cells of a third type, comprising step (a) culturing the cells of the first type in the presence of a first set of reprogramming factors, wherein the first A set of reprogramming factors comprises glycogen synthase kinase 3 (GSK3) inhibitor, transforming growth factor (TGF ⁇ ) inhibitor and cyclic AMP inducer, and step (b) culturing in the presence of the second set of reprogramming factors from step (a) Cells obtained wherein the second set of reprogramming factors comprises a TGF[beta] inhibitor and a casein kinase 1 inhibitor.
  • GSK3 glycogen synthase kinase 3
  • TGF ⁇ transforming growth factor
  • the present disclosure provides a method of reprogramming cells of a first type into cells of a third type, comprising culturing cells of the first type in the presence of a first set of reprogramming factors and a second set of reprogramming factors cells, wherein the first group of reprogramming factors comprises glycogen synthase kinase 3 (GSK3) inhibitors, transforming growth factor (TGF ⁇ ) inhibitors, and cyclic AMP inducers, and the second group of reprogramming factors comprises TGF ⁇ inhibitors and phenol protein kinase 1 inhibitor.
  • GSK3 glycogen synthase kinase 3
  • TGF ⁇ transforming growth factor
  • the second group of reprogramming factors comprises TGF ⁇ inhibitors and phenol protein kinase 1 inhibitor.
  • the first set of reprogramming factors further comprises bFGF.
  • the second set of reprogramming factors further comprises BMP4 and/or DNA methyltransferase inhibitors.
  • culturing the first type of cells in the presence of the first and second sets of reprogramming factors results in 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 cells become third type of cells.
  • the method further comprises washing the cells obtained from step (a) before starting step (b). In certain embodiments, there is no washing step between steps (a) and (b).
  • the present disclosure provides populations of NCC-like cells produced according to the methods provided herein. In certain embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the population are NCC-like cells.
  • the present disclosure provides a method for generating patient-specific neural crest cell-like cells.
  • the cells of the first type are obtained from a subject suffering from a neurological disorder.
  • the present disclosure provides populations of corneal endothelial cell-like cells (CEC-like cells) produced according to the methods provided herein. In certain embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in a population of the present disclosure are CEC-like cells.
  • NCC-like cells or CEC-like cells produced according to the methods provided herein are purified ex vivo.
  • the methods for reprogramming yield NCC-like cells or CEC-like cells of high purity and do not require purification.
  • the reprogramming methods provided herein result in NCC-like cells comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more or cellular composition of CEC-like cells.
  • the reprogramming methods provided herein yield NCC-like cells or CEC-like cells of low or less than desired purity, and require purification.
  • the 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.
  • other cells in the composition may include undifferentiated NCC-like cells and/or NCC-like cells that differentiate into an undesired cell lineage or phenotype.
  • the present disclosure provides a composition comprising NCC-like cells or CEC-like cells produced according to the methods provided herein.
  • the composition may include one or more pharmaceutically acceptable carriers and diluents.
  • compositions disclosed herein can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers , antioxidants, anesthetics, suspending/dispersing agents, sequestering/chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof .
  • compositions described herein may also include components to facilitate transplantation.
  • the compositions described herein can be pyrogen-free or substantially pyrogen-free and pathogen-free, wherein pathogens include bacterial contaminants, mycoplasma contaminants, and viruses.
  • compositions comprising the NCC-like cells or CEC-like cells described herein may further comprise an immunosuppressive or immune tolerizing agent.
  • the present disclosure relates to therapeutic uses of the CEC-like cells provided herein.
  • the CEC-like cells of the present invention can be used in cell therapy as grafts for the treatment of diseases requiring transplantation of the corneal endothelium, such as bullous keratopathy, corneal edema, leukoplakia, and the like.
  • the present disclosure provides a method of treating a disease or condition associated with dysfunctional or damaged corneal endothelial cells comprising administering to a subject in need thereof an effective amount of a CEC-like cell provided herein or comprising Composition of CEC-like cells.
  • the administration of corneal endothelial cells induces an ocular healing process.
  • corneal endothelial cells are administered to replenish diseased tissue.
  • administration of corneal endothelial cells has a regenerative effect on damaged or diseased ocular tissue.
  • 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, implantation at the ocular site, and the like.
  • the particular mode of administration selected will depend on the particular treatment, the disease state or condition of the patient, the nature or route of administration of other drugs or therapeutic agents administered to the subject, and the like.
  • corneal endothelial cells may be administered to a subject in a single dose or in multiple doses at selected time intervals, eg, in titrated doses. When multiple doses are administered, the doses may be spaced apart from each other, eg, by one week, one month, one year, or ten years.
  • One or more growth factors, hormones, interleukins, cytokines, small molecules, or other cells can also be administered before, during, or after administration of the cells to further bias the cells toward a particular cell type.
  • an effective amount broadly refers to an amount of a compound or cell that, when administered to a patient to treat a disease, is sufficient to effect such treatment of a disease.
  • An effective amount may be a prophylaxis effective amount and/or a prophylactically effective amount.
  • An effective amount can be an amount effective to reduce signs/symptoms, effective to prevent the occurrence of signs/symptoms, reduce the severity of the occurrence of signs/symptoms, eliminate the occurrence of signs/symptoms, slow the development of the occurrence of signs/symptoms, prevent the occurrence of signs/symptoms development and/or effect of prevention and treatment of signs/symptoms.
  • an “effective amount” may vary depending on the disease and its severity, as well as the age, weight, medical history, susceptibility and pre-existing conditions of the patient to be treated.
  • the term “effective amount” is synonymous with “therapeutically effective amount.”
  • treating/treatment encompasses the treatment of a disease or medical condition described herein in a subject, such as a human, animal or mammal, and includes: (i) inhibiting the disease or condition, i.e., arresting its progression; (ii) alleviating the disease or condition, i.e., causing regression of the condition; (iii) slowing the progression of the condition; and/or (iv) inhibiting, alleviating one or more symptoms of the disease or medical condition or slowing the disease or progression of one or more symptoms of a medical condition.
  • the term "subject” is not limited to a particular species or sample type.
  • the term “subject” can refer to a patient, and often refers to a human patient.
  • this term is not limited to humans and thus encompasses a variety of mammalian species, such as non-human veterinary mammals such as dogs, cats, rabbits, pigs, rodents, horses or monkeys.
  • the CEC-like cells provided herein are cell clumps, such as clumps obtained by concentration and filtration, and analogs thereof, and analogs thereof are used as agents of the present invention.
  • protective agents such as glycerol, DMSO (dimethyl sulfoxide), propylene glycol, acetamide, etc.
  • the agent may be subjected to treatment under conditions that cause pathological protein denaturation, such as heat treatment, radiation treatment, etc., while preserving the function of the corneal endothelial cells.
  • CEC-like cells can be administered in combination with surgery.
  • the procedure may be Descemet's stripping with endothelial keratoplasty (DSEK), which includes removal of Descemet's membrane and corneal endothelium, and subsequent transplantation of donor tissue.
  • DSEK Descemet's stripping with endothelial keratoplasty
  • PEP penetrating keratoplasty
  • Other procedures may include lamellar keratoplasty, Descemet's Membrane Endothelial Keratoplasty (DMEK), DSAEK, and DLEK.
  • Disease or condition associated with dysfunctional or damaged corneal endothelial cells includes any disease or condition amenable to treatment by administration of CEC-like cells, including a subject whose CECs are reduced in number or die, decrease in density, or otherwise become Dysfunctional disease.
  • Primary diseases affecting the corneal endothelium include Fucker's dystrophy, iridocorneal endothelial syndrome, posterior polymorphic dystrophy, and congenital hereditary endothelial dystrophy.
  • Effective treatments may include replacement of the corneal endothelium for secondary diseases or conditions including age-related macular degeneration (AMD), retinitis pigmentosa, glaucoma, corneal dystrophy, contact lens use, cataract surgery, and advanced endothelial failure in corneal transplantation.
  • AMD age-related macular degeneration
  • retinitis pigmentosa retinitis pigmentosa
  • glaucoma corneal dystrophy
  • contact lens use cataract surgery
  • advanced endothelial failure in corneal transplantation may include replacement of the corneal endothelium for secondary diseases or conditions including age-related macular degeneration (AMD), retinitis pigmentosa, glaucoma, corneal dystrophy, contact lens use, cataract surgery, and advanced endothelial failure in corneal transplantation.
  • Corneal endothelial cell disease additionally includes any damage to the cornea, such as damage caused by chemical irritation, damage caused by contact lens use, reaction, or sensitivity (eg, to contact lens care solutions, cosmetics, eye drops, pharmaceuticals, smoke, etc.), scratches, scratches, abrasions, contusions, foreign objects in the eyes (eg, sand or dust), or exposure to ultraviolet light (from eg sunlight, fluorescent lamps, snow reflections, water reflections, or arc welding or other exposures).
  • the disease or condition associated with dysfunctional or damaged corneal endothelial cells results in vision loss in the subject.
  • the subject's vision loss is permanent or irreversible.
  • the corneal endothelial cells are immunocompatible (eg, allogeneic or autologous) with the subject.
  • the method of treatment may further comprise administering to the subject an immunosuppressive or immunotolerant agent.
  • the immunosuppressive or immune tolerance agent can be administered in an amount sufficient to reduce the risk of rejection of the CEC-like cells.
  • the immunosuppressant or immune tolerance agent may comprise one or more of the following: anti-lymphocyte globulin (ALG) polyclonal antibody, anti-thymocyte globulin (ATG) polyclonal antibody, azathioprine, (anti-lL-2Ra receptor antibody), cyclosporin (cyclosporin A), (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, (anti-CD20 antibody), sirolimus, tacrolimus, mycophenolate mofetil, and corticosteroids.
  • ALG anti-lymphocyte globulin
  • ATG anti-thymocyte globulin
  • azathioprine anti-lL-2Ra receptor antibody
  • cyclosporin cyclosporin A
  • anti-IL-2Ra receptor antibody anti-IL-2Ra receptor antibody
  • the immunosuppressive agent can be administered at least about 1, 2, 4, 5, 6, 7, 8, 9 or 10 mg/kg.
  • an immunosuppressive agent When an immunosuppressive agent is used, it can be administered systemically or locally, and it can be administered prior to the administration of the CEC-like cells, concomitantly with the CEC-like cells, or after the administration of the CEC-like cells.
  • Immunosuppressive therapy can continue for weeks, months, years, or indefinitely following cell administration. For example, a patient may be administered 5 mg/kg of cyclosporine for 6 weeks following CEC-like cell administration.
  • the present disclosure provides use of the CEC-like cells or compositions provided herein in the manufacture of a medicament for the treatment of a disease or condition associated with dysfunctional or damaged corneal endothelial cells.
  • the present disclosure provides a method of treating a disease or condition associated with dysfunctional or damaged corneal endothelial cells comprising administering to a subject in need thereof an effective amount of the NCC-like cells provided herein or comprising Composition of NCC-like cells.
  • the present disclosure provides use of the NCC-like cells or compositions provided herein for the manufacture of a medicament for the treatment of a disease or condition associated with dysfunctional or damaged corneal endothelial cells.
  • the NCC-like cells provided herein can be used to treat neurological diseases.
  • Neurode disease as used herein is defined as a disorder of the nervous system, and includes those involving the central nervous system (cerebrum, brainstem, and cerebellum), peripheral nervous system (including cranial nerves), and autonomic nervous system (parts of which are located centrally and peripherally) disorders of the nervous system).
  • central nervous system Cerebrum, brainstem, and cerebellum
  • peripheral nervous system including cranial nerves
  • autonomic nervous system parts of which are located centrally and peripherally
  • neurological disease includes any disease in which the function of neural crest cells or Schwann cells is impaired, altered or destroyed.
  • Examples of neurological diseases associated with Schwann cells are demyelinating diseases, multiple sclerosis, myelopathy, experimental allergic encephalomyelitis (EAE), acute disseminated encephalomyelitis ; ADEM), post-infectious or post-vaccination encephalomyelitis, peripheral neuropathy, Schwannomatosis, Charcot-Marie-Tooth disease, Guillain-Barré syndrome ( Guillain-Barre Syndrome), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).
  • demyelinating diseases multiple sclerosis, myelopathy, experimental allergic encephalomyelitis (EAE), acute disseminated encephalomyelitis ; ADEM), post-infectious or post-vaccination encephalomyelitis, peripheral neuropathy, Schwannomatosis, Charcot-Marie-Tooth disease, Guillain-Barré syndrome ( Guillain-Barre Syndrome), chronic inflammatory demyelinating polyradiculoneuropathy
  • the present disclosure also provides methods for drug discovery and/or drug screening. Assays for drug discovery and/or drug screening are also provided.
  • the methods include administering the drug candidate to NCC-like cells or CEC-like cells, and detecting the response of the cells to the drug candidate. Detection of responses can identify whether a drug candidate has suitable properties (eg, toxicity or therapeutic efficacy).
  • methods can be used to determine cell health and viability in the presence of a drug candidate.
  • methods can be used to test drug candidates for toxicity.
  • methods can be used to assess changes in the phenotype of a cell population in the presence of a drug candidate.
  • the present disclosure provides a method of using NCC-like cells to screen for drugs that reverse, inhibit or prevent neurological side effects of neurological diseases or agents (eg, diabetic agents).
  • neurological diseases or agents eg, diabetic agents
  • the present disclosure provides a kit for reprogramming cells of a first type into cells of a second type, wherein the kit comprises a first set of reprogramming factors, and the first set of reprogramming factors comprises a carbohydrate Pro-synthase kinase 3 (GSK3) inhibitors, transforming growth factor (TGF ⁇ ) inhibitors, and cyclic AMP inducers.
  • GSK3 carbohydrate Pro-synthase kinase 3
  • TGF ⁇ transforming growth factor
  • cyclic AMP inducers a carbohydrate Pro-synthase kinase 3
  • the present disclosure provides a kit for reprogramming cells of a second type into cells of a third type, wherein the kit comprises a second set of reprogramming factors, and the second set of reprogramming factors comprises TGF ⁇ inhibitors and casein kinase 1 inhibitors.
  • the present disclosure provides a kit for reprogramming cells of a first type into cells of a third type, wherein the kit comprises a first set of reprogramming factors and a second set of reprogramming factors, wherein the first set of reprogramming factors
  • One group of reprogramming factors includes glycogen synthase kinase 3 (GSK3) inhibitors, TGF ⁇ inhibitors and cyclic AMP inducers
  • a second group of reprogramming factors includes TGF ⁇ inhibitors and casein kinase 1 inhibitors.
  • the kit may further comprise instructions for use, and packaging that separates each of the components of the kit.
  • Oct4-GFP transgenic allele-carrying mice (CBA/CaJ ⁇ C57BL/6J) were from Jackson Laboratory (Jackson Laboratory); Wnt1-cre and Fsp1-Cre mice were from Jackson Laboratory (BALB/c-Tg(S100a4 -cre)lEgn/YunkJ);ROSA26-tdTomato mice were from Jackson Laboratory (Gt(ROSA)26Sortm14(CAG-tdTomato)Hze). 129Sv/Jae and C57BL/6 mice were from Shanghai 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 Figure 4A).
  • New Zealand white rabbits were 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°C ⁇ 2°C) with a 12 hour dark/light cycle.
  • MEFs Primary mouse embryonic fibroblasts
  • mouse embryos Liu, C., Hu, X., Li, Y., Lu, W. ., Li, W., Cao, N., Zhu, S., Cheng, J., Ding, S., and Zhang, M. (2019).
  • "Chemical conversion of mouse fibroblasts into oligodendrocytes Conversion of mouse fibroblasts into oligodendrocyte progenitor-like cells through a chemical approach.
  • Fibroblasts were grown in cells supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM GlutaMAX (Gibco), 0.1 mM non-essential amino acids (Sigma), 100 units/mL penicillin, and 100 mg/mL streptomycin (Gibco). Fibroblasts cultured in DMEM (Gibico) in fibroblast medium. All fibroblasts were expanded for two passages and subsequently used for further experiments. To prepare Wnt1 - MEFs, fibroblasts derived from Wnt1-Cre/ROSA26 tdTomato mouse embryos were sorted by FACS against tdTomato- cells.
  • Mouse primary corneal endothelial cells (CP-M179) and culture medium (CM-M179) were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China).
  • Mouse primary CECs were cultured in DMEM containing 10% FBS (Gebeco), 0.1 mM non-essential amino acids (Sigma), 2 mM GlutaMAX (Gebeco), and 1% penicillin-streptomycin (Gebeco).
  • mESCs were maintained in ESC medium consisting of 10% FBS (Gebeco), LIF, 0.1 mM non-essential amino acids (Sigma), 2 mM GlutaMAX (Gebeco), 1% penicillin-streptomycin (Gebeco) Co), 0.1 mM 2-mercaptoethanol (Gibco), CHIR99021 (3 mM) and PD0325901 (1 mM) in DMEM.
  • HEF Human embryonic fibroblasts
  • HNF human neonatal fibroblasts
  • HNF adult fibroblasts
  • MSCs Human umbilical cord mesenchymal stem cells
  • FBS FBS
  • GlutaMAX Gebee
  • NEAA NEAA
  • UC Human exfoliated renal epithelial cells
  • tdMEFs To prepare tdMEFs, the resulting fibroblasts were sorted by FACS against tdTomato + /p75 ⁇ cells as previously described.
  • Primary MEFs were isolated from E13.5 mouse embryos with a genetic background of Fsp1-Cre/Rosa26 tdTomato (Fsp1-Cre mice x Rosa26 tdTomato mice). MEFs at passage 2 were dissociated with 0.25% trypsin for 5 minutes at 37°C and neutralized with MEF medium. Those MEFs were stained with antibodies specific for p75 and subjected to FACS sorting against tdTomato + /p75 ⁇ cells.
  • tdMEFs Matrigel-coated 24-well culture plates were pre-warmed at 37°C for at least 30 minutes before seeding with tdMEFs. Immediately after FACS sorting, tdMEFs were seeded at 15,000 cells/well into pre-warmed Matrigel-coated 24-well culture plates supplemented with 1 ⁇ M thiazole at 37°C in 5% CO and 20% O. (Thiazovivin) (Tzv) in MEF medium for 5 hours to allow MEFs to attach to the plate. After 5 h, the medium was changed to Tzv-free MEF medium, and tdMEFs were cultured overnight at 37 °C in 5% CO .
  • Thiazovivin Thiazovivin
  • Small molecules were obtained from Sigma, including GSK3b inhibitor CHIR99021 (SML1046), TGFb inhibitor SB431542 (S4317), DNA methylation inhibitor 5-aza-dC (A3656), cyclic AMP inducer forskolin (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.
  • NC canonical neural crest
  • a 30-gauge needle (Terumo, Tokyo, Japan) was used to create the center of the cornea for De Smith.
  • a 6.0 mm diameter circular orifice for descemetorhexis, and Descemet's membrane is removed from the anterior chamber of the eye.
  • Corneal endothelium was mechanically scraped from De Smith's membrane using a lacrimal irrigator (Shandong Weigao) as previously described.
  • Fsp-ciCECs were dissociated using 0.25% trypsin-EDTA, resuspended in minimal medium at a density of 1 x 107 cells/ml and kept on ice.
  • the anterior chamber was washed three times with PBS. Following this procedure, 1 ⁇ 10 6 cultured ciCECs suspended in 100 ⁇ l basic DMEM containing 100 ⁇ M ROCK inhibitor Y-27632 (Selek) were injected into the anterior chamber of the right eye using a 26 gauge needle. Thereafter, rabbits in the cryo (cryoinjury alone), CE eye down (cryoinjury and CE injection), and spheroid eye down (cryoinjury and spheroid injection) groups were kept in an eye down position for 24 hours, allowing cells to pass through. The rabbits in the spheroid eye-up group were kept in the eye-up position under deep anesthesia for 24 hours.
  • Proliferation rates of ciCECs cultured in differentiation medium alone or in M5 alone were determined by Click-iT TM Ethynyldeoxyuridine (EdU) Alexa Fluor 488 Imaging Kit (Invitrogen) according to the manufacturer's instructions. Briefly, passaged CECs were seeded onto slides at a lower density of 5 x 103 cells/cm 2 and cultured for 24 hours.
  • RNA from each sample was isolated with TRIzol reagent and purified using the RNeasy 23 mini kit (QIAGEN) according to the manufacturer's instructions. RNA quality and quantity were assessed using NanoDrop 2000, Agilent 2100 Bioanalyzer, and Agilent RNA 6000 Nano Kit. RNA library construction and RNA sequencing were performed by Annoroad Gene Technology. Sequencing libraries were generated using the NEB Next Ultra RNA Library Prep Kit (NEB) for Illumina24, following the manufacturer's recommendations, and library clustering was performed using the HiSeq PE Cluster Kit v4-cBot-HS (Enlightening). After cluster generation, the library was sequenced on the TusHoldings platform and 150 bp paired-end reads were generated. Initial data analysis was performed on BMKCloud (http://www.biocloud.net/).
  • fibroblasts with the desired genotype were cultured in MEF medium until they reached more than 80% confluency. Cells were washed twice with IX PBS and treated with 0.25% trypsin for 5 minutes at 37°C. After harvest, cells were passed through a 70- ⁇ m filter, washed twice with pre-cooled buffer (1 ⁇ PBS, 1.5% FBS, 0.5% BSA), and resuspended in the buffer. Cells were incubated with FITC-conjugated P75 antibody (Albocine) or isotype control (BD) at recommended concentrations for 30 minutes on ice, or 45 minutes at room temperature, followed by six washes with FACS buffer. Cells were then resuspended in FACS buffer and sorted with BD FACSAria II.
  • FITC-conjugated P75 antibody Albocine
  • BD isotype control
  • DMEM/F12/Glutamax Containing DMEM/F12/Glutamax (Gebeco), 10% KSR (Gebeco), 10% FBS (Gebeco), 1% NEAA (Gebeco), 0.1 mM 2-mercaptoethanol (Gebeco) basal medium supplemented with small molecules Repsox (10 ⁇ M), Chir99021 (10 ⁇ M), forskolin (10 ⁇ M), and bFGF (10 ng/ml).
  • the basal medium may also contain DMEM/F12/Glutamax (Gebeco), 0.075% bovine serum albumin (BSA) (Gebeco), 1% NEAA (Gebeco) before adding the first set of reprogramming factor molecules ) and 0.1 mM 2-mercaptoethanol (Gebeco), and produced comparable or better effects on cell reprogramming (data not shown).
  • DMEM/F12/Glutamax (Gebeco), 10% KSR (Gebeco), 1% NEAA (Gebeco), 0.1 mM 2-mercaptoethanol (Gebeco), supplemented with 5 ⁇ M SB431542 and 5 ⁇ M CKI-7 .
  • FACS fluorescence-activated cell sorting
  • TGF-beta signaling inhibitors such as Repsox(R), which inhibit mesoderm and endoderm specification
  • GSK3 inhibitors such as CHIR99021 (C)
  • cyclic AMP inducers such as forskolin (F).
  • bFGF basic fibroblast growth factor
  • RCF chemically defined medium
  • MEFs were seeded in 6-well culture plates at 50,000 cells/well. After overnight incubation, MEFs were treated in RCF chemically defined reprogramming medium. In RCF medium, many small dense cell clusters with sharp edges appeared rapidly within twelve days (Fig. 1B).
  • ciNCCs chemically induced neural crest cells
  • Wnt1 - MEFs were used to observe tdTomato expression. The results show that RCF compounds can reprogram MEFs into tdTomato-positive neural crest cell-like cells (Fig. ID).
  • RCF-treated MEFs were fixed, immunostained, and analyzed. These cells expressed HNK1, P75, and AP2 ⁇ (FIG. IF). Those fibroblast-derived, highly proliferative and self-regenerating Sox10-positive cells are hereafter referred to as ciNCCs.
  • FIG. 3A shows the labelling of stage II-induced ciCECs (Na + -K + ATPase, AQP1, vimentin, N-cadherin laminin, and AQP1 ) detected by immunofluorescence staining at day 14. Tight junctions of corneal endothelial cells were also observed under transmission electron microscopy.
  • FIG. 4B To avoid possible contamination of neural crest cells (ciNCCs) in starting MEFs, we performed lineage tracing experiments to trace the origin of ciNCCs and ciCECs (Fig. 4B). In Fsp1-Cre-RosaTomato mice, tdTomato was faithfully expressed in ciNCC and ciCEC under the control of the Fsp1 gene. Expression of ciNCC markers P75, Hnk1, AP2 ⁇ , and SOX10 was observed by immunocytochemistry in Fsp1-ciNCC (see Figure 4C). Figure 4D shows representative morphological changes of Fsp1-Cre:R26RtdTomato MEFs and ciCECs induced from these MEFs.
  • Human cells human embryonic skin fibroblasts (HEF), human neonatal fibroblasts (HNF), adult fibroblasts (HAF), human umbilical cord mesenchymal stromal cells
  • HEF human embryonic skin fibroblasts
  • HNF human neonatal fibroblasts
  • HNF adult fibroblasts
  • UC human umbilical cord mesenchymal stromal cells
  • the chemical factors added in Phase I are Repsox (10 ⁇ M), Chir99021 (10 ⁇ M), Forskolin (10 ⁇ M), 5-azacytidine (5 ⁇ M), VPA (500 ⁇ M) and BMP4 (10 ng/ml)
  • the chemical factors added in Phase II were 5 ⁇ M SB431542 and 5 ⁇ M CKI-7, which is consistent with what was mentioned above. Induced ciNCCs and ciCECs derived from the above cells are shown in FIG. 5 .
  • Example 3 Characterization of ciNCC and ciCEC obtained in Example 2
  • 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 is then partially degraded by heating, and the second strand cDNA is synthesized using DNA polymerase. Double-stranded DNA was then amplified using single-primer isothermal amplification (SPIA).
  • SPIA single-primer isothermal amplification
  • SPIA is a linear cDNA amplification process in which RNase H degrades RNA in a DNA/RNA heteroduplex at the 5' end of the double-stranded DNA, after which the SPIA primer binds to the cDNA and the polymerase acts on the primer's 5' end. Replication is initiated at the 3' end by replacing the existing forward strand. The second strand cDNA is then linearly amplified using random hexamers. Finally, a library from SPIA amplified cDNA was made using the Ultralow V2 library kit (NuGEN). RNA sequencing libraries were analyzed by a bioanalyzer and quantified by QPCR (KAPA).
  • RNA-sequencing libraries were pooled in each lane of paired-end 100 bp sequencing on a HiSeq 2500 instrument (Enlightening Inc.). Reads were adjusted for known linkers and low-quality regions using Fastq-mcf. Sample QC was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). 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.) Align the reads with the mouse reference assembly mm9. By Subread featureCounts (Liao et al. 2014.
  • FeatureCounts an efficient general-purpose program for assigning sequence reads to genomic features). Bioinformatics Bioinformatics" 30, 923-930.), using the Ensembl gene annotation of mm9, combined with gene level expression. Before dealing with 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 that did not have at least two samples with CPM (counts per million) values between .5 and 5000 were filtered out. Differential expression P value using edgeR (Robinson, M.D. et al., 2010.
  • edgeR a Bioconductor package for differential expression analysis of digital gene expression data
  • Bioinformatics 26, 139-140 Computation. Use the built-in R function "p.adjust" to use the Benjamini-Hochberg method (Benjamini and Hochberg, (1995). Controlling the False Discovery Rate: A Practical and Powerful Multiple Testing Method (Controlling the false discovery rate: a practical and powerful approach to multiple testing)”. "Journal of the Royal Statistical Society Series B” 57, 289-300) to calculate FDR. Gene ontology analysis was done by DAVID Bioinformatics Resources 6.7 or ToppGene. Generate heatmaps with Cluster 3.0 and view them with Java Treeview. Transcriptome analysis revealed that these cells were very similar to mouse NCs, but not MEFs.
  • ciNCCs For maintenance of ciNCCs after sorting and purification by FACS, cells were grown on poly-D-lysine/laminin-coated plates in neural crest medium supplemented with 1 ⁇ N2, 1 ⁇ B27, 10 ⁇ g/ml bFGF, 10 ⁇ g/ml EGF, and 10 ng/ml BMP4 in neural matrix medium (Gibico).
  • ciNCCs To characterize the differentiation potential of expanded ciNCCs, they were first cultured under differentiation conditions. Peripheral neurons are generated by inducing neuronal differentiation by withdrawal from FGF2/EGF and exposure to BDNF, GDNF, NGF, and dibutyryl cyclic AMP (dbcAMP). By immunostaining, ciNCCs produced Tuj1+ and perikinin+ neurons at passage 5, and this differentiation potential was well maintained during long-term culture (Fig. 2A). Schwann cell differentiation as assessed by S100b and GFAP+ expression was induced in the presence of CNTF, neuregulin lb and dbcAMP.
  • melanocyte differentiation medium EBM2 basal medium, 5% (v/v) FBS, 100 ng/ml SCF (Life Technologies), 200 nM endothelin 3 ( EDN3, Sigma), 50ng/ml WNT1, 10ng/ml FGF2, 5 ⁇ g/ml insulin, 1pM cholera toxin, 10nM 12-O-tetra-decanoylphorbol-13-acetate (TPA, Sigma) and 10 ⁇ M SB431542 (Sigma)). After 3-4 weeks of treatment, melanocytes were observed (Figure 2A).
  • melanocyte differentiation medium EBM2 basal medium, 5% (v/v) FBS, 100 ng/ml SCF (Life Technologies), 200 nM endothelin 3 ( EDN3, Sigma), 50ng/ml WNT1, 10ng/ml FGF2, 5 ⁇ g/ml insulin, 1pM cholera toxin, 10nM 12-O-tetra-decanoylphorbol-13-acetate (TPA, Sigma
  • ciNCC-derived neurons 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 10-20 days of co-culture with rat cortical neurons under differentiation conditions. Cultured neurons were transferred to a perfusion stage on an Olympus BX51WI upright microscope and perfused with artificial cerebrospinal fluid (aCSF) containing the following substances (in mM) at 2.5 mL/min at room temperature : NaCl 119, KCl 2.5, NaH2PO4 1, NaHCO3 26.2 and glucose 11, CaCl2 2.5 and MgSO4 1.3, and the osmotic pressure was adjusted to 300osm L-1.
  • aCSF cerebrospinal fluid
  • Electrode solutions used for current clamp experiments contained the following (in mM): Potassium Gluconate 123, KCl 10, MgCl 2 1, HEPES 10, EGTA 1, CaCl 2 0.1, K 2 ATP 1, Na 4 GTP 0.2 and Glucose 4, pH adjusted to 7.2 with KOH.
  • the membrane potential was held at approximately -70 mV and step currents of -20 to 50 pA were injected at 10-pA intervals.
  • Whole-cell currents were recorded at a holding potential of -70 mV, with voltage steps ranging from -70 mV to +30 mV delivered in 20-mV increments.
  • Spontaneous postsynaptic currents were recorded in whole-cell voltage-clamp mode.
  • Whole cell electrode solutions for synaptic current recordings contained the following (in mM): CsCl 135, HEPES 10, EGTA 1, Mg-ATP 4 , Na4GTP 0.4 and QX-314 10, pH 7.4.
  • ciCECs were subcutaneously injected into NOD-SCID mice, and teratomas formed at 4 to 8 weeks.
  • ciCECs were injected into ICR blastocysts and transplanted into pseudopregnant ICR females. Germline transmission of the resulting chimeric mice was determined by mating F2 mice with ICR mice. All animal experiments were approved and performed according to the guidelines of the Animal Care and Use Committee of the Guangzhou Institutes of Biomedicine and Health. There was no tumor formation within 6 months after transplantation with ciCECs, whereas large teratomas appeared after 4-8 weeks in recipients transplanted with mESCs (data not shown).
  • ciCECs have minimal, if any, tumorigenic potential.
  • ciCEC maintained a normal karyotype during 30 serial passages in vitro (data not shown).
  • ciCECs were transplanted into the eyes of NOD/SCID mice. After 4-8 weeks, after transplantation with ciCEC, the transplanted ciCEC did not form tumors within 6 months (data not shown).
  • a 12 o'clock centered 6 mm scleral corneal incision was performed with a slit knife (Alcon Surgical, Shanghai, China), and viscoelastic (Healon; Amersham Pharmacia Biotech AB) was infused into the anterior in the room.
  • a marker Devon Industries, Madrid, Spain
  • a 30-gauge needle (Termo, Tokyo, Japan) was used to create a 6.0 mm diameter circle in the center of the cornea for descemetorhexis The orifice is removed, and De Smith's membrane is removed from the anterior chamber of the eye.
  • the corneal endothelium was mechanically scraped from De Smith's membrane with a lacrimal irrigator (Shandong Weigao).
  • Fsp-ciCECs were dissociated using 0.25% trypsin-EDTA and resuspended at a density of 1 x 107 cells/ml in PBS and kept on ice.
  • the anterior chamber was washed three times with PBS.
  • a 26-gauge needle was used to resuspend 1 x 10 6 in 100 ⁇ l of basic DMEM containing 100 ⁇ l of ROCK inhibitor Y-27632 (which promotes cell adhesion to the implantation site) (ROCK inhibitor, Celec) ciCECs were injected into the anterior chamber of the right eye (FIG. 6B, first image).
  • Example 4 Generation of ciNCC and ciCEC from fibroblasts according to another example
  • the basal medium contained knockout DMEM (Gebeco), 10% KSR (Gebeco), 10% FBS (Gebeco), 1% NEAA (Gebeco), and 0.1 mM 2-mercaptoethanol (Gebeco) ), supplemented with small molecules Chir99021 (3 ⁇ M), SB431542 (5 ⁇ M), forskolin (10 ⁇ M), VPA (500 mM), EPZ004777 (5 ⁇ M) and 5-aza-dC (0.5 ⁇ M). The medium was shaken for 30 minutes to ensure complete dissolution of all components.
  • DMEM/F12/GlutaMAX (Gebeco), 10% KSR (Gebeco), 1% NEAA (Gebeco), and 0.1 mM 2-mercaptoethanol (Gebeco) supplemented with SB431542 (5 ⁇ M) and CKI-7 (5 ⁇ M).
  • MEFs were seeded at 5 x 104 cells/well in fibroblast medium on 6-well tissue culture plates. Plates were pre-coated with fibronectin or laminin for more than two hours. After overnight incubation, 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 7-10 days of induction, FACS sorting was performed to collect Wnt1 + cells.
  • Approximately 5 x 103 ciNCCs were seeded on laminine-coated glass coverslips in 24-well culture plates containing NCSC medium for the first 24 hours. After 24 hours, cells were subjected to differentiation conditions. For neuronal differentiation, switch the medium to neuronal differentiation medium (NCC medium without bFGF and EGF supplemented with 200 ⁇ M ascorbic acid, 2 ⁇ M db-cAMP, 25 ng/ml BDNF, 25 ng/ml NT3, and 50 ng/ml GDNF ). Change half of the medium every 2-3 days. Specific neuronal markers were analyzed by day 10 to day 20 after differentiation.
  • cells were cultured for 1 day in the presence of 5 ⁇ M retinoic acid and 200 ng/ml Shh and in the presence of 20 ng/ml PDGF-AA, 20 ng/ml bFGF and 200 ng/ml SHH for 3-5 days; then, they were cultured in differentiation medium containing 40ng/ml T3, 200ng/ml Shh, 1nM LDN193189, 5mM db-cAMP and 10ng/ml NT3 for 8-12 days. The medium was refreshed every other day. For astrocyte differentiation, 50ng/ml BMP4 was added to the differentiation medium for 8-12 days, and the medium was changed every other day.
  • the corneal endothelium originates from NCC
  • the first step was to direct the chemical reprogramming of mouse embryonic fibroblasts (MEFs) into ciNCCs.
  • MEFs mouse embryonic fibroblasts
  • Wnt1-Cre transgenic mice have been demonstrated as a lineage tracing reporter model for NC development.
  • Wnt1-Cre/ROSA26tdTomato mice In Wnt1-Cre/ROSA26tdTomato mice, tdTomato protein was faithfully expressed in NCC. Therefore, MEFs were isolated from Wnt1-Cre/ROSA26tdTomato mice at E13.5. Because the NCC populations were tagged with tdTomato, we performed fluorescence-activated cell sorting (FACS) to collect tdTomato - populations to exclude any NCC or progenitor cells (purified cells are referred to below as Wnt1-tdTomato - MEFs; Figure 15B). We confirmed that Wnt1-tdTomato - MEFs were also negative for other NCC markers including Sox10, P75, Pax3, Hnk1 and AP2 ⁇ (Fig. 15C,D). Additionally, these Wnt1-tdTomato - MEFs were negative for typical NSC markers including Sox2, Pax6 and Nestin (data not shown).
  • FACS flu
  • Example 5 Characterization of ciNCC and ciCEC obtained in Example 4 and additional studies
  • the reprogramming process of ciNCC has two phases: an initial phase (days 0-7) and an expansion phase (days 7-12).
  • the first stage is to grow MEFs in reprogramming medium to initiate epigenetic activation. A few small NCC-like clusters with sharp edges appeared.
  • the second stage is to grow epigenetically activated cells in a defined small-molecule medium. Most of the clusters expanded in the second stage and grew gradually.
  • ciNCCs To obtain ciNCCs, we performed FACS to collect Wnt1-tdTomato + cells.
  • the established ciNCCs were serially propagated in conventional NCC expansion medium containing N2, B27, bFGF and EGF. Morphologically, M6-induced cells at P3 maintained typical NCC characteristics in monolayer culture ( Figure 9A). After passaging, ciNCC became morphologically uniform.
  • M6-induced Wnt1-tdTomato + cells To further characterize M6-induced Wnt1-tdTomato + cells, we sought to examine their gene expression. Our results show that ciNCCs express multiple NCC markers, including P75, HNK1, AP2 ⁇ , and nestin (Fig. 9B).
  • ciNCCs have differentiation potential towards peripheral neurons, Schwann cells and others, among others.
  • these cells were cultured in different lineage differentiation media. After 2-4 weeks in culture, differentiated cells were examined by assessing marker expression by immunostaining.
  • ciNCC also generated cells expressing markers specific for neurons, including Tuj1 and peripherins (Fig. 9C).
  • melanocyte differentiation we observed melanocytes 2-3 weeks after induction (Fig. 9C).
  • Our immunostaining results show that ciNCCs can differentiate into Schwann cells. Induced Schwann cells were GFAP + and S100 ⁇ + cells (Fig. 9D).
  • ciCECs have the ability to regenerate corneal endothelium in vivo
  • ciCECs were injected into the anterior chamber of the eye in combination with a ROCK inhibitor (Y27636) ( Figure 14A). Each recipient received 1 ⁇ 10 6 ciCECs. Contralateral eyes (normal) and untreated eyes (PBS injected) were used as experimental controls.
  • Corneal edema was reduced much earlier after ciCEC transplantation compared to untreated eyes.
  • the corneas of the transplanted eyes became clear, while corneal opacity and stromal edema remained poor in the untreated eyes.
  • Slit-lamp examination results showed that the transparency of the cornea of the transplanted eye was also significantly improved after injection, and pupil and iris texture were clearly visible (Figure 14C, Figure 19A).
  • Fsp1-Cre fibroblast-specific protein 1
  • Figure 12A Fsp1-Cre mice were crossed with ROSA26tdTomato mice.
  • MEFs were isolated from transgenic mice at E13.5 (Fspl-Cre/ROSA26tdTomato) and fibroblasts specifically expressed tdTomato; these cells are hereinafter designated tdMEFs (FIG. 16A).
  • Fsp1-tdTomato + ciNCCs could differentiate into tdTomato + ciCECs (Fig. 16D).
  • differentiated CEC-like cells co-expressed Na + /K + -ATPase, AQP1, laminin, ZO-1, Na + /K + -ATPase, and tdTomato (Fig. 12E).
  • all these ciCECs also expressed tdTomato, which demonstrated transformation from fibroblasts.

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Abstract

提供了一种用于在一种或多种重编程因子存在下重编程细胞的方法、使用该方法获得的重编程细胞及其用途，以及包含重编程因子的试剂盒。

Description

用于重编程细胞的方法 发明领域
本公开大体上涉及用于重编程细胞的方法。
背景技术
正常作用的角膜内皮细胞(CEC)维持角膜的透明度和适当流体水平，例如在流体“泄漏”到基质中与从基质连续主动泵送以将流体移动到眼的前房中之间保持平衡。
已报道的是，角膜内皮细胞几乎没有或没有体内增殖能力，因此其在受损或以其它方式损失时无法被自然地替换。在人体内，角膜内皮细胞层在出生时最密集地聚集，并且在其后细胞密度随着眼睛生长快速降低(使得相同数目的细胞覆盖了更大的面积)。其后，角膜细胞密度随着年龄增长逐渐降低，明显反映了未被替换的细胞的逐渐损失。随着细胞密度降低，每个细胞展开并且覆盖更大的面积以维持细胞层的屏障和泵功能。然而，一旦细胞密度降到过低(低于约500到1000个细胞/平方毫米)，则其功能受到损害，引起角膜混浊、基质水肿、视敏度(visual acuity)损失以及最终失明。
虽然开发了不同的治疗方法，但非常需要新的角膜内皮重建技术。
发明内容
本公开提供了一种用于将第一类型的细胞重编程成第二类型的细胞的方法，其包含在第一组重编程因子存在下培养第一类型的细胞，其中第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子-β(TGFβ)抑制剂以及环状AMP诱导剂。
在某些实施例中，第一组重编程因子进一步包含碱性成纤维细胞生长因子(bFGF)、DNA甲基转移酶抑制剂、组蛋白甲基转移酶抑制剂(例如，DOT1L抑制剂)、组蛋白脱乙酰基酶(HDAC)抑制剂、BMP4或其组合。
在某些实施例中，第一组重编程因子由以下组成：(a)GSK3抑制剂、TGFβ抑制剂以及环状AMP诱导剂，(b)GSK3抑制剂、TGFβ抑制剂、环状AMP诱导剂以及bFGF或(c)GSK3抑制剂、TGFβ抑制剂、环状AMP诱导剂、DNA甲基转移酶抑制剂、组蛋白甲基转移酶抑制剂(例如，DOT1L抑制剂)以及组蛋白脱乙酰基酶抑制剂。
在某些实施例中，GSK3抑制剂选自由以下组成的组：CHIR99021、LiCl、Li 2CO 3以及BIO((2’Z,3’E)-6-溴靛玉红(Bromoindirubin)-3’-肟)、TD114-2、坎帕罗酮(Kenpaullone)、TWS119、CBM1078、SB216763、3F8(TOCRIS)、AR-A014418、FRATide、靛玉红3’-肟以及L803。
在某些实施例中，TGFβ抑制剂选自由以下组成的组：SB431542、Repsox、616452、LDN193189、A8301、GW788388、SD208、SB525334、LY364947、D4476、SB505124以及曲尼司特(Tranilast)。
在某些实施例中，环状AMP诱导剂是毛喉素(forskolin)、IBMX、咯利普兰(Rolipram)、8BrcAMP、前列腺素E2(PGE2)、NKH477、二丁酰一环腺苷酸(DBcAMP)、Sp-8-Br-cAMPs。
在某些实施例中，DNA甲基转移酶抑制剂选自由以下组成的组：5-氮杂-dC、5-氮杂胞苷(Azacytidine)以及RG108。
在某些实施例中，DOT1L抑制剂是EPZ004777。
在某些实施例中，组蛋白脱乙酰基酶抑制剂选自由以下组成的组：丙戊酸(VPA)、曲古霉素A(trichostatin A；TSA)、伏立诺他(vorinostat)、缩酚酸肽(depsipeptide)、Trapoxin、Depudecin、FR901228以及丁酸盐。
在某些实施例中，第一类型的细胞是体细胞。在某些实施例中，体细胞衍生自中胚层、外胚层或内胚层。
在某些实施例中，体细胞是成纤维细胞。在某些实施例中，成纤维细胞选自由以下组成的组：小鼠胚胎成纤维细胞(MEF)、小鼠尾尖成纤维细胞(TTF)、人胚胎成纤维细胞(HEF)、人新生成纤维细胞(HNF)、成人成纤维细胞(HAF)、人包皮成纤维细胞(HFF)以及其混合物。
在某些实施例中，体细胞是人脱落肾上皮细胞。
在某些实施例中，第一类型的细胞是干细胞。在某些实施例中，干细胞选自由以下组成的组：人脐带间充质干细胞、人胚胎干细胞以及诱导多能干细胞(iPSC)。
在某些实施例中，第二类型的细胞是干细胞。在某些实施例中，干细胞是神经嵴细胞样细胞(NCC样细胞)。在某些实施例中，NCC样细胞呈P75、Hnk1、AP2α以及Sox10阳性。
在某些实施例中，第一类型的细胞在第一组重编程因子存在下培养(a)至少1、2、3、4、5、6、7、8、9、10、11或12天或(b)不超过20、19、18、17、16、15、14、13或12天。
本公开提供了一种将第二类型的细胞重编程成第三类型的细胞的方法，其包含在第二组重编程因子存在下培养第二类型的细胞，其中第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
在某些实施例中，第二组重编程因子进一步包含BMP4和/或DNA甲基转移酶抑制剂。在某些实施例中，酪蛋白激酶1抑制剂是CKI-7。在某些实施例中，DNA甲基转移酶抑制剂选自由以下组成的组：5-氮杂胞苷、5-氮杂-dC以及RG108。
在某些实施例中，第三类型的细胞是体细胞。在某些实施例中，体细胞是角膜内皮细胞(CEC)样细胞(CEC样细胞)。在某些实施例中，CEC样细胞呈ZO-1和Na +/K +-ATP酶阳性。
在某些实施例中，第二类型的细胞在第二组重编程因子存在下培养(a)至少1、2、3、4、5、6、7、8、9、10、11或12天或(b)不超过20、19、18、17、16、15、14、13或12天。
本公开提供了一种将第一类型的细胞重编程成第三类型的细胞的方法，其包含步骤(a)在第一组重编程因子存在下培养第一类型细胞，其中第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子(TGFβ)抑制剂以及环状AMP诱导剂，和步骤(b)在第二组重编程因子存在下培养从步骤(a)获得的细胞，其中第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
在某些实施例中，方法进一步包含在开始步骤(b)之前洗涤从步骤(a)获得的细胞。在某些实施例中，步骤(a)与步骤(b)之间不存在洗涤步骤。
本公开提供了一种将第一类型的细胞重编程成第三类型的细胞的方法，其包含在第一组重编程因子和第二组重编程因子存在下培养第一类型的细胞，其中第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子(TGFβ)抑制剂以及环状AMP诱导剂，并且第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
本公开提供了根据本文所提供的方法产生的NCC样细胞群。
本公开提供了根据本文所提供的方法产生的角膜内皮细胞样细胞(CEC样细胞)群。
本公开提供了一种包含本文所提供的NCC样细胞或CEC样细胞的组合物。
本公开提供了一种治疗与功能失调或受损角膜内皮细胞相关的疾病或病状的方法，其包含向有需要的受试者施用有效量的本文所提供的CEC样细胞或本文所提供的组合物。在某些实施例中，受试者是人。
在某些实施例中，疾病或病状选自由以下组成的组：富克氏营养不良(Fuch’s dystrophy)、虹膜角膜内皮综合征、后部多态性营养不良、先天性遗传性内皮营养不良、年龄相关性黄斑变性(AMD)、视网膜色素变性、青光眼、角膜营养不良、隐形眼镜使用、白内障手术以及角膜移植中的晚期内皮衰竭。
本公开提供了一种用于将第一类型的细胞重编程成第二类型的细胞的试剂盒，其中试剂盒包含第一组重编程因子，并且第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、TGFβ抑制剂以及环状AMP诱导剂。
本公开提供了一种用于将第二类型的细胞重编程成第三类型的细胞的试剂盒，其中试剂盒包含第二组重编程因子，并且第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
本公开提供了一种用于将第一类型的细胞重编程成第三类型的细胞的试剂盒，其中试剂盒包含第一组重编程因子和第二组重编程因子，其中第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、TGFβ抑制剂以及环状AMP诱导剂，并且第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
本公开提供了一种鉴别影响NCC、化学诱导的NCC(ciNCC)或化学诱导的CEC(ciCEC)的效应的药物的方法，其包含向NCC、ciNCC或ciCEC施用药物候选物，并且检测细胞对药物候选物的反应，从而鉴别药物。
附图说明
图1展示通过化学成分确定的条件将成纤维细胞转化成ciNCC。A)示出从小鼠成纤维细胞开始的诱导方法的示意图。B)展示在神经嵴细胞样细胞诱导期间不同时间点处的形态变化。C)展示指定条件下生成的ciNCC集落数。数据为平均值±SD，n＝3独立实验。D)展示荧光显微镜原位拍摄的SOX10 +集落的图像。E)展示指定条件下生成的SOX10 +集落数。数据为平均值±SD，n＝3独立实验。F)展示第12天时ciNCC标记P75、Hnk1以及AP2α的免疫染色。
图2展示从成纤维细胞转化的ciNCC的分化潜能。A)展示免疫细胞化学分析，该分析展示ciNCC可分化成外周神经元(如Tuj1和外周蛋白(peripherin)的免疫细胞化学所指示的)，并且还可分化成雪旺细胞(schwann cell)(如S100β和GFAP的免疫细胞化学所指示的)。右侧明场图像展示从ciNCC分化的黑色素细胞。B)展示ciNCC可进一步分化成软骨细胞、脂肪细胞以及骨细胞(如分别由阿尔新蓝(Alcian blue)染色、油红O(Oil Red O)染色以及茜素红(Alizarin Red)染色所示)。
图3展示ciNCC可进一步分化成角膜内皮细胞样细胞(或化学诱导的CEC、ciCEC)。图3A展示角膜内皮标记(包括Na+-K+ATP酶、AQP1、波形蛋白(Vimentin)、N-钙粘蛋白(N-cadherin)、层粘连蛋白(laminin)以及AQP1)的免疫荧光染色。细胞核用DAPI染色。比例尺，50μm。图3B展示透射电子显微镜图像，该图像展示角膜内皮细胞的紧密连接。图3C展示用于从成纤维细胞功能地生成角膜内皮细胞的两步谱系重编程策略的方案。
图4展示成纤维细胞朝角膜内皮细胞样细胞(或ciCEC)重编程的谱系追踪(Lineage tracing)。A)展示用于追踪从Fsp1-Cre:R26R tdTomato MEF重编程的角膜内皮细胞样细胞的起源的遗传命运图谱(genetic fate mapping)方法的示意图。B)展示从Fsp1-Cre:R26R tdTomato MEF重编程的角膜内皮细胞样细胞的起源的谱系追踪。C)展示免疫细胞化学分析，该分析展示FSP1-ciNCC呈P75、Hnk1、AP2α以及SOX10阳性。D)展示从这些MEF诱导的Fsp1-Cre:R26R tdTomato MEF和ciCEC的代表性形态变化。
图5展示通过小分子从不同人体细胞类型生成角膜内皮细胞样细胞。人胚胎皮肤成纤维细胞(HEF)、人新生成纤维细胞(HNF)、成人成纤维细胞(HAF)、人脐带间充质基质细胞(MSC)以及尿液细胞(UC)。
图6展示兔中的ciCEC组和对照组的不同天数时的临床观察结果。A)展示第10代(P10)时ciCEC的明场图像。第10代时的ciCEC表达Na+-K+ATP酶和ZO-1。B)展示裂隙灯照片，该照片展示在ciCEC移植的第7天注射之后，ciCEC组角膜的透明度显著改良(第1图)，而通过裂隙灯照片分析的未处理对照组中角膜混浊和基质水肿仍然严重(第3图)。C)展示通过visante OCT分析的CEC样细胞组与对照组中的显著角膜厚度差异。D)展示共聚焦显微镜图像，该图像证实了ciCEC组中德斯密氏膜(Descemet’s membrane)上多角形细胞的全覆盖。E)展示在低剂量(1×10 6个细胞/毫升ciCEC)、高剂量(2×10 6个细胞/毫升ciCEC)以及经PBS处理的对照组中临床观察期间的角膜厚度变化。B)-D)从 左到右的图像：第1图像：经ciCEC处理的组，第2图像：完好的对侧眼，第3图像：经PBS处理的对照组，第4图像：正常眼组。
图7展示兔中的ciCEC组和对照组的不同天数时的裂隙灯照片。裂隙灯照片展示注射之后ciCEC组角膜的透明度显著改良，并且可看见瞳孔和虹膜纹理(ciCEC组，上方的图)。仅约14天之后，角膜变得明显透明，而对照组中角膜混浊和基质水肿仍然严重(对照组，上方的图)。ciCEC组和对照组两者的下方的图展示通过裂隙灯检测到的角膜反射，并且下方的图中的每个图像对应于其上方的图中的图像。
图8展示通过小分子诱导将MEF转化为ciNCC。A)从MEF重编程NCC的示意图。B)各个化学品对ciNCC生成的效应。数据为平均值±SD，n＝3独立实验。C)各个化学品对ciNCC生成的促进效应(数据为平均值±SD，n＝3独立实验)。D)我们将MEF转化成ciNCC的策略的示意性图示。E)使用小分子混合物(cocktail)从MEF生成Wnt1 +ciNCC。F)组合筛选中候选混合物所诱导的ciNCC簇中Wnt1 +细胞数的定量(独立实验，n＝3)。G)ciNCC诱导处理期间不同天数时的形态变化(比例尺，50μm)。H)不同天数时候选混合物所诱导的Wnt1 -tdTomato +细胞的百分比(独立实验，n＝3)。
图9展示对M6诱导的ciNCC的表征。A)M6诱导的ciNCC的形态(比例尺，400μm)。B)展示MEF源的ciNCC表达P75、HNK1、AP2a以及巢蛋白(Nestin)的免疫染色(比例尺，50μm)。C)染色有外周神经元标记的分化的ciNCC的代表性图像(比例尺，50μm)。D)ciNCC向雪旺细胞和黑色素细胞的分化以及标记物表达(比例尺，50μm)。E)ciNCC分化成间充质谱系并且进一步分化成脂肪细胞、软骨细胞以及骨细胞(比例尺，100μm)。
图10展示通过小分子诱导从成纤维细胞生成小鼠ciCEC。A)从MEF化学重编程ciCEC的示意图。B)初始MEF、重编程ciNCC集落以及ciCEC的明场图像(比例尺，400μm)。C)从Wnt1 -tdTomato +ciNCC诱导加工ciCEC期间不同天数时的形态变化(比例尺，400μm)。D)ciCEC针对角膜内皮标记Na +/K +-ATP酶、AQP1、波形蛋白、N-钙粘蛋白、层粘连蛋白以及ZO-1的免疫荧光染色(比例尺，50μm)。E)ciCEC中的LDL摄入功能(比例尺，50μm)。F)指定时间点处样品中差异表达基因的热图。热图下方的这些编号指示独立的生物学重复。红色和蓝色分别指示上调和下调的基因。G)展示紧密连接的ciCEC的TEM(比例尺，5μm)。
图11展示ciNCC和ciCEC的基因表达谱分析。A)展示指定时间点处NC基因表达的qRT-PCR分析。基因表达(log2)相对于MEF中的基因表达归一化。B)对不同传代数 的MEF源的ciCEC、MEF以及pCEC中指定NC细胞基因表达的qRT-PCR分析。C)指定时间点处样品中差异表达基因的热图。热图下方的编号指示独立的生物学重复(n＝2-3)。红色和蓝色分别指示上调和下调的基因。D)来自重编程、ciCEC以及对照pCEC的第0天(D0)、第7天(D7)以及第12天(D12)的样品的主成分分析。
图12展示证实从成纤维细胞诱导ciCEC的谱系追踪。A)示出遗传谱系追踪策略的示意图。通过分选从衍生自Fsp1-Cre/ROSA26 tdTomato背景的E13.5小鼠胚胎的MEF的p75 -/tdTomato +细胞获得MEF。B)展示来自具有Fsp1-Cre/ROSA26 tdTomato遗传背景的MEF的p75 -/tdTomato +细胞的FACS分选结果。C)展示p75 -/tdTomato +细胞中Sox10、P75、Olig2、Hnk1、AP2、Sox2以及Pax6结果阴性的免疫染色分析(比例尺，100μm)。D)p75 -/tdTomato +细胞朝向ciNCC和ciCEC的分化(比例尺，400μm)。e)展示Fsp1-tdTomato-MEF源的ciCEC中ZO-1、层粘连蛋白、Na +/K +-ATP酶以及AQP1阳性的免疫染色分析(比例尺，50μm)。
图13展示小分子促进ciCEC长期扩增。A)ciCEC在无血清对照培养基中扩增3天(比例尺，200μm)。B)添加SB431542和CKI-7的无血清培养基中ciCEC的连续扩增(比例尺，200μm)。C)在含或不含SB431542和CK1-7的培养基中培养的ciCEC的平均群体倍增时间(平均值±SD，n＝3；***p<0.001)。D)处于P5的ciCEC的明场图像，ciCEC在添加SB431542和CKI-7的培养条件下扩增3天(比例尺，50μm)。E)处于P30的这些ciCEC被固定并且针对Na +/K +-ATP酶、AQP1以及ZO-1染色(比例尺，50μm)。
图14展示ciCEC体内移植。A)描绘将ciCEC以及ROCK抑制剂移植到模型兔中的图。B)移植之后，经移植眼中的角膜透明度显著改良，而未处理对照中角膜混浊和基质水肿仍然严重。C)裂隙灯显微图像，其展示移植之后，经移植角膜的透明度显著改良，而未处理对照中角膜混浊和基质水肿仍然存在。D)展示存活tdTomato +ciCEC连接到德斯密氏膜的免疫组织化学(比例尺，100μm)。E)展示经移植眼中改善的角膜水肿(减小的角膜厚度)的Visante OCT。F)移植之后角膜厚度的趋势。未处理对照与经移植对照之间存在角膜厚度上的显著差异。结果为生物学重复(n＝9)的平均值和SEM。G)角膜内皮的实时共聚焦成像，其证实了经移植眼中德斯密氏膜上多角形细胞的全覆盖。
图15展示Wnt1 -MEF的表征。A)示出遗传谱系追踪策略的示意图。通过分选从衍生自Wnt1 -Cre/ROSA26tdTomato背景的E13.5小鼠胚胎的MEF的tdTomato -细胞获得Wnt1 -MEF。B)展示来自MEF的tdTomato -细胞的FACS分选结果。C)展示Wnt1 -MEF 中P75、HNK1、Sox10以及Ap2阴性结果的免疫染色分析(比例尺，100μm)。D)展示Wnt1 -MEF和原代NCC中指定神经嵴基因表达的RT-PCR分析。Gapdh充当对照。E)展示Wnt1 -MEF源的ciNCC集落的代表性图像(比例尺，400μm)。
图16展示M6将tdMEF转化成ciNCC。A)tdMEF和tdMEF源的ciNCC的形态(比例尺，400μm)。B)ciNCC分化成ciCEC的代表性图像(比例尺，400μm)。C)展示tdMEF源的ciNCC表达NC细胞标记(P75、HNK1、Sox10以及Ap2α)的免疫染色分析(比例尺，50μm)。D)FSP1-tdTomato +ciCEC的分化过程。
图17展示通过小分子从成纤维细胞生成ciCEC，其不通过iPSC阶段。A)从MEF诱导ciCEC期间不同时间点处的形态变化(比例尺，400μm)。B)MEF源的ciNCC集落针对NC标记Sox10进行的免疫荧光染色(比例尺，400μm)。C)从OG-MEF诱导ciCEC期间不同时间点处的形态变化(比例尺，400μm)。D)通过FACS分析测得，对于ciCEC诱导，不存在Oct4-GFP阳性细胞。E)典型ciCEC核型(第10代)。
图18展示ciCEC的增殖潜能。A)处于P3的ciCEC和处于P3的pCEC的代表图像(比例尺，400μm)。B)处于P3的ciCEC和处于P3的pCEC中Ki67和ZO-1表达的免疫荧光图像。C)通过流式细胞术分析的不同传代数的ciCEC和pCEC的EdU并入实验。D)细胞周期(G1、S以及G2期)中ciCEC和pCEC的分布(比例尺，400μm)。刮划处于P3的ciCEC和处于P3的pCEC，并且在8小时和20小时后观察到迁移，并拍摄图像。E)处于P3的pCEC和处于P3、P15、P30的ciCEC的划痕分析(scratch wound assay)(比例尺，200μm)。8小时和20小时后观察到迁移。F)展示对划痕闭合的定量。
图19展示对兔模型的经移植眼的观察结果。A)在基线(细胞注射之前)和补充有ROCK抑制剂的ciCEC注射之后不同天数时获得的受试者#10中经移植眼的裂隙灯显微(中间)和共聚焦显微镜图像(右侧)。B)展示不同天数时经ciCEC移植的眼中的角膜厚度的Visante OCT。C)展示连接到德斯密氏膜的存活tdTomato +ciCEC的免疫染色分析。D)不同天数时角膜厚度的变化。
具体实施方式
以下对本公开的描述仅旨在说明本公开的各种实施例。因此，所讨论的具体修改不应被解释为对本公开范围的限制。对于所属领域的技术人员显而易见的是，在不脱离本公开的范围的情况下可以作出各种等效物、改变和修改，并且应理解，此类等效实施例将包括 在本文中。本文引用的所有参考文献，包括出版物、专利和专利申请，均以全文引用的方式并入本文中。
A.一般定义
除非上下文另外明确指示，否则单数术语“一(a/an)”和“所述”包括复数指代物。作为实例，提及“一细胞”是指一种或多种细胞，并且提及“所述方法”包括提及本文所公开和/或所属领域的技术人员已知的等效步骤和方法，以此类推。类似地，除非上下文另外明确指示，否则词语“或”打算包括“和”。虽然与本文所描述的方法和材料相似或等效的方法和材料可用于实践或测试本公开，但在下文描述合适的方法和材料。缩写“例如(e.g.)”源自于拉丁文exempli gratia，并且在本文中用于指示非限制性实例。因此，缩写“例如(e.g.)”与术语“例如(for example)”同义。
如本文所用，术语“包含(comprising/comprises)”在提及组合物、方法以及对所述方法或组合物来说必不可少的其相应组分时使用，但对于包括未指明的要素，无论是否必要，仍然是开放的。
术语“由……组成”是指如本文所描述的组合物、方法和其相应组分，其排除未在所述实施例的描述中叙述的任何要素。
术语“约”或“大致”意指给定值或范围的20％内，优选10％内，并且更优选5％内。
如本文所用的术语“细胞”是指单个细胞、细胞系或衍生自此类细胞的培养物。
如本文所用，术语“重编程(reprogram/reprogramming/reprogrammed)”或其等效物是指相比于细胞在不进行重编程的相同条件下将具有的分化状态，在培养物中或体内改变或逆转细胞的分化状态的过程。换句话说，在本公开的情形下，“重编程”包括分化、脱分化以及转分化。如本文所用，术语“分化”是指一种细胞过程，较低特化的细胞通过所述细胞过程变为较高特化的细胞类型。相比之下，术语“脱分化”是指一种细胞过程，其中部分或终末分化细胞恢复为较早发育阶段，如具有多能性或多潜能性的细胞。进一步相比，术语“转分化”是指将一种分化细胞类型转化成另一分化细胞类型的细胞过程。因此，如本文所用，术语“较低分化状态”或“较低特化”或“较早发育阶段”是相对术语并且包括完全脱分化状态(或完全脱分化)和部分分化状态(或部分分化)。为与上述细胞发育区分，“未分化细胞”是能够向许多方向分化的细胞，即其能够分化成两种或更多种类型的特化细胞。未分化细胞的典型实例是干细胞。
细胞类型在分化期间通过各种水平潜能，如全能性、多能性以及多潜能性。短语“全能干细胞”是指能够分化成构成生物体的所有细胞的细胞，如由卵细胞和精细胞的融合产生的细胞。由受精卵的前几次分裂产生的细胞也可能是全能的。这些细胞可分化成胚胎和胚外细胞类型。多能干细胞，例如ES细胞，可产生任何胚儿和成体细胞类型。然而，其无法单独发育成胚儿或成体动物，因为其缺乏发育胚外组织的潜能。胚外组织部分衍生自胚外内胚层，并且可进一步分类成腔壁内胚层(parietal endoderm)(赖歇特氏膜(Reichert's membrane))和内脏内胚层(visceral endoderm)(形成卵黄囊的部分)。腔壁内胚层和内脏内胚层两者支持胚胎发育但本身并不形成胚胎结构。还存在其它胚外组织，包括胚外中胚层和胚外外胚层。如本文所用，“多能干细胞”或具有“多能性”的细胞或等效物是指可分化成所有三种胚层(例如，内胚层、中胚层以及外胚层)的细胞群。多能细胞表达多种多能细胞特异性标记，具有未分化细胞的细胞形态特征(即，致密集落、高核质比以及明显的核仁)，并且在引入到免疫低下动物，如SCID小鼠中时形成畸胎瘤。畸胎瘤典型地含有所有三种胚层的细胞或组织特征。所属领域的一般技术人员可通过使用所属领域中常用的技术评定这些特征。参见例如Thomson等人,《科学(Science)》282:1145-1147(1998)。多能细胞能够在细胞培养中增殖并且朝向展现多潜能特性的多种谱系限制细胞群分化。多潜能干细胞或具有多潜能性或等效性的细胞相对于多能干细胞分化程度更高，但未终末分化。多能干细胞因此具有比多潜能干细胞更高的潜能。
B.重编程细胞
在一个方面，本公开提供了一种用于将第一类型的细胞重编程成第二类型的细胞的方法，其包含在第一组重编程因子存在下培养第一类型的细胞，其中第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子-β(TGFβ)抑制剂以及环状AMP诱导剂。
在一些实施例中，在第一组重编程因子存在下培养第一类型的细胞使至少50％、55％、60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多细胞变为第二类型的细胞。
在某些实施例中，第一类型的细胞是衍生自中胚层、外胚层或内胚层的哺乳动物细胞，如人或小鼠。
外胚层、中胚层以及内胚层是胚胎发育期间形成的三个胚层，其中中胚层作为中间层，外胚层作为外层并且内胚层作为内层。中胚层形成间充质、间皮、非上皮血细胞以及体腔细胞，其构成肌肉(平滑肌和横纹肌)、骨、软骨、结缔组织、脂肪组织、循环系统、淋 巴系统、真皮、泌尿生殖系统、浆膜以及脊索。内胚层形成咽、食管、胃、小肠、结肠、肝脏、胰腺、膀胱、气管和支气管上皮部分、肺、甲状腺以及甲状旁腺。外胚层形成表面外胚层、神经嵴以及神经管，其中表面外胚层发育成表皮、毛发、指甲、眼的晶状体、皮脂腺、角膜、牙釉质、口和鼻的上皮；外胚层的神经嵴发育成周围神经系统、肾上腺髓质、黑色素细胞、面部软骨以及牙本质；并且外胚层的神经管发育成脑、脊髓、脑垂体后叶、运动神经元以及视网膜。
在某些实施例中，第一类型的细胞是体细胞。如本文所用，术语“体细胞”是指除生殖系细胞(例如，精子和卵，以及制造其的细胞(配子母细胞))和未分化干细胞外的任何细胞。内脏、皮肤、骨、血液以及结缔组织均由体细胞构成。体细胞可以是任何起源的任何类型的体细胞。举例来说，体细胞可包括但不限于成纤维细胞、上皮细胞、支持细胞、内皮细胞、上皮粒层、神经元、胰岛细胞、表皮细胞、肝细胞、毛囊细胞、角质形成细胞、造血细胞、黑色素细胞、软骨细胞、淋巴细胞(B和T淋巴细胞)、红细胞、巨噬细胞、单核细胞(monocyte)、单个核细胞(mononuclear cell)、心肌细胞以及其它肌细胞。
在某些实施例中，第一类型的细胞是中胚层细胞。中胚层细胞的标记是所属领域已知的，例如CD56和APJ。在某些实施例中，中胚层细胞包括成纤维细胞、上皮细胞、白细胞、脂肪细胞以及角质形成细胞。在某些实施例中，第一类型的细胞是成纤维细胞，包括但不限于小鼠胚胎成纤维细胞(MEF)、小鼠新生成纤维细胞(MNF)、小鼠尾尖成纤维细胞(TTF)、人胚胎皮肤成纤维细胞(HEF)、人新生成纤维细胞(HNF)、成人成纤维细胞(HAF)、人胚胎肺成纤维细胞、人包皮成纤维细胞(HFF)以及其混合物。
成纤维细胞可从任何合适的来源获得，例如从商业来源或从各种器官组织或皮肤组织获得。优选的成纤维细胞是肺成纤维细胞、包皮成纤维细胞以及成体真皮成纤维细胞。在某些实施例中，成纤维细胞从患者获得，例如通过皮肤活检(例如，用确定的因子重编程人体细胞达到多能性(reprogramming of human somatic cells to pluripotency with defined factors).George Q.Daley等人《自然(Nature)》2008；用于从人皮肤单一钻取活检分离并且连续繁殖角质形成细胞、内皮细胞以及成纤维细胞的方法(a method for the isolation and serial propagation of keratinocytes,endothelial cells,and fibroblasts from a single punch biopsy of human skin),Normand等人《体外细胞与发育生物学-动物(In Vitro Cellular & Developmental Biology-Animal)》,1995)。
在某些实施例中，第一类型的细胞是上皮细胞，如人脱落肾上皮细胞。上皮细胞可从 尿液样品获得。
在某些实施例中，第一类型的细胞是白细胞(leucocyte)。白细胞可从血液样品获得。白细胞是可通常由以下差异分类成其亚群(淋巴细胞、单核细胞、中性粒细胞、嗜酸性粒细胞以及嗜碱性粒细胞)的白细胞(white blood cell)：RF信号强度(高频下电阻抗的变化)、DC信号强度(悬浮粒子与粒子悬浮于其中的液体介质之间的电导性差异所引起的直流电流变化)、荧光强度、散射光强度、吸光度、散射光去极化等等(参见US5618733A)。
在某些实施例中，第一类型的细胞是脂肪细胞或角质形成细胞。脂肪细胞和角质形成细胞也可容易地通过皮肤活检或采集的毛发得到(从皮肤或采集的毛发分离并且培养人角质形成细胞以便生成诱导多能干细胞(isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells),Belmonte等人《自然实验手册(Nature Protocols)》2010)。
在其它实施例中，第一类型的细胞是成体细胞。如本文所用，术语“成体细胞”是指胚胎发育之后在整个身体中发现的细胞。在某些实施例中，第一类型的细胞可以是干细胞，如胚胎干细胞、诱导多能干细胞(iPSC)以及成体干细胞，包括但不限于造血干细胞、血管内皮干细胞、心脏干细胞、肌源干细胞、间充质干细胞、表皮干细胞、脂肪源干细胞、肠干细胞、神经干细胞、肾上皮干细胞、尿道上皮干细胞以及肝脏干细胞。
在一些实施例中，干细胞是指未分化细胞，其能够增殖并产生更多祖细胞，祖细胞能够生成大量母细胞，母细胞可继而产生分化或可分化子细胞。干细胞可不对称分裂，其中一个子细胞保留干细胞状态并且另一子细胞表达一些独特的其它特定功能和表型。可替代地，群体中干细胞中的一些可对称分裂成两个干细胞，因此在整体上在群体中维持一些干细胞，而群体中的其它细胞仅产生分化的子代。子细胞本身可被诱导以增殖并且产生子代，子代随后分化成一种或多种成熟细胞类型，同时也保留一种或多种具有亲本发育潜能的细胞。在其它实施例中，术语“干细胞”是指具有在特定环境下分化为更特化或分化表型的能力或潜能的祖细胞亚群，其保留有在某些情况下增殖而大体不分化的能力。在一个实施例中，术语干细胞一般是指天然存在的母细胞，其后代(子代)通过分化(例如通过获取完全个体特征)通常在不同方向上特化，如在胚胎细胞和组织的渐进多样化中发生。分化的细胞可衍生自多潜能细胞，该多潜能细胞自身衍生自多潜能细胞，以此类推。尽管这些多潜能细胞中的每一个都可视为干细胞，但每一个可产生的细胞类型的范围可显著改变。在许多生物个例中，干细胞也是“多潜能”的，因为其可产生多于一种独特细胞类型的子 代，但这不是“干细胞特性(stem-ness)”所需的。自我更新是干细胞定义的另一典型部分。理论上，自我更新可通过两大机制中的任一个进行。
术语“胚胎干细胞”用于指胚胎囊胚的内部细胞团块的多能干细胞(参见美国专利第5,843,780号、第6,200,806号，其以引用的方式并入本文中)。胚胎干细胞的区别性特征限定胚胎干细胞表型。因此，如果细胞具有使得所述细胞可以与其它细胞区别开来的胚胎干细胞独特特征中的一个或多个，那么所述细胞具有胚胎干细胞的表型。示例性胚胎干细胞区别性特征包括但不限于基因表达谱、增殖能力、分化能力、核型、对特定培养条件的反应性等等。
术语“成体干细胞”或“ASC”用于指衍生自非胚胎组织(包括胎儿、幼体以及成体组织)的任何多潜能干细胞。成体干细胞已从广泛多种成体组织分离，包括血液、骨髓、脑、嗅上皮、皮肤、胰、骨骼肌以及心肌。这些干细胞中的每一种都可基于在培养物中的基因表达、因子反应性以及形态来表征。如上文所示，已发现干细胞驻留于几乎每个组织中。因此，从本文所描述的技术可得知干细胞群可从几乎任何动物组织分离。
在某些实施例中，第一类型的细胞可以是间充质干细胞、间充质基质细胞、人胚胎干细胞或诱导多能干细胞(iPSC)。
如本文所用，术语“iPS细胞”、“iPSC”以及“诱导多能干细胞”可互换使用，且指人工衍生(例如，通过完全或部分逆转诱导)自分化的体细胞(即，衍生自非多能细胞)的多能细胞。多能细胞可分化为三种发育胚层的所有细胞。
间充质干细胞(MSC)或间充质基质细胞是传统上发现于骨髓中的成体干细胞。然而，间充质干细胞也可从其它组织(包括脐带血、外周血、输卵管和胎儿肝脏以及肺)分离。MSC可分化成多种细胞类型，包括成骨细胞(骨细胞)、软骨细胞(chondrocyte/cartilage cell)、肌细胞(肌肉细胞)以及脂肪细胞(adipocyte)(产生骨髓脂肪组织的脂肪细胞(fat cell))。
在某些实施例中，第一类型的细胞是外胚层细胞。在某些实施例中，第一类型的细胞是内胚层细胞。
在某些实施例中，第二类型的细胞从第一类型的细胞转分化。举例来说，中胚层细胞重编程成外胚层细胞。在某些实施例中，第二类型的细胞是外胚层细胞。在某些实施例中，多潜能干细胞是神经嵴细胞(NCC)或神经嵴细胞样细胞。在某些实施例中，第二类型的 细胞是神经嵴细胞(NCC)或神经嵴细胞样细胞。
“神经嵴细胞”或“NCC”一般是指具有产生共表达黑素体标记和HMB45的色素细胞的发育潜能的神经祖细胞。神经嵴细胞可通过表达本文所鉴别和所属领域中已知的标记而被鉴别。为区分本文所提供的化学诱导的NCC(ciNCC)与原代NCC，所得ciNCC也被命名为神经嵴细胞样细胞(NCC样细胞)。在一些实施例中，所得NCC的NCC样细胞展现与原代NCC表型一致的一种或多种生物标记。在一些实施例中，所得NCC样细胞缺乏与NCC表型一致的一种或多种生物标记的表达或其表达较低(例如PAX6的表达)。存在情况与NCC表型一致的示例性NC生物标记可包括巢蛋白(Nestin)、SOX10、SOX9、HNK-1、P75(NGFR)、AP2α、PAX3、PAX7、SNAI2、Snail、Twistl、Krox20、CD271、FoxD3、AN2以及Ki67，和/或至少一种多能性标记NANOG、ZNF206或OCT4。在一些实施例中，NC生物标记是P75、Hnk1、AP2α和/或SOX10。在实施例中，相对于产生NCC样细胞的第一类型的细胞，NC生物标记的表达的提高超过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倍或更多。
在某些实施例中，第一类型的细胞是成纤维细胞并且第二类型的细胞是NCC样细胞。在某些实施例中，第一类型的细胞是小鼠胚胎成纤维细胞(MEF)并且第二类型的细胞是NCC样细胞。在某些实施例中，第一类型的细胞是小鼠尾尖成纤维细胞(TTF)并且第二类型的细胞是NCC样细胞。在某些实施例中，第一类型的细胞是人胚胎皮肤成纤维细胞(HEF)并且第二类型的细胞是NCC样细胞。在某些实施例中，第一类型的细胞是人胚胎肺成纤维细胞、人包皮成纤维细胞(HFF)并且第二类型的细胞是NCC样细胞。在某些实施例中，第一类型的细胞是人新生成纤维细胞(HNF)或成人成纤维细胞(HAF)，并且第二类型的细胞是NCC样细胞。
在某些实施例中，第一类型的细胞是上皮细胞(例如人脱落肾上皮细胞)并且第二类型的细胞是NCC样细胞。在某些实施例中，第一类型的细胞是白细胞(例如淋巴细胞、单核细胞、中性粒细胞、嗜酸性粒细胞以及嗜碱性粒细胞)并且第二类型的细胞是NCC样细胞。在某些实施例中，第一类型的细胞是脂肪细胞并且第二类型的细胞是NCC样细胞。在某些实施例中，第一类型的细胞是角质形成细胞并且第二类型的细胞是NCC样细胞。在某些实施例中，第一类型的细胞是MSC、人胚胎干(ES)细胞或iPSC并且第二类 型的细胞是NCC样细胞。
在某些实施例中，第二类型的细胞是从另一类型的干细胞(第一类型的细胞)(如间充质干细胞、人胚胎干细胞或诱导多能干细胞(iPSC))重编程的某些类型的多潜能干细胞。
标记的表达可通过所属领域中已知的任何方法检测，包括但不限于蛋白质印迹法、基于mRNA扩增的方法(例如，PCR、等温扩增等，其可包括逆转录并且可应用于检测来自单一细胞或多个细胞的表达)、RNA印迹法、免疫染色等。另外，所述标记的表达可通过报导子构建体(reporter construct)(如表达可被视觉检测的荧光蛋白、表达可通过在抗生素存在下的细胞存活被检测的抗生素抗性基因等)的表达来推断，该报导子构建体处于赋予细胞类型特异性表达的基因元件(如前述标记中的一种或其片段的启动子)的控制下。示例性报导子构建体是pOCT4-GFP和pOCT4-LUC基因，其分别驱动GFP和荧光素酶在ES细胞中的表达，GFP和荧光素酶中的任一个的表达可容易地使用常规方法检测。可使用的检测标记表达的其它方法是所属领域中已知的。一般参见Ausubel,《现代分子生物学实验技术(Current Protocols in Molecular Biology)》(实验室指南(Current Protocols),1988)；Ausubel等人,《分子生物学简短方案(Short Protocols in Molecular Biology)》(实验室指南；第5版,2002)；Sambrook等人,《分子克隆：实验指南(Molecular Cloning:A Laboratory Manual)》(冷泉港实验室出版社(Cold Spring Harbor Laboratory Press),第3版,2001)；Sambrook等人,《来自〈分子克隆：实验指南〉的精简方案(The Condensed Protocols from Molecular Cloning:A Laboratory Manual)》(冷泉港实验室出版社,2006)，所述文献中的每一种以全文引用的方式并入本文中。
所属领域中已知的任何细胞培养系统可用于本公开。在某些实施例中，贴壁培养系统用于本公开的方法。术语“贴壁培养”是指一种细胞培养系统，细胞借此在固体表面上培养，固体表面又可用基质涂布。细胞可以或可以不紧密粘附于固体表面或基质。用于贴壁培养的基质可进一步包含例如以下中的任一种或组合：聚苯乙烯、聚酯、聚碳酸酯、聚(N-异丙基丙烯酰胺)、聚鸟氨酸、层粘连蛋白、聚赖氨酸、纯化胶原蛋白、明胶、纤维素、胞外基质、纤维连接蛋白、粘合素(tenacin)、玻璃连结蛋白(vitronectin)、聚糖酵解酸(PGA)、聚乳酸(PLA)、聚乳酸-乙醇酸(PLGA)、基质胶、羟基磷灰石以及羊膜。
在某些实施例中，悬浮培养可用于本公开的方法中。如本文所用的术语“悬浮培养”是指使得细胞不粘附于固体载体或培养容器的细胞培养方式。为将细胞转移到悬浮培养物 中，细胞例如通过细胞刮棒从培养容器移出并且转移到含有培养基的无菌低粘附培养板中，所述培养板不允许细胞粘附到培养板表面。因此，细胞可悬浮培养，而不粘附到基质或培养皿底部。
适用于培养细胞的培养基是适用于在培养皿中生长某一细胞类型的任何培养基。该培养基包括如汉姆氏(Ham’s)F10(西格玛(Sigma))、汉姆氏F12培养基、最小必需培养基(Minimal Essential Medium；MEM)(西格玛)、RPMI-1640(西格玛)和杜氏改良伊格尔培养基(Dulbecco’s Modified Eagle’s Medium；DMEM)(西格玛)、IMDM培养基、培养基199、伊格尔最低必需培养基(Eagle's Minimum Essential Medium；EMEM)、aMEM培养基、费舍尔氏培养基(Fischer's medium)、神经基质培养基(Neurobasal medium)(生命技术公司(Life Technologies Corporation))以及这些培养基的混合物。另外，Ham等人,《酶学方法(Meth.Enz.)》58:44(1979)，Barnes等人,《分析生物化学(Anal.Biochem.)》102:255(1980)，美国专利第4,767,704号；第4,657,866号；第4,927,762号；第4,560,655号；或第5,122,469号；WO 90/03430；WO 87/00195；或美国专利再颁号30,985中所描述的培养基中的任一种可用作培养基。这些培养基中的任一种可按需要补充所需的盐(如氯化钠、钙盐、镁盐以及磷酸盐)、缓冲剂(如HEPES)、核苷酸(如腺苷和胸苷)、抗生素(如GENTAMYCIN TM药物)、微量元素(被定义为通常以微摩尔范围内的最终浓度存在的无机化合物)、葡萄糖或等效能量来源、白蛋白、胰岛素、转铁蛋白、硒、脂肪酸、2-巯基乙醇、硫醇甘油、脂质、氨基酸、L-谷氨酰胺、非必需氨基酸、维生素、生长因子、低分子量化合物、抗氧化剂、丙酮酸、细胞因子等等。还可包括所属领域的技术人员已知的适当浓度的任何其它必需补充剂。培养条件(如温度、pH等等)是先前用于细胞培养的培养条件，并且是一般熟练技术人员所显而易见的。
在本文所描述的细胞重编程方法中，细胞在补充有一种或多种本文所描述的重编程因子的基本培养基中培养。举例来说，在添加第一组重编程因子之前，基本培养基可含有DMEM/F12/Glutamax(吉毕科(GIBCO))、10％敲除血清替代(KnockOut Serum Replacement；KSR)(吉毕科)、1％NEAA(吉毕科)、10％FBS(吉毕科)以及0.1mM 2-巯基乙醇(吉毕科)。在另一实施例中，在添加第一组重编程因子之前，基本培养基可含有DMEM/F12/Glutamax(吉毕科)、0.075％牛血清白蛋白(BSA)(吉毕科)、1％NEAA(吉毕科)以及0.1mM 2-巯基乙醇(吉毕科)。对于另一实例，在添加第二组重编程因子之前，基本培养基可含有DMEM/F12/Glutamax(吉毕科)、10％敲除血清替代(KSR)(吉毕科)、 1％NEAA(吉毕科)以及0.1mM 2-巯基乙醇(吉毕科)。在某些实施例中，用于第二组重编程因子的基本培养基不含血清。所属领域的技术人员应了解，其它必需补充剂(如所描述的)可添加到培养基中。
对于培养温度来说，已证实在35.0℃或更高的温度下培养能促进细胞重编程。培养温度是不损害细胞的温度，如优选地35.0℃到42.0℃，或更优选地36.0℃到40.0℃，或更优选地37.0℃到39.0℃。
重编程因子是在与细胞接触时(例如，由细胞表达、转化到细胞中以便表达、外源地提供给细胞等)可以单独或与其它分子组合引起重编程的分子。重编程因子可从外源来源提供，例如通过添加到培养基中，并且可通过所属领域中已知的方法引入到细胞中，如通过与细胞进入肽偶合、蛋白质或核酸转染剂、脂质体转染、电穿孔、生物弹道粒子递送系统(biolistic particle delivery system)(基因枪(gene gun))、微注射等等。在某些实施例中，重编程因子添加到培养基中而不与任何其它组分偶合。
在某些实施例中，可用于将第一类型的细胞重编程为第二类型的细胞的第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子-β(TGFβ)抑制剂以及环状AMP诱导剂。在其它实施例中，第一组重编程因子进一步包含碱性成纤维细胞生长因子(bFGF)、DNA甲基转移酶抑制剂、组蛋白甲基转移酶抑制剂(例如，DOT1L抑制剂)、组蛋白脱乙酰基酶抑制剂、BMP4或其组合。
在某些实施例中，第一组重编程因子由GSK3抑制剂、TGFβ抑制剂以及环状AMP诱导剂组成。在某些实施例中，第一组重编程因子由GSK3抑制剂、TGFβ抑制剂、环状AMP诱导剂以及bFGF组成。在某些实施例中，第一组重编程因子由GSK3抑制剂、TGFβ抑制剂、环状AMP诱导剂、DNA甲基转移酶抑制剂、组蛋白脱乙酰基酶抑制剂以及BMP4组成。在某些实施例中，第一组重编程因子由GSK3抑制剂、TGFβ抑制剂、环状AMP诱导剂、DNA甲基转移酶抑制剂、组蛋白甲基转移酶抑制剂(例如，DOT1L抑制剂)以及组蛋白脱乙酰基酶抑制剂组成。
在某些实施例中，重编程因子有时可由其各自家族内的旁系同源物(paralog)在功能上置换。
如本文所用，术语“抑制剂”是指可使被靶向的表达产物(例如编码靶标的mRNA或靶标多肽)的表达和/或活性降低例如至少10％或更高(例如10％或更高、50％或更高、70％ 或更高、80％或更高、90％或更高、95％或更高或98％或更高)的试剂。可例如通过测量表达产物的水平和/或靶标的活性测定抑制剂的功效(例如其降低靶标水平和/或活性的能力)。用于测量给定mRNA和/或多肽的水平的方法是所属领域的技术人员已知的，例如RT-PCR可用于测定RNA水平，并且利用抗体的蛋白质印迹法可用于测定多肽水平。靶标活性可使用所属领域中已知并且在本文中描述的方法，例如转录活性分析测定。在一些实施例中，抑制剂可以是抑制性核酸；适体；抗体或其结合片段；或小分子。
GSK3(糖原合酶激酶3)是丝氨酸/苏氨酸蛋白激酶，在与糖原产生、凋亡、干细胞维持等等相关的许多信号路径中涉及。GSK3包括由不同基因编码并且在氨基酸水平上具有高同源性的同工型(GSK3α和GSK3β)。GSK3抑制剂的实例包括GSK3α抑制剂和GSK3β抑制剂。GSK3抑制剂的具体实例包括针对编码GSK3的基因的siRNA、抗GSK3抗体、CHIR98014(贝德福密理博(Milipore,Bedford))、CHIR99021(贝德福密理博)、坎帕罗酮(Kenpaullone)(贝德福密理博)、AR-AO144-18(圣克鲁兹的圣克鲁兹生物技术(Santa Cruz Biotechnology,Santa Cruz))、TDZD-8(美国剑桥艾博抗(Abcam,Cambridge,US))、SB216763(美国剑桥艾博抗)、BIO((2’Z,3’E)-6-溴靛玉红-3’-肟)(美国明尼阿波利斯安迪生物公司(R&D Systems,Minneapolis,US))、TWS-119(美国剑桥艾博抗)、SB415286(美国剑桥艾博抗)、Ro3303544(US6479490)LiCl、Li 2CO 3等等。所有这些都是市售的，或也可由所属领域的一般技术人员参照已知文献制备。
在某些实施例中，GSK3抑制剂选自由以下组成的组：CHIR99021、LiCl、Li 2CO 3以及BIO。在某些实施例中，GSK3抑制剂是CHIR99021。在某些实施例中，GSK3抑制剂是BIO。培养基中GSK3抑制剂的浓度根据将使用的抑制剂的种类适当地确定。在CHIR99021或BIO情况下，浓度一般是0.1-10μM，优选1-5μM，更优选约3μM。一种或多种类别的GSK3抑制剂可组合使用。
TGFβ(转化生长因子β)是属于转化生长因子超家族的多功能细胞因子。TGFβ包括三种不同哺乳动物同工型(TGFβ1到3，HGNC符号TGFBETA1、TGFBETA2、TGFBETA3)。TGFβ可由包括巨噬细胞的许多细胞类型分泌，呈潜伏形式，其中TGFβ与其它两种多肽(潜伏TGFβ结合蛋白(LTBP)和潜伏相关肽(LAP))复合。本发明中将使用的TGFβ抑制剂的来源不受特别限制，只要其可有效抑制TGFβ功能即可。TGFβ抑制剂是市售的，或也可由所属领域的一般技术人员参照已知文献制备。TGFβ抑制剂的具体实例包括针对编码GSK3的基因的siRNA、抗TGFβ抗体以及化学拮抗剂。
在某些实施例中，TGFβ抑制剂选自由以下组成的组：SB431542(英国布里斯托尔托克里斯生命科学(Tocris Bioscience,Bristol,UK))、Repsox(英国布里斯托尔托克里斯生命科学)、LDN193189(英国布里斯托尔托克里斯生命科学)以及曲尼司特(利喘平(Rizaben))。在某些实施例中，TGFβ抑制剂是SB431542。培养基中TGFβ抑制剂的浓度是根据将使用的抑制剂的种类适当地确定的。在SB431542情况下，浓度一般是0.1-20μM，优选1-10μM，更优选约5μM。在Repsox情况下，浓度一般是0.1-20μM，优选1-15μM，更优选约10μM。
如本文所用，术语“环状AMP诱导剂”是指在有效浓度下使细胞中cAMP的细胞内浓度提高至少2％，优选至少5％，更优选至少10％，最优选至少20％的任何化合物。用于测量细胞内cAMP水平的方法是所属领域的技术人员已知的。优选环状AMP诱导剂包括异丁基甲基黄嘌呤和毛喉素。毛喉素的浓度一般是1-20μM，优选5-15μM，更优选约10μM。
bFGF(碱性成纤维细胞生长因子)是体内固有存在的一种类型的蛋白质，且已知其可控制细胞生长和分化，并且在各种组织和器官中具有如血管生成、平滑肌细胞增殖、伤口治疗、组织修复、造血、神经细胞分化等等的功能。本发明中将使用的bFGF的由来不受特别限制，只要其对于重编程有效即可。bFGF是市售的，或也可由所属领域的一般技术人员参照已知文献制备。举例来说，其可基于已知碱基序列和氨基酸序列合成，例如氨基酸序列可根据NCBI登记号AAA52448.1(人)和AAA37621.1(小鼠)获得。培养基中bFGF的浓度一般是1-50ng/ml，优选约1-20ng/ml，更优选约10ng/ml或约20ng/ml。
组蛋白乙酰化是可逆的修饰，脱乙酰化由被称为组蛋白脱乙酰基酶(HDAC)的酶家族催化。HDAC抑制剂包括丙戊酸(VPA)、曲古霉素A(TSA)、伏立诺他(辛二酰苯胺异羟肟酸，SAHA，默克公司(Merck & Co.,Inc.))、缩酚酸肽(罗米地辛(Romidepsin)，FK-228，格洛斯特制药公司(Gloucester Pharmaceutical Inc.))、Trapoxin、Depudecin、FR901228(藤泽药品工业(Fujisawa Pharmaceuticals))以及丁酸盐。在某些实施例中，HDAC抑制剂是VPA。培养基中HDAC抑制剂的浓度根据将使用的抑制剂的种类适当地确定。在VPA情况下，浓度一般是1-2000μM，优选10-1000μM，更优选约500μM。
补充有第一组重编程因子的基本培养基每1、2或3天更新一次。
在某些实施例中，第一类型的细胞在第一组重编程因子存在下培养至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30天、1.5个月或2个月。在某些实施例中，第一类型的细胞在第一组重编程因子存在下培养不超过2个月、 1.5个月、30、25、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2或1天。在某些实施例中，第一类型的细胞在第一组重编程因子存在下培养1天到2个月、1天到1个月、1天到25天、1天到20天、1天到19天、1天到18天、1天到17天、1天到16天、5天到16天、7天到16天、8天到16天、9天到16天、10天到16天、11天到16天、12天到16天、1天到15天、1天到14天、1天到13天、1天到12天、2天到12天、3天到12天、4天到12天、5天到12天、6天到12天、7天到12天。
在一个方面，本公开提供了一种用于将第二类型的细胞重编程成第三类型的细胞的方法，其包含在第二组重编程因子存在下培养第二类型的细胞，其中第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
酪蛋白激酶1(CK1)是在大部分真核细胞类型中充当信号转导路径调节子的丝氨酸/苏氨酸选择性酶。CK1同工型在Wnt信号传导、昼夜节律、转录因子核质穿梭、DNA修复以及DNA转录中涉及。本发明中将使用的CK1抑制剂的由来不受特别限制，只要其对于重编程有效即可。CK1抑制剂是市售的，或也可由所属领域的一般技术人员参照已知文献制备。CK1抑制剂的具体实例包括针对编码CK1的基因的siRNA、抗CK1抗体以及化学拮抗剂。优选地，CK1抑制剂是CKI-7。培养基中CK1抑制剂的浓度根据将使用的抑制剂的种类适当地确定。在CKI-7情况下，浓度一般是0.1-20μM，优选1-10μM，更优选约5μM。酪蛋白激酶1的其它抑制剂包括PF 670462(美国明尼阿波利斯安迪生物公司)、D4476(美国明尼阿波利斯安迪生物公司)、(R)-CR8(美国明尼阿波利斯安迪生物公司)、(R)-DRF053二盐酸盐(美国明尼阿波利斯安迪生物公司)、TAK 715(美国明尼阿波利斯安迪生物公司)、PF 4800567盐酸盐(美国明尼阿波利斯安迪生物公司)、LH 846、CKI 7二盐酸盐(美国明尼阿波利斯安迪生物公司)、SR 3029(美国明尼阿波利斯安迪生物公司)、Epiblastin A(美国明尼阿波利斯安迪生物公司)、PF 5006739(美国明尼阿波利斯安迪生物公司)。
在某些实施例中，第二类型的细胞是外胚层细胞。在某些实施例中，外胚层细胞是神经嵴细胞(NC)或NCC样细胞。在某些实施例中，第二类型的细胞是NC或NCC样细胞。NC或NCC样细胞可例如使用如本文所描述或如WO/2010/096496中所描述的双重SMAD抑制剂从人胚胎干细胞(hES细胞)分化。NC或NCC样细胞可使用Wnt激动剂(例如Wnt3a和/或(2’Z,3’E)-6-溴靛玉红-3’-肟(BIO))与SMAD抑制剂(如SB431542和/或头蛋白(Noggin))的组合从hES细胞分化；参见Menendez等人,《美国国家科学院院刊 (PNAS)》2011年11月29日,第108卷第48期19240-19245。举例来说，已报道过使hES细胞与SB431542和(2’Z,3’E)-6-溴靛玉红-3’-肟(BIO)(在存在或不存在头蛋白的情况下)或Wnt3a和SB431542接触之后对NC或NCC样细胞的高效诱导。NC也可获自神经花环(rosette)培养，例如通过在MS5基质饲养细胞上培养hES细胞(参见Lee等人,《干细胞(Stem Cells)》25(8),1931-1939(2007)，其以全文引用的方式并入本文中)。NC也可获自大量组织，包括在发育胚胎中、神经管、坐骨神经、肠道以及背根神经节中；以及幼体和成体中、背根神经节、骨髓、皮肤、心脏、角膜、齿以及颈动脉体中。参见Nagoshi等人,《细胞生物化学杂志(Journal of Cellular Biochemistry)》107:1046-1052(2009)；Crane和Trainor,《细胞和发育生物学年度评论(Annu.Rev.Cell Dev.Biol.)》2006.22:267-86；以及Blum,《脑研究通报(Brain Research Bulletin)》83(2010)189-193，所述文献中的每一种以全文引用的方式并入本文中。在某些实施例中，NCC样细胞通过根据本申请重编程第一类型的细胞而获得。
在一些实施例中，在第二组重编程因子存在下培养第二类型的细胞，引起至少50％、55％、60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多细胞变为第三类型的细胞。
在某些实施例中，第三类型的细胞是外胚层细胞。在某些实施例中，第三类型的细胞是神经体细胞。在某些实施例中，第三类型的细胞是角膜内皮细胞样细胞(CEC样细胞)。
“角膜内皮细胞”或“CEC”一般是指在活生物体中排列在角膜后表面并且面向眼的前房的富线粒体细胞。为区分本文所提供的化学诱导的CEC(ciCEC)与原代CEC，所得CEC也被命名为角膜内皮细胞样细胞(CEC样细胞)。通过本文所公开的重编程方法获得的CEC样细胞可通过其展现如以下的内源性CEC属性中的一种或多种而被鉴别或识别：表达CEC标记、形成主要六角形的均一大小细胞单层的能力、形成“泄漏泵”(允许溶质和营养素从房水泄漏到角膜更浅表的层，并同时以相反方向从基质到房水主动泵送水)的能力。示例性CEC标记包括但不限于：Na +/K +ATP酶、紧密连接蛋白1(TJPl/ZO-1)、KLF13、AQP1、胶原蛋白VIII、SLC 16A3、CFTR、NBC1、CA2、AE2/SCL4A2、SCL16A1、CA12、CA4、FoxCl。举例来说，CEC通常表达胶原蛋白VIII、Na +K +ATP酶泵以及ZO-1，并且不表达vWF和CD31(后者存在于血管内皮细胞中)。另外，CEC可表达一种或多种角膜内皮泵标记(其包括AQP1、CA2、CA4、CA12、SCL14A2、SLC 16A1、SLC 16A3、SLC 16A7、CFTR、NHE1、ADC Y10、电压依赖性阴离子通道VDAC2和VDAC3、氯离子通道蛋白 CLCN2和CLC)、眼周神经嵴标记(其包括PITX2和FOXCl)和/或细胞粘附和基质蛋白(其包括闭合蛋白(Occludin)、连接蛋白(Connexin)43、9.3E抗原、胶原蛋白III、胶原蛋白IV、N钙粘蛋白、VE钙粘蛋白、E钙粘蛋白、β连环蛋白、层粘连蛋白α4、巢蛋白(Nidogen)-2以及轴突引导因子(Netrin)4)。举例来说，CEC可表达至少一种角膜内皮泵标记、至少一种眼周神经嵴标记以及至少一种细胞粘附和基质蛋白。
在一些实施例中，所得CEC样细胞展现与原代CEC表型一致的一种或多种生物标记。在一些实施例中，所得CEC样细胞表达紧密连接蛋白1(TJPl/ZO-1)、N-钙粘蛋白以及Na+/K+ATP酶。在一些实施例中，相对于产生CEC样细胞的NCC或NCC样细胞，CEC生物标记的表达提高超过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倍或更多。
标记可通过所属领域中已知的方法检测或测量。举例来说，由N-钙粘蛋白进行的粘附连接可通过检查蛋白质水平(例如，利用抗原抗体反应的方法)或基因水平(例如，利用RT-PCR的方法)上的表达而证实。细胞的Na +/K +-ATP酶泵送功能可例如根据《眼科研究与视觉科学(Investigative Ophthalmology & Visual Science)》,2010第51卷,第8期,3935-3942，和《当前眼研究(Current Eye Research)》,2009第34卷,347-354中所描述的方法并且使用尤斯室(Ussing chamber)测量。
在某些实施例中，第二组重编程因子进一步包含BMP4和/或DNA甲基转移酶抑制剂。
骨形态发生蛋白(BMP)是一组促进骨和软骨形成的生长因子。BMP与被称作骨形态发生蛋白受体(BMPR)的细胞表面上的特异性受体相互作用。通过BMPR的信号转导引起SMAD家族蛋白成员的动员。涉及BMP、BMPR以及Smad的信号传导路径在心脏、中枢神经系统和软骨发育以及出生后骨发育中很重要。其在胚胎发育期间对胚胎模式化和早期骨骼形成具有重要作用。因此，干扰BMP信号传导会影响发育胚胎的形体模式(body plan)。BMP抑制剂的实例包括但不限于DMH2(美国贝德福密理博)、德索莫啡(Dorsomorphin)(美国剑桥艾博抗)、LDN193189(英国布里斯托尔托克里斯生命科学)、DMH-1(英国布里斯托尔托克里斯生命科学)、K 02288(英国布里斯托尔托克里斯生命科学)以及ML 347(英国布里斯托尔托克里斯生命科学)。培养基中BMP抑制剂的浓度根据将使用的抑制剂的种类适当地确定，如0.1nM-10μM、0.1nM-5μM、0.1nM-2.5μM、0.1 nM-2μM、0.5nM-2μM、1nM-2μM、1nM-1.5μM M、1nM-1000nM、5nM-1000nM、10nM-1000nM、50nM-1000nM、50nM-500nM、50nM-200nM、100nM-200nM、100nM-150nM或100nM。
BMP4(骨形态发生蛋白4)是作为转化生长因子-β超家族的一部分的骨形态发生蛋白家族的成员。BMP4发现于腹缘区中和眼、心血以及听泡中的早期胚胎发育中。本发明中将使用的BMP4抑制剂的由来不受特别限制，只要其对于重编程有效即可。BMP4抑制剂是市售的，或也可由所属领域的一般技术人员参照已知文献制备。BMP4抑制剂的具体实例包括针对编码BMP4的基因的siRNA、脊索蛋白(Chordin)、头蛋白以及抗BMP4抗体。培养基中BMP4抑制剂的浓度根据将使用的抑制剂的种类适当地确定，如0.01-100μg/ml、0.01-50μg/ml、0.01-25μg/ml、0.01-10μg/ml、0.01-5μg/ml、0.01-1μg/ml、0.01-0.5μg/ml、0.01-0.1μg/ml、0.01-0.05μg/ml、0.05-10μg/ml、0.1-10μg/ml、0.1-5μg/ml、0.1-4μg/ml、0.1-3μg/ml或0.1-2μg/ml或10ng/ml。
DNMT(DNA甲基转移酶)家族酶可催化甲基到DNA的转移。DNA甲基化提供广泛多种生物功能。本发明中将使用的DNMT抑制剂的由来不受特别限制，只要其对于重编程有效即可。DNMT抑制剂是市售的，或也可由所属领域的一般技术人员参照已知文献制备。DNMT抑制剂的具体实例包括针对编码DNMT的基因的siRNA、抗DNMT抗体以及化学拮抗剂。
在某些实施例中，DNMT抑制剂选自由以下组成的组：地西他滨(decitabine)(英国布里斯托尔托克里斯生命科学)、5-氮杂胞苷(英国布里斯托尔托克里斯生命科学)、5-氮杂-dC(英国布里斯托尔托克里斯生命科学)以及RG108(英国布里斯托尔托克里斯生命科学)。培养基中DNMT抑制剂的浓度根据将使用的抑制剂的种类适当地确定，如0.1-100μM、0.1-50μM、0.1-25μM、0.1-10μM、0.5-10μM、1-10μM、5μM。
DOT1L(端粒沉默干扰子1样蛋白)是一类组蛋白甲基转移酶(HMT)，其催化染色质结构(例如端粒染色质)内组蛋白3的第37赖氨酸残基的甲基化。本发明中将使用的DOT1L抑制剂不受特别限制，只要其对于重编程有效即可。DOT1L抑制剂是市售的，或也可由所属领域的一般技术人员参照已知文献制备。DOT1L抑制剂的具体实例包括针对编码DOT1L的基因的siRNA、抗DOT1L抗体以及化学拮抗剂。在某些实施例中，DOT1L抑制剂是小分子DOT1L抑制剂。在不希望受理论束缚的情况下，已知DOT1L可使用腺苷甲硫氨酸(AdoMet)作为辅因子通过DOT1L上的AdoMet结合位点催化H3K27甲基化。 因此，可将AdoMet从其在DOT1L上的结合位点移开的模拟AdoMet分子结构的任何化学品作为DOT1L抑制剂在本公开的预期内。在某些实施例中，DOT1L抑制剂选自由以下组成的组：EPZ004777、EPZ5676(也称为皮诺司他(pinometostat))、SGC 0946以及SYC-522。
在某些实施例中，第二类型的细胞在第二组重编程因子存在下培养至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、25、30天、1.5个月或2个月。在某些实施例中，第二类型的细胞在第二组重编程因子存在下培养不超过2个月、1.5个月、30、25、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2或1天。在某些实施例中，第二类型的细胞在第二组重编程因子存在下培养1天到2个月、1天到1个月、1天到25天、1天到20天、1天到19天、1天到18天、1天到17天、1天到16天、5天到16天、7天到16天、8天到16天、9天到16天、10天到16天、11天到16天、12天到16天、1天到15天、1天到14天、1天到13天、1天到12天、2天到12天、3天到12天、4天到12天、5天到12天、6天到12天、7天到12天。
在一个方面，本公开提供了一种将第一类型的细胞重编程成第三类型的细胞的方法，其包含步骤(a)在第一组重编程因子存在下培养第一类型细胞，其中第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子(TGFβ)抑制剂以及环状AMP诱导剂，和步骤(b)在第二组重编程因子存在下培养从步骤(a)获得的细胞，其中第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
在一个方面，本公开提供了一种将第一类型的细胞重编程成第三类型的细胞的方法，其包含在第一组重编程因子和第二组重编程因子存在下培养第一类型的细胞，其中第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子(TGFβ)抑制剂以及环状AMP诱导剂，并且第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
在某些实施例中，第一组重编程因子进一步包含bFGF。在某些实施例中，第二组重编程因子进一步包含BMP4和/或DNA甲基转移酶抑制剂。
在一些实施例中，在第一组和第二组重编程因子存在下培养第一类型的细胞使至少50％、55％、60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多细胞变为第三类型的细胞。
在某些实施例中，方法进一步包含在开始步骤(b)之前洗涤从步骤(a)获得的细胞。在某些实施例中，步骤(a)与步骤(b)之间不存在洗涤步骤。
C.医药组合物和治疗方法
在一个方面，本公开提供了根据本文所提供的方法产生的NCC样细胞群。在某些实施例中，群中的至少50％、至少60％、至少70％、至少80％或至少90％是NCC样细胞。
在一个方面，本公开提供了一种用于产生患者特异性神经嵴细胞样细胞的方法。在一个实施例中，第一类型的细胞从罹患神经疾病的受试者获得。
在一个方面，本公开提供了根据本文所提供的方法产生的角膜内皮细胞样细胞(CEC样细胞)群。在某些实施例中，本公开的群中至少50％、至少60％、至少70％、至少80％或至少90％的细胞是CEC样细胞。
在一些实施例中，根据本文所提供的方法产生的NCC样细胞或CEC样细胞离体纯化。在一些实施例中，用于重编程的方法产生高纯度的NCC样细胞或CEC样细胞，并且不需要纯化。举例来说，在一些实施例中，本文所提供的重编程方法可得到包含至少50％、60％、70％、80％、90％、95％、98％、99％或更多NCC样细胞或CEC样细胞的细胞组合物。在一些实施例中，本文所提供的重编程方法产生低纯度或低于所需的纯度的NCC样细胞或CEC样细胞，并且需要纯化。在一些实施例中，NCC样细胞或CEC样细胞通过将NCC样细胞或CEC样细胞与组合物中的其它细胞大体上的分离来纯化。在CEC样细胞纯化的情况下，组合物中的其它细胞可包括未分化NCC样细胞和/或分化成非所需细胞谱系或表型的NCC样细胞。
在一个方面，本公开提供一种组合物，其包含根据本文所提供的方法产生的NCC样细胞或CEC样细胞。组合物可包括一种或多种药学上可接受的载剂和稀释剂。
术语“药学上可接受”指示指定的载剂、媒剂、稀释剂、赋形剂和/或盐一般在化学和/或物理上与构成配制物的其它成分相容，并且与其接受者生理相容。用于本文所公开的医药组合物的药学可接受载剂可包括例如药学上可接受的液体、凝胶或固体载剂、水性媒剂、非水性媒剂、抗菌剂、等渗剂、缓冲剂、抗氧化剂、麻醉剂、悬浮剂/分散剂、螯合(sequestering/chelating)剂、稀释剂、佐剂、赋形剂或无毒性辅助物质、所属领域中已知的其它组分或其各种组合。
本文所描述的组合物还可包括便于移植的组分。本文所描述的组合物可以是无热原或基本上无热原并且无病原体的，其中病原体包含细菌污染物、支原体污染物以及病毒。
包含本文所描述的NCC样细胞或CEC样细胞的组合物可进一步包含免疫抑制剂或免 疫耐受剂。
在另一方面，本公开涉及本文所提供的CEC样细胞的治疗用途。举例来说，本发明的CEC样细胞可在细胞疗法中作为用于治疗需要移植角膜内皮的疾病的移植物使用，所述疾病例如大泡性角膜病变、角膜水肿、角膜白斑等等。
在一个方面，本公开提供了一种治疗与功能失调或受损角膜内皮细胞相关的疾病或病状的方法，其包含向有需要的受试者施用有效量的本文所提供的CEC样细胞或包含CEC样细胞的组合物。
在一些实施例中，施用角膜内皮细胞诱导眼治愈过程。在一些实施例中，施用角膜内皮细胞补充病变组织。在一些实施例中，施用角膜内皮细胞对受损或病变眼组织具有再生效应。
施用途径可包括任何适合的手段，包括但不限于局部施加到眼部位、注射到眼部位中、在眼部位处移植等等。在一些实施例中，所选择的特定施用模式将取决于特定治疗、患者的疾病状态或病状、向受试者施用的其它药物或治疗剂的性质或施用途径等。在一些实施例中，角膜内皮细胞可以单次剂量或在所选择的时间间隔内以多次剂量向受试者施用，例如以滴定剂量。当施用多次剂量时，剂量可彼此隔开例如一周、一个月、一年或十年。也可在施用细胞之前、期间或之后施用一种或多种生长因子、激素、白细胞介素、细胞因子、小分子或其它细胞，以使所述细胞进一步偏向特定细胞类型。
如本文所用，术语“有效量”泛指当向患者施用以便治疗疾病时足以实现针对疾病的此类治疗的化合物或细胞的量。有效量可以是防治(prophylaxis)有效量和/或预防有效量。有效量可以是可有效减少体征/症状的量、可有效防止体征/症状发生、降低体征/症状发生的严重程度、消除体征/症状发生、减慢体征/症状发生的发展、防止体征/症状发生的发展和/或实现对体征/症状发生的防治的量。“有效量”可根据疾病和其严重程度以及待治疗的患者的年龄、体重、病史、易感性和先前存在的病状而不同。出于本发明的目的，术语“有效量”与“治疗有效量”同义。
如本文所用，“治疗(treating/treatment)”涵盖对受试者，例如人、动物或哺乳动物的本文所描述的疾病或医学病状的治疗，并且包括：(i)抑制疾病或病症，即，遏制其发展；(ii)缓解疾病或病症，即，引起病症消退；(iii)减慢病症进展；和/或(iv)抑制、缓解疾病或医学病状的一种或多种症状或减慢疾病或医学病状的一种或多种症状的进展。
如本文所用，术语“受试者”不限于特定物种或样品类型。举例来说，术语“受试者”可以指患者，并且常常指人患者。然而，这一术语不限于人并且因此涵盖多种哺乳动物物种，如非人兽医哺乳动物，如狗、猫、兔、猪、啮齿动物、马或猴。
本文所提供的CEC样细胞是细胞团，如通过浓缩和过滤获得的团块和其类似物，并且其类似物用作本发明的药剂。此外，也有可能向药剂中添加保护剂，如甘油、DMSO(二甲亚砜)、丙二醇、乙酰胺等等，并且冷冻保存混合物。为了更安全地利用药剂，药剂可经历处于引起病理蛋白变性的条件下的处理，如热处理、辐射处理等等，同时保留角膜内皮细胞的功能。
在一些实施例中，CEC样细胞可与手术组合施用。在一些实施例中，手术可以是德斯密氏剥除联合内皮角膜移植(Descemet's stripping with endothelial keratoplasty；DSEK)，其包括去除德斯密氏膜和角膜内皮，并且后续移植供体组织。替代地，手术可以是穿透性角膜移植(penetrating keratoplasty；PKP)，其中整个角膜被去除并且替换。其它手术可包括板层角膜移植(lamellar keratoplasty)、德斯密氏膜内皮角膜移植(Descemet's Membrane Endothelial Keratoplasty；DMEK)、DSAEK以及DLEK。
“与功能失调或受损角膜内皮细胞相关的疾病或病状”包括适合于通过施用CEC样细胞治疗的任何疾病或病状，包括受试者的CEC数目减少或死亡、密度降低或以其它方式变为功能失调的疾病。影响角膜内皮的原发疾病包括富克氏营养不良、虹膜角膜内皮综合征、后部多态性营养不良以及先天性遗传性内皮营养不良。有效治疗可包括替换角膜内皮的继发疾病或病状包括年龄相关性黄斑变性(AMD)、视网膜色素变性、青光眼、角膜营养不良、隐形眼镜使用、白内障手术以及角膜移植中的晚期内皮衰竭。角膜内皮细胞疾病另外包括对角膜的任何损伤，例如由化学刺激引起的损伤，因以下导致的损伤：隐形眼镜使用、反应或敏感(例如，对隐形眼镜护理液、化妆品、滴眼液、药剂、烟气等)、刮擦、刮伤、擦伤、挫伤、眼中异物(例如，砂或粉尘)或暴露于紫外光(来自例如日光、日光灯、雪反射、水反射或弧焊或其它暴露)。在某些实施例中，与功能失调或受损角膜内皮细胞相关的疾病或病状导致受试者视力丧失。在某些实施例中，受试者的视力丧失是永久性或不可逆的。
在某些实施例中，角膜内皮细胞与受试者免疫相容(例如同种异体或自体)。
在某些实施例中，治疗方法可进一步包含向所述受试者施用免疫抑制剂或免疫耐受剂。免疫抑制剂或免疫耐受剂可以以足以降低对所述CEC样细胞的排斥反应风险的量施 用。
免疫抑制剂或免疫耐受剂可包含以下中的一种或多种：抗淋巴细胞球蛋白(ALG)多克隆抗体、抗胸腺细胞球蛋白(ATG)多克隆抗体、硫唑嘌呤(azathioprine)、
Figure PCTCN2020141524-appb-000001
(抗lL-2Ra受体抗体)、环孢素(cyclosporin)(环孢素A)、
Figure PCTCN2020141524-appb-000002
(抗IL-2Ra受体抗体)、依维莫司(everolimus)、霉酚酸(mycophenolic acid)、
Figure PCTCN2020141524-appb-000003
(抗CD20抗体)、西罗莫司(sirolimus)、他克莫司(tacrolimus)、霉酚酸酯以及皮质类固醇。
免疫抑制剂可给药至少约1、2、4、5、6、7、8、9或10mg/kg。当使用免疫抑制剂时，其可全身或局部施用，并且其可在CEC样细胞施用之前施用、与CEC样细胞伴随施用或在CEC样细胞施用之后施用。免疫抑制性疗法可在细胞施用之后持续数周、数月、数年或无限期。举例来说，患者可在CEC样细胞施用之后施用5mg/kg环孢素持续6周。
在一个方面，本公开提供了本文所提供的CEC样细胞或组合物的用途，其用于制造用于治疗与功能失调或受损角膜内皮细胞相关的疾病或病状的药剂。
在一个方面，本公开提供了一种治疗与功能失调或受损角膜内皮细胞相关的疾病或病状的方法，其包含向有需要的受试者施用有效量的本文所提供的NCC样细胞或包含NCC样细胞的组合物。
在一个方面，本公开提供了本文所提供的NCC样细胞或组合物的用途，其用于制造用于治疗与功能失调或受损角膜内皮细胞相关的疾病或病状的药剂。
本文所提供的NCC样细胞可用于治疗神经疾病。
如本文所用的“神经疾病”被定义为神经系统的病症，并且包括涉及中枢神经系统(大脑、脑干和小脑)、周围神经系统(包括脑神经)以及自主神经系统(其部分位于中枢和周围神经系统中)的病症。特别地，神经疾病包括其中神经嵴细胞或雪旺细胞功能受损、改变或破坏的任何疾病。与雪旺细胞有关的神经疾病的实例是脱髓鞘疾病、多发性硬化、脊髓病、实验性变态反应性脑脊髓炎(Experimental allergic encephalomyelitis；EAE)、急性播散性脑脊髓炎(acute disseminated encephalomyelitis；ADEM)、感染后或接种后脑脊髓炎、周围神经病变、雪旺细胞瘤病(Schwannomatosis)、夏-马-图三氏病(Charcot-Marie-Tooth disease)、吉兰-巴雷综合征(Guillain-Barre Syndrome)、慢性炎症性脱髓鞘性多发性神经根神经病(Chronic inflammatory demyelinating polyradiculoneuropathy；CIDP)。
本公开还提供了用于药物发现和/或药物筛选的方法。还提供了用于药物发现和/或药物筛选的分析。在某些实施例中，这些方法包括向NCC样细胞或CEC样细胞施用药物候选物，并且检测细胞对药物候选物的反应。检测反应可鉴别药物候选物是否具有合适特性(例如，毒性或治疗有效)。在一些实施例中，方法可用于确定在药物候选物存在下的细胞健康和活力。在一些实施例中，方法可用于测试药物候选物的毒性。在一些实施例中，方法可用于评定在药物候选物存在下细胞群表型的变化。
在一个方面，本公开提供了一种使用NCC样细胞以筛选逆转、抑制或预防神经疾病或药剂(例如糖尿病药剂)的神经副作用的药物的方法。
D.试剂盒
在一个方面，本公开提供了一种用于将第一类型的细胞重编程成第二类型的细胞的试剂盒，其中试剂盒包含第一组重编程因子，并且第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子(TGFβ)抑制剂以及环状AMP诱导剂。
在一个方面，本公开提供了一种用于将第二类型的细胞重编程成第三类型的细胞的试剂盒，其中试剂盒包含第二组重编程因子，并且第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
在一个方面，本公开提供了一种用于将第一类型的细胞重编程成第三类型的细胞的试剂盒，其中试剂盒包含第一组重编程因子和第二组重编程因子，其中第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、TGFβ抑制剂以及环状AMP诱导剂，并且第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
试剂盒可进一步包含使用说明书，和将试剂盒中的组分中的每一种组分分隔开来的包装。
本说明书中所引用的所有出版物和专利均以全文引用的方式并入本文中。
实例
实例1：材料和方法
1.动物
所有动物实验均经中国温州的温州医科大学动物伦理委员会(Animal Ethics Committee of Wenzhou Medical University,Wenzhou,China)批准。Oct4-GFP转基因等位基因携载小鼠 (CBA/CaJ×C57BL/6J)来自杰克逊实验室(Jackson Laboratory)；Wnt1-cre和Fsp1-Cre小鼠来自杰克逊实验室(BALB/c-Tg(S100a4-cre)1Egn/YunkJ)；ROSA26-tdTomato小鼠来自杰克逊实验室(Gt(ROSA)26Sortm14(CAG-tdTomato)Hze)。129Sv/Jae和C57BL/6小鼠来自上海维通利华实验动物技术有限公司(Beijing Vital River Laboratory)。Wnt1-Cre/ROSA26 tdTomato和Fsp1-Cre/ROSA26 tdTomato小鼠分别通过将Wnt1-Cre和Fsp1-Cre小鼠与ROSA26-tdTomato小鼠杂交获得(参见图4A)。新西兰白兔(New Zealand white rabbit)来自中国苏州的昭衍(苏州)新药研究中心有限公司(JOINN Laboratories(Suzhou)Inc.,Suzhou,China)。所有动物根据视觉与眼科研究协会(Association for Research in Vision and Ophthalmology，ARVO)眼科和视觉研究中使用动物的声明(Statement for the Use of Animals in Ophthalmic and Vision Research)进行处理。所有动物在具有12小时暗/光循环的稳定条件(21℃±2℃)下关养。
2.细胞培养
如先前所描述，原代小鼠胚胎成纤维细胞(MEF)从胚胎第13.5天(E13.5)的小鼠胚胎分离(Liu,C.,Hu,X.,Li,Y.,Lu,W.,Li,W.,Cao,N.,Zhu,S.,Cheng,J.,Ding,S.和Zhang,M.(2019).《通过化学方法将小鼠成纤维细胞转化成少突胶质细胞祖细胞样细胞(Conversion of mouse fibroblasts into oligodendrocyte progenitor-like cells through a chemical approach)》.《分子细胞生物学杂志(Journal of molecular cell biology)》；Wang,H.,Cao,N.,Spencer,C.I.,Nie,B.,Ma,T.,Xu,T.,Zhang,Y.,Wang,X.,Srivastava,D.和Ding,S.(2014).《小分子使得能够用单一因子Oct4进行小鼠成纤维细胞的心脏重编程(Small molecules enable cardiac reprogramming of mouse fibroblasts with a single factor,Oct4)》.《细胞报道(Cell reports)》6,951-960)。简单来说，将胚胎的头、尾、肢体以及内脏组织小心地取出且弃去。将其余组织切片成小片，通过0.25％胰蛋白酶/EDTA(吉毕科)胰蛋白酶化并且接种到10-cm培养皿上。将成纤维细胞在含有补充有10％胎牛血清(FBS，吉毕科)、2mMGlutaMAX(吉毕科)、0.1mM非必需氨基酸(西格玛)、100单位/毫升青霉素以及100mg/mL链霉素(吉毕科)的DMEM(吉毕科)的成纤维细胞培养基中培养。所有成纤维细胞扩增两代并且随后用于进一步实验。为制备Wnt1 -MEF，将衍生自Wnt1-Cre/ROSA26 tdTomato小鼠胚胎的成纤维细胞针对tdTomato -细胞通过FACS进行分选。
小鼠原代角膜内皮细胞(CEC)(CP-M179)和培养基(CM-M179)购自普诺赛生命科技有限公司(Procell Life Science & Technology Co.,Ltd)(中国武汉)。将小鼠原代CEC 在含有10％FBS(吉毕科)、0.1mM非必需氨基酸(西格玛)、2mMGlutaMAX(吉毕科)以及1％青霉素链霉素(吉毕科)的DMEM中培养。将mESC维持在ESC培养基中，ESC培养基由含10％FBS(吉毕科)、LIF、0.1mM非必需氨基酸(西格玛)、2mMGlutaMAX(吉毕科)、1％青霉素链霉素(吉毕科)、0.1mM 2-巯基乙醇(吉毕科)、CHIR99021(3mM)以及PD0325901(1mM)的DMEM构成。
人胚胎成纤维细胞(HEF)、人新生成纤维细胞(HNF)以及成人成纤维细胞(HAF)购自ScienCell Research Laboratories。人脐带间充质干细胞(MSC)由安徽中盛溯源生物科技有限公司(Nuwacell Ltd.)(中国合肥)友情提供。将所述细胞维持在含有补充有10％FBS(吉毕科)、1％GlutaMAX(吉毕科)以及1％NEAA(吉毕科)的DMEM(吉毕科)的成纤维细胞培养基中。人脱落肾上皮细胞(UC)来源于来自正常人的100-300mL尿液样品，如先前报道Zhou,T.等人(2012).《从尿液样品生成人诱导多能干细胞(Generation of human induced pluripotent stem cells from urine samples)》.《自然实验手册》7,2080-2089)。
3.用以产生tdMEF的FACS分选
为制备tdMEF，如先前所描述将所得成纤维细胞针对tdTomato +/p75 －细胞通过FACS分选。将原代MEF从具有Fsp1-Cre/Rosa26 tdTomato(Fsp1-Cre小鼠×Rosa26 tdTomato小鼠)的遗传背景的E13.5小鼠胚胎分离。将处于第2代的MEF用0.25％胰蛋白酶在37℃下解离5分钟，并且用MEF培养基中和。将那些MEF用针对p75的特异性抗体染色，并且经历针对tdTomato +/p75 －细胞的FACS分选。在FACS分选期间，将基质胶涂布的24孔培养板在37℃下预温热至少30分钟，随后接种tdMEF。在FACS分选之后，立即在37℃下在5％CO2和20％O2中将tdMEF以15,000个细胞/孔接种到预温热的基质胶涂布的24孔培养板中的补充有1μM噻唑维(Thiazovivin)(Tzv)的MEF培养基中持续5小时，以使MEF连接到板。5小时后，将培养基改变为不含Tzv的MEF培养基，并且将tdMEFs在37℃下在5％CO 2中培养过夜。
4.小分子化合物和文库
小分子从西格玛获得，包括GSK3b抑制剂CHIR99021(SML1046)、TGFb抑制剂SB431542(S4317)、DNA甲基化抑制剂5-氮杂-dC(A3656)、环状AMP诱导剂毛喉素(F6886)以及CKI-7(C0742)。bFGF从派普泰克(Peprotech)获得。DOT1L抑制剂EPZ004777(S7353)和ROCK抑制剂Y-27632(S1049)从赛莱克(Selleck)获得。
5.免疫细胞化学
为进一步研究典型神经嵴(NC)细胞标记的表达，对重编程MEF进行固定、免疫染色以及分析。简单来说，将细胞用1×PBS洗涤一次并且用4％多聚甲醛在室温下固定10分钟，接着用0.2％Triton X-100的1×PBS溶液透性化10分钟，并且用7.5％BSA封闭至少1小时。将所有一抗(primary antibody)在7.5％BSA中稀释，并且在4℃下进行培育过夜。将细胞在室温下用1×PBS洗涤10分钟，持续五次。二抗(secondary antibody)Alexa-488、Alexa-555以及Alexa-647购自英杰公司(Invitrogen)，其稀释到7.5％BSA中，并且在室温下持续1小时培育，接着用1×PBS进行五次10分钟洗涤。细胞核用DAPI染色。这一研究中使用的抗体在表2中列出。
表1：这一研究中使用的抗体
Figure PCTCN2020141524-appb-000004
Figure PCTCN2020141524-appb-000005
6.统计分析
所有实验独立进行至少三次。结果表示为平均值±SD。数据通过未配对双尾学生t检验(unpaired two-tailed Student’s t-test)分析以便比较两个组，并且通过单因素方差分析(one-way ANOVA)以及图基检验(Tukey’s test)或邓尼特多重比较检验(Dunnett’s multiple comparisons test)分析以便比较多个组。所有分析使用SPSS Statistics 19.0软件进行。P值<0.05则视为显著。
7.细胞周期分析
使用阿库酶(Accutase)溶液将细胞小心地解离成单细胞悬浮液，用PBS洗涤两次，并且随后用冷的70％乙醇固定过夜。将固定的细胞用PBS洗涤两次，接着在37℃下进行核糖核酸酶(100μg/mL，西格玛)处理和碘化丙啶(50μg/mL，西格玛)染色30分钟。使用FACSCanto II(BD公司(Becton Dickinson))分析大致1×10 6个细胞以确定细胞周期分布模式。使用ModFit 4.1(Verity Software House)分析处于细胞周期的G1、S以及G2/M期的细胞的百分比。
8.移植
将所有重量为2.0-2.5kg的兔用氯胺酮盐酸盐(60mg/kg)和甲苯噻嗪(10mg/kg，德国慕尼黑拜耳(Bayer,Munich,Germany))肌肉内麻醉。将兔分成2个组(每组n＝10)，并且其右眼用于这一实验。在对操作部位进行消毒和无菌覆盖之后，用开缝刀进行12点钟居中的6mm角膜切割，并且将粘弹性剂(Healon；Amersham Pharmacia Biotech AB)输注到前房中。在角膜表面用记号笔(西班牙马德里(Madrid,Spain)Devon Industries公司)划线之后，用30号针头(日本东京泰尔茂(Terumo,Tokyo,Japan))在角膜中心产生用于德斯密氏膜去除术(descemetorhexis)的6.0mm直径圆形孔口，并且从眼的前房去除德斯密氏膜。如先前所描述的，用泪道冲洗器(山东威高(Shandong Weigao))从德斯密氏膜机械刮擦角膜内皮。使用0.25％胰蛋白酶-EDTA将Fsp-ciCEC解离，以1×10 7个细胞/毫升密度在基本培养基中再悬浮并且在冰上保持。前房用PBS洗涤三次。在这一程序之后，使用26号针头来将悬浮于100μl含有100μMROCK抑制剂Y-27632(赛莱克)的基本DMEM中的1×10 6个培养的ciCEC注射到右眼的前房中。其后，冷冻(单独的冷冻损伤)、CE眼向下(冷冻损伤和CE注射)以及球体眼向下(冷冻损伤和球体注射)组中的兔保持眼向下位置24小时，使得细胞可通过重力而连接，并且球体眼向上组中的兔在深度麻醉下保持眼向上位置24小时。每个手术眼通过外部检验，一周检查两次或三次，并且在注射之后第3、7、14和28天拍摄照片。在手术之后第0.5、1、3、7、14、21和28天，用超声波角膜测厚仪(ultrasound pachymeter)测量中央角膜厚度，并且用气压眼压计(pneumatic tonometer)测量眼内压。取三次读数的平均值。
9.细胞增殖分析
通过Click-iT TM乙炔基脱氧尿苷(EdU)Alexa Fluor 488成像试剂盒(英杰公司)，根据制造商的说明书测定单独的分化培养基或单独的M5中培养的ciCEC的增殖速率。简单来说，将传代CEC以5×10 3个细胞/平方厘米的较低密度接种到载玻片上，并且培养24小时。
10.透射电子显微术(TEM)分析
对于TEM分析，将细胞在2.5％EM级戊二醛(赛维尔生物(Servicebio))中在4℃下固定2-4小时，用0.1M磷酸盐缓冲液(pH 7.4)洗涤，在1％四氧化锇中、在4℃下后固定处理2-4小时，洗涤并且随后在乙醇连续液(50-100％)中脱水到100％丙酮中的最终冲洗，接着在1:1丙酮/Pon 812(SPI)中培育2小时，并且在1:2丙酮/Pon 812中培育过夜。 将样品包埋于Pon 812中，在60℃下聚合48小时，并且随后用金刚石刀(Daitome)切片(60-80nm)。将切片用2％醋酸铀染色，接着用柠檬酸铅染色，并且使用HT7700透射电子显微镜(日立(HITACHI))可视化。
11.核型分析
用0.1μg/mL秋水仙胺(吉毕科)在37℃下将细胞处理2小时，胰蛋白酶化，再悬浮并且在0.075M氯化钾中在37℃下培育15分钟，用3:1甲醇:乙酸固定，并且随后滴到载玻片上以使染色体分散。染色体通过吉姆萨(赛维尔生物)染色可视化。
12.RNA测序和分析
根据制造商的说明书，用TRIzol试剂将每个样品的总RNA分离并且使用RNeasy 23微型试剂盒(凯杰)纯化。使用NanoDrop 2000、安捷伦2100生物分析仪和安捷伦RNA 6000Nano试剂盒评定RNA质量和数量。通过安诺优达基因科技(Annoroad Gene Technology)进行RNA文库构筑和RNA测序。遵循制造商的建议，使用用于Illumina24的NEB Next Ultra RNA文库制备试剂盒(NEB)生成测序文库，并且使用HiSeq PE簇试剂盒v4-cBot-HS(启迪公司)进行文库成簇。簇生成之后，在启迪公司平台上对文库进行测序并且生成150bp双端读段。在BMKCloud(http://www.biocloud.net/)上进行初始数据分析。
13.FACS细胞测量术
对于MEF制备，将具有所需基因型的成纤维细胞在MEF培养基中培养，直到其达到超过80％的融合度。将细胞用1×PBS洗涤两次并且用0.25％胰蛋白酶在37℃下处理5分钟。收获之后，使细胞通过70-μm过滤器，用预冷却缓冲液(1×PBS，1.5％FBS，0.5％BSA)洗涤两次，并且再悬浮于所述缓冲液中。在推荐浓度下，将细胞与FITC缀合P75抗体(艾博抗)或同型对照(BD)在冰上一起培育30分钟，或室温培育45分钟，接着用FACS缓冲液洗涤六次。随后使细胞再悬浮于FACS缓冲液中并且用BD FACSAria II分选。
实例2：从成纤维细胞生成ciNCC和ciCEC
1.培养基制备
阶段I培养基制备
含有DMEM/F12/Glutamax(吉毕科)、10％KSR(吉毕科)、10％FBS(吉毕科)、1％NEAA(吉毕科)、0.1mM 2-巯基乙醇(吉毕科)的基础培养基，其补充有小分子Repsox (10μM)、Chir99021(10μM)、毛喉素(10μM)、以及bFGF(10ng/ml)。
在添加第一组重编程因子分子之前，基础培养基还可含有DMEM/F12/Glutamax(吉毕科)、0.075％牛血清白蛋白(BSA)(吉毕科)、1％NEAA(吉毕科)以及0.1mM 2-巯基乙醇(吉毕科)，并且在细胞重编程方面产生可比的或更好的效果(数据未展示)。
此外，小分子Repsox(10μM)、Chir99021(10μM)以及毛喉素(10μM)的组合可达成类似诱导效应(数据未展示)。将培养基振荡30分钟以确保完全溶解。
阶段II培养基制备
DMEM/F12/Glutamax(吉毕科)、10％KSR(吉毕科)、1％NEAA(吉毕科)、0.1mM 2-巯基乙醇(吉毕科)，补充有5μM SB431542和5μM CKI-7。
2.从成纤维细胞化学诱导ciCEC
为研究小分子是否可以化学方式将成纤维细胞重编程成角膜内皮细胞，我们设计了两步骤方法以直接将小鼠成纤维细胞重编程成神经嵴细胞，并且分化成角膜内皮细胞(图1A)。在Wnt1-Cre-Rosa Tomato小鼠中，tdTomato在Wnt1基因的控制下在神经嵴(NC)中如实表达。因此，从处于胚胎期E13.5的Wnt1-Cre/ROSA26 tdTomato小鼠分离MEF，并且神经嵴群体用tdTomato表达标记。我们进行荧光激活细胞分选(FACS)来收集tdTomato -群体，以排除任何神经嵴或祖细胞。这些MEF对于典型NC标记(包括Sox10、P75、Hnk1以及AP2α)呈阴性(数据未展示)。另外，这些MEF也对典型神经干细胞(NSC)标记(包括Sox2、Pax6以及Olig2)呈阴性(数据未展示)。尚未报道过用于通过小分子从成纤维细胞诱导ciNCC的方法。为研究小分子是否可以化学方式将成纤维细胞重编程成ciNCC，我们选择了超过20种小分子作为我们的潜在候选分子，这些候选分子用于基于靶向表观遗传修饰和调节神经嵴发育信号传导进行神经嵴谱系重编程。候选小分子聚焦于包含三种小分子的三个主要类别的重编程因子：(1)TGF-β信号传导抑制剂，如Repsox(R)，其抑制中胚层和内胚层特化；(2)GSK3抑制剂，如CHIR99021(C)，其促进神经发育，以及(3)环状AMP诱导剂，如毛喉素(F)。这三种化合物与碱性成纤维细胞生长因子(bFGF)组合作为化学成分确定的培养基(以下称为RCF)，以将小鼠胚胎成纤维细胞(MEF)重编程成ciNCC。
简单来说，将MEF以50,000个细胞/孔接种于6孔培养板中。过夜培养之后，在RCF化学成分确定的重编程培养基中处理MEF。在RCF培养基中，许多具有清晰边缘的小的 致密细胞簇在十二天内快速出现(图1B)。为鉴别足以将小鼠成纤维细胞重编程成化学诱导的神经嵴细胞(ciNCC)的RCF的组合，使用Wnt1 －MEF来观察tdTomato表达。结果展示RCF化合物可将MEF重编程成tdTomato阳性神经嵴细胞样细胞(图1D)。为进一步研究典型NC标记的表达，对RCF处理的MEF进行固定、免疫染色以及分析。这些细胞表达HNK1、P75以及AP2α(图1F)。那些成纤维细胞起源的、高增殖性并且可自我再生的Sox10阳性细胞其后被称作ciNCC。
在重编程过程期间，许多具有清晰边缘的小的致密细胞簇在7天内快速出现(图1B和图1C)。经Repsox、Chir99021、毛喉素以及bFGF处理之后，我们在10天的处理之后观察到了tdTomato +细胞(图1D和图1E)。第12天时，观察到神经嵴细胞(NC)的标记(HNK1、P75以及AP2α)(参见图1F)。然而，在没有bFGF的情况下，小分子Repsox(10μM)、Chir99021(10μM)以及毛喉素(10μM)的组合可达成类似诱导效应(数据未展示)。
在用SB431542和CKI-7分化培养基诱导ciNCC14天之后，ciNCC分化成角膜内皮细胞样细胞(ciCEC)(参见图3C)。图3A展示了第14天时通过免疫荧光染色检测到的阶段II诱导的ciCEC的标记(Na +-K +ATP酶、AQP1、波形蛋白、N-钙粘蛋白层粘连蛋白以及AQP1)。还在透射电子显微镜下观察到角膜内皮细胞的紧密连接。
为避免可能出现的起始MEF中神经嵴细胞(ciNCC)的污染，我们进行谱系追踪实验以追踪ciNCC和ciCEC的起源(图4B)。在Fsp1-Cre-RosaTomato小鼠中，tdTomato在Fsp1基因的控制下在ciNCC和ciCEC中如实表达。在Fsp1-ciNCC中通过免疫细胞化学观察到ciNCC标记P75、Hnk1、AP2α以及SOX10的表达(参见图4C)。图4D展示Fsp1-Cre:R26RtdTomato MEF的代表性形态变化和从这些MEF诱导的ciCEC。
使用如上文所描述的相同程序，通过化学因子诱导人细胞(人胚胎皮肤成纤维细胞(HEF)、人新生成纤维细胞(HNF)、成人成纤维细胞(HAF)、人脐带间充质基质细胞(MSC)以及尿液细胞(UC))。优选地，在阶段I中添加的化学因子是Repsox(10μM)、Chir99021(10μM)、毛喉素(10μM)、5-氮杂胞苷(5μM)、VPA(500μM)以及BMP4(10ng/ml)。阶段II中添加的化学因子是5μM SB431542和5μM CKI-7，这与上文所提及的一致。衍生自以上细胞的诱导的ciNCC和ciCEC展示于图5中。
实例3：对实例2中所获得的ciNCC和ciCEC的表征
1.1 RNA制备和RT-PCR
为了证实NC基因(包括Sox10、P75、Pax3以及Msx1)的表达。使用RNeasy Plus微型试剂盒(凯杰)提取总RNA。简单来说，1μg总RNA用于与iScript cDNA合成试剂盒(伯乐(Bio-Rad))进行逆转录反应，并且将所得cDNA在H 2O中稀释五次以供PCR使用。对于半定量PCR，1μl 1/5稀释的cDNA用作PCR程序的模板：95℃5分钟，和35个95℃30秒、60℃30秒以及72℃30秒循环，接着72℃10分钟。遵循FAST SYBR Green主混合物(ABI)的方案进行定量PCR。所有PCR重复进行三次，并且将个别基因的表达相对于Gapdh的表达归一化。引物序列在表2中列出。
表2：用于qRT-PCR的引物
Figure PCTCN2020141524-appb-000006
1.2 RNA测序和分析管线
为了验证ciNCC的转录组分析，进行RNA测序。用ovation RNA测序系统v2试剂盒(NuGEN)制备RNA测序文库。将总RNA(50ng)逆转录以用随机六聚体和聚T嵌合引物的组合合成第一链cDNA。随后将RNA模板通过加热部分降解，并且使用DNA聚合酶合成第二链cDNA。随后使用单引物等温扩增(SPIA)扩增双链DNA。SPIA是一个线性cDNA扩增过程，该过程中核糖核酸酶H在双链DNA的5′端处降解DNA/RNA异源双链体中的RNA，之后SPIA引物结合到cDNA并且聚合酶在引物的3′端处通过置换现有正向链开始复制。随后使用随机六聚体来线性扩增第二链cDNA。最后，使用Ultralow V2文库 试剂盒(NuGEN)制得来自SPIA扩增cDNA的文库。将RNA测序文库通过生物分析仪分析并且通过QPCR(KAPA)定量。在HiSeq 2500仪器(启迪公司)上，将三个RNA测序文库汇集在双端100bp测序的每个通路上。使用Fastq-mcf对读段的已知接头和低质量区进行调整。使用FastQC(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)评定样品QC。使用Tophat 2.0.13(Kim,D.等人2011.《TopHat 2：在插入、缺失和基因融合存在下准确比对转录组(TopHat 2:accurate alignment of transcriptomes in the presence of insertions,deletions and gene fusions)》.《基因组生物学(Genome Biology)》14.)将读段与小鼠参考组装体mm9比对。通过Subread featureCounts(Liao等人2014.《FeatureCounts：一种用于序列读段指配到基因组特征的高效通用程序(FeatureCounts:an efficient general-purpose program for assigning sequence reads to genomic features)》.《生物信息学(Bioinformatics)》30,923-930.)，使用mm9的Ensembl基因注解，联合基因水平表达。在处理差异表达之前，我们使用RUVSeq以调节批次效应(David Risso等人,2014,《使用对对照基因或样品的因子分析使RNA测序数据归一化(Normalization of RNA-seq data using factor analysis of control genes or samples)》.《自然·生物技术(nature Biotechnology)》32,896-902)。不具有CPM(每百万计数)值在.5与5000之间的至少两个样品的基因被滤除。差异表达P值使用edgeR(Robinson,M.D.等人,2010.《edgeR：一种用于数字基因表达数据的差异表达分析的Bioconductor包(edgeR:a Bioconductor package for differential expression analysis of digital gene expression data)》.《生物信息学》26,139-140)计算。使用内置R函数“p.adjust”以用本亚明-霍赫贝格方法(Benjamini-Hochberg method)(Benjamini和Hochberg,(1995).《控制错误发现率：实用且强大的多重测试方法(Controlling the false discovery rate:a practical and powerful approach to multiple testing)》.《皇家统计学会杂志B系列(Journal of the Royal Statistical Society Series B)》57,289-300)计算FDR。通过DAVID Bioinformatics Resources 6.7或ToppGene完成基因本体分析。通过Cluster 3.0生成热图，并且通过Java Treeview查看。转录组的分析展现了这些细胞与小鼠NC非常类似，但与MEF不同。
1.3蛋白质印迹法
为了证实NC特定标记和CEC特定标记的表达，进行蛋白质印迹法。简单来说，在磷酸酶抑制剂(赛信通技术公司(Cell Signaling Technology))存在下收集细胞，并且将其与同等体积的含DTT的2×SDS-PAGE样品缓冲液混合。将样品煮沸并且通过离心机澄清。 在电泳之后，将蛋白质转移到PVDF膜上。通过将膜在丽春红(Ponceau)染色剂中短暂染色确定转移的效率。将膜用5％BSA在室温下封闭至少1小时，并且与在5％BSA的TBST溶液中稀释的所需抗体一起在4℃下培育过夜。将膜用TBST洗涤五次(每次10分钟)，随后与在TBSA中稀释的HRP缀合二抗一起在室温下培育1小时。用TBST洗涤五次之后，通过ECL外加检测试剂盒使印迹显色。对于将成纤维细胞重编程成ciNCC和ciCEC，这些结果共同展现化学混合物具有稳健且普遍的效果(数据未展示)。
2.ciNCC的维持和分化潜能
对于在通过FACS分选纯化之后维持ciNCC，将细胞在神经嵴培养基中的聚D-赖氨酸/层粘连蛋白涂布的培养板上培养，所述培养基由补充有1×N2、1×B27、10μg/ml bFGF、10μg/ml EGF以及10ng/ml BMP4的神经基质培养基(吉毕科)组成。
为表征扩增ciNCC的分化潜能，首先将其在分化条件下培养。通过戒断FGF2/EGF并且暴露于BDNF、GDNF、NGF以及二丁酰基环状AMP(dbcAMP)诱导神经元分化，产生外周神经元。通过免疫染色，ciNCC在第5代时产生Tuj1+和外周蛋白+神经元，并且这种分化潜能在长期培养期间很好地维持(图2A)。如通过S100b和GFAP+表达评定的雪旺细胞分化在CNTF、神经调节蛋白(neuregulin)1b以及dbcAMP存在下被诱导。
对于黑色素细胞分化，在7天的诱导处理之后，将ciNCC在黑色素细胞分化培养基(EBM2基础培养基，5％(v/v)FBS、100ng/ml SCF(生命技术)、200nM内皮素3(EDN3，西格玛)、50ng/ml WNT1、10ng/ml FGF2、5μg/ml胰岛素、1pM霍乱毒素、10nM 12-O-四-癸酰佛波醇-13-乙酸酯(TPA，西格玛)以及10μM SB431542(西格玛))中培养。在3-4周的处理之后，可观察到黑色素细胞(图2A)。
为检查朝向间充质谱系的分化潜能，我们在MSC培养条件下培养ciNCC。在这些条件下，出现具有间充质形态和标记表达(CD105+)的细胞(数据未展示)。再培养一个月之后，大部分细胞表达CD105和一组间充质干细胞所特有的表面标记。我们使用已建立的间充质干细胞分化方案，并且展示由ciNCC生成的间充质前体细胞能够进行脂肪细胞、软骨以及成骨(图2B)分化。
3.对ciNCC源的神经元的功能表征
为了确定ciNCC源的神经元的功能，我们检查其电生理特性。在分化条件下，与大鼠皮层神经元共培养10-20天之后，从ciNCC源的神经元获取全细胞膜片钳记录(Whole-cell  patch-clamp recording)。将培养的神经元转移到奥林巴斯(Olympus)BX51WI正置显微镜上的灌注台上，并且用含有以下物质(以mM为单位)的人工脑脊液(aCSF)在室温下以2.5毫升/分钟灌注：NaCl 119、KCl 2.5、NaH2PO4 1、NaHCO3 26.2和葡萄糖11、CaCl2 2.5以及MgSO4 1.3，并且渗透压调整到300osm L-1。在整个记录过程中将aCSF用95％O 2和5％CO 2鼓泡。数据通过MultiClamp 700B放大器(Axon Instruments)汇集、在2kHz下过滤并且在10kHz下数字化。离线分析在Igor Pro(Wavemetrics)中进行。动作电位在电流钳全细胞配置下记录。用于电流钳实验的电极液含有以下物质(以mM为单位)：葡萄糖酸钾123、KCl 10、MgCl 2 1、HEPES 10、EGTA 1、CaCl 2 0.1、K 2ATP 1、Na 4GTP 0.2以及葡萄糖4，pH用KOH调整到7.2。膜电位保持在大约-70mV并且-20到50pA的阶跃电流以10-pA间隔注入。全细胞电流在-70mV的保制电位下记录，-70mV到+30mV范围内的电压阶跃以20-mV增量递送。自发突触后电流以全细胞电压钳模式记录。用于突触电流记录的全细胞电极液含有以下物质(以mM为单位)：CsCl 135、HEPES 10、EGTA 1、Mg-ATP 4、Na 4GTP 0.4以及QX-314 10，pH 7.4。为对兴奋性和抑制性电流进行采样，将1mM谷氨酸和100μM GABA在10p.s.i.下喷气100ms，并且保持电压分别为-70mV和0mV。正如期望，其生成通过以电流钳模式对膜进行去极化诱发的重复序列的动作电位，表明ciNCC源神经元具有正常神经元活性(数据未展示)。
4.细胞增殖分析
为了测量ciCEC的增殖能力，进行细胞增殖分析。通过Click-iT TM乙炔基脱氧尿苷(EdU)Alexa Fluor 488成像试剂盒(英杰公司/生命技术)，按照制造商的说明书测定在阶段II培养基中培养的ciCEC的增殖速率。简单来说，将传代CEC以5×10 3个细胞/平方厘米的较低密度接种到FNC涂布的载玻片上，并且培养24小时。结果表明这些细胞具有高增殖能力(数据未展示)。
5.通过畸胎瘤形成测试验证的ciCEC安全性
为评估肿瘤发生的潜在风险，将1×10 6个ciCEC皮下注射到NOD-SCID小鼠中，并且畸胎瘤在4到8周形成。为了生成嵌合体，将ciCEC注射到ICR囊胚中并且移植到假孕ICR雌性中。通过使F2小鼠与ICR小鼠交配确定所得嵌合小鼠的生殖系传递。所有动物实验经广州生物医药与健康研究院动物保护和使用委员会(Animal Care and Use Committee of the Guangzhou Institutes of Biomedicine and Health)批准且根据其准则进行。在用ciCEC移植之后6个月内无肿瘤形成，而在用mESC移植的接受者中在4-8周之后出现大畸胎瘤(数 据未展示)。这表明ciCEC具有极小(如果存在的话)致瘤潜能。此外，在体外30次连续传代期间ciCEC维持正常核型(数据未展示)。为更好地理解体内分化，将ciCEC移植到NOD/SCID小鼠的眼中。在4-8周之后，用ciCEC移植之后，6个月内移植的ciCEC没有形成肿瘤(数据未展示)。
6.ciCEC向兔受损眼中的移植
为评定ciCEC是否具有体内移植和扩增的能力，我们将其移植到通过从德斯密氏膜机械刮擦的角膜内皮诱导为大泡性角膜病变的兔模型中。将兔分成2个组(每组n＝10)，并且右眼用于这一实验。将ciCEC注射到前房中(图6B第1图像)。每个接受者接受1×10 6个(低剂量)ciCEC或2×10 6个(高剂量)ciCEC。未处理的正常的兔(图6B第4图像)和仅注射PBS(图6B第3图像)的兔用作对照。
在对操作部位进行消毒和无菌覆盖之后，用开缝刀(中国上海Alcon Surgical)进行12点钟居中的6mm巩膜角膜切割，并且将粘弹性剂(Healon；Amersham Pharmacia Biotech AB)输注到前房中。在角膜表面用记号笔(西班牙马德里Devon Industries公司)划线之后，用30号针头(日本东京泰尔茂)在角膜中心产生用于德斯密氏膜去除术(descemetorhexis)的6.0mm直径圆形孔口，并且从眼的前房去除德斯密氏膜。用泪道冲洗器(山东威高)从德斯密氏膜机械刮擦角膜内皮。将Fsp-ciCEC使用0.25％胰蛋白酶-EDTA解离，并且以1×10 7个细胞/毫升密度在PBS中再悬浮且在冰上保持。前房用PBS洗涤三次。
在这一程序之后，使用26号针头来将悬浮于100μl含有100μMROCK抑制剂Y-27632(其促进细胞粘附到植入部位)(ROCK抑制剂，赛莱克)的基本DMEM中的1×10 6个ciCEC注射到右眼的前房中(图6B，第1图像)。
在所述程序之后，将兔以俯卧位置放置2-3小时。每个手术眼通过外部检验一周检查两次或三次，并且在手术之后第1、3、5、7、14、21、35和42天拍摄照片。裂隙灯照片展示注射之后CEC样细胞组(ciCEC组)角膜的透明度显著改良，并且可看见瞳孔和虹膜纹理。仅约7天之后，角膜变得明显透明，而对照组中角膜混浊和基质水肿仍然严重(图6B从左起分别第1图像和第3图像)。Visante OCT也展示在注射CEC样细胞之后角膜厚度快速减小(图6C，每个图像分别对应于上方图6B的图像)。共聚焦显微镜图像证实ciCEC组中德斯密氏膜上多角形细胞的全覆盖(图6D，第1图像。图6D中的每个图像分别对应于上方图6B和图6C的图像)。可发现ciCEC组中第1、3、5、7、14、21、35和42天时 的平均角膜厚度显著小于未处理对照组中的厚度(图6E)。裂隙灯照片展示在第1、3、7、14、21和28天时ciCEC组角膜的透明度显著改良(图7)。
实例4：根据另一实施例从成纤维细胞生成ciNCC和ciCEC
M6重编程培养基制备
基础培养基含有敲除DMEM(吉毕科)、10％KSR(吉毕科)、10％FBS(吉毕科)、1％NEAA(吉毕科)以及0.1mM 2-巯基乙醇(吉毕科)，并补充有补充有小分子Chir99021(3μM)、SB431542(5μM)、毛喉素(10μM)、VPA(500mM)、EPZ004777(5μM)以及5-氮杂-dC(0.5μM)。将培养基振荡30分钟以确保所有组分完全溶解。
分化培养基制备
DMEM/F12/GlutaMAX(吉毕科)、10％KSR(吉毕科)、1％NEAA(吉毕科)以及0.1mM 2-巯基乙醇(吉毕科)补充有SB431542(5μM)和CKI-7(5μM)。
从成纤维细胞化学转化NCC
将MEF以5×10 4个细胞/孔接种在6孔组织培养板上成纤维细胞培养基中。将培养板用纤维连接蛋白或层粘连蛋白预涂布超过两个小时。在过夜培养之后，将培养基用M6化学培养基更换，其每2天更新。NCC样细胞在第3-5天出现且增加。在7-10天的诱导之后，进行FACS分选以收集Wnt1 +细胞。
从小鼠ciNCC化学诱导CEC样细胞
在第12-16天，将M6化学培养基用SB431542和CKI-7培养基替换，其每2天更新。内皮样细胞簇在第8天出现且增加，并且Oct4-GFP阳性簇在第12天出现。CEC样细胞早在第20天出现。在第30天到第35天期间，对CEC样细胞集落进行计数或进一步检测。
ciNCC分化
将大致5×10 3个ciNCC接种在含有NCSC培养基的24孔培养板中的昆布氨酸涂布的玻璃盖玻片上，持续第一个24小时。在24小时后，使细胞经历分化条件。对于神经元分化，将培养基切换为神经元分化培养基(不含bFGF和EGF的NCC培养基，其添加200μM抗坏血酸、2μM db-cAMP、25ng/ml BDNF、25ng/ml NT3以及50ng/ml GDNF)。每2-3天更换一半的培养基。到分化之后第10天到第20天分析特异性神经元标记。为分化成少突胶质细胞，在5μM维甲酸和200ng/ml Shh存在下，将细胞培养1天并且在20ng/ml  PDGF-AA、20ng/ml bFGF以及200ng/ml SHH存在下培养3-5天；随后，将其在含有40ng/ml T3、200ng/ml Shh、1nM LDN193189、5mM db-cAMP以及10ng/ml NT3的分化培养基中培养8-12天。培养基每隔一天更新一次。对于星形胶质细胞分化，将50ng/ml BMP4添加到分化培养基中持续8-12天，并且培养基每隔一天更换。
结果
鉴于角膜内皮起源于NCC，我们设计了使用小分子将小鼠成纤维细胞重编程成CEC样细胞的两步骤方法。第一步骤是引导小鼠胚胎成纤维细胞(MEF)化学重编程成ciNCC。为筛选具有将成纤维细胞转化为ciNCC的潜能的小分子，我们进行谱系追踪实验以追踪转化过程并且从起始MEF处排除任何NCC或祖细胞(图8A，图15A)。Wnt1-Cre转基因小鼠已被证实为NC发育的谱系追踪报道模型。在Wnt1-Cre/ROSA26tdTomato小鼠中，tdTomato蛋白在NCC中如实表达。因此，从处于E13.5的Wnt1-Cre/ROSA26tdTomato小鼠分离出MEF。因为NCC群体标记有tdTomato，所以我们进行荧光激活细胞分选(FACS)来收集tdTomato -群体以排除任何NCC或祖细胞(纯化细胞在下文中称为Wnt1-tdTomato -MEF；图15B)。我们证实Wnt1-tdTomato -MEF还对其它NCC标记(包括Sox10、P75、Pax3、Hnk1以及AP2α)呈阴性(图15C、D)。另外，这些Wnt1-tdTomato -MEF对典型NSC标记(包括Sox2、Pax6以及巢蛋白)呈阴性(数据未展示)。
据报道，一些用于增强重编程的小分子可促进谱系重编程。为从MEF生成ciNCC，基于(1)对NCC发育的表观遗传调节和信号传导调节和(2)增强的神经谱系重编程，我们选择一组16种小分子作为候选物。最初，用于NC谱系重编程的小分子候选物成功地聚焦于包含Chir99021(GSK3抑制剂)、SB431542(TGF-β抑制剂)以及毛喉素(cAMP激动剂)的三个类别的重编程因子(图8B)。对于后续筛选和优化，我们发现VPA(HDAC抑制剂)、EPZ004777(DOT1L抑制剂)以及5-氮杂-dC(DNA甲基化抑制剂)进一步增强对Wnt1-tdTomato +细胞的诱导(图8C)。在这一研究中，我们使用化学成分确定的培养基与上述六种小分子的混合物的组合(以下称为M6)来将MEF重编程成ciNCC(图8D)。在使用M6培养基处理的情况下，早在第3天就观察到各个细胞中Wnt1-tdTomato的表达(图8E)。M6重编程培养基以3.97％有效地诱导Wnt1-tdTomato +细胞(图8F)。在第5-7天，在M6重编程培养基中观察到诱导的Wnt1-tdTomato +集落(图8G、图15E)。这些Wnt1-tdTomato +细胞和集落具有类似于原代NCC(pNCC)的典型形态。大致第12天时，小集落中Wnt1-tdTomato +细胞数显著增加(图8H)。尽管Wnt1-tdTomato +NCC生成的效 率仅与使用TFs31转化人成纤维细胞一样，但这一研究中仅2-5％的细胞呈Wnt1-tdTomato阳性。这些结果在不同批次MEF(n＝8)中可再现，并且具有不同遗传背景(C57BL/6、129×C57BL/6以及129)的MEF也可通过M6条件转化成ciNCC。综合起来，这些结果表明M6可将MEF重编程成ciNCC。
实例5：对实例4和额外研究中所获得的ciNCC和ciCEC的表征
对转化的ciNCC的表征
ciNCC的重编程过程具有两个阶段：初始阶段(第0-7天)和扩增阶段(第7-12天)。第一阶段是在重编程培养基中培养MEF以启动表观遗传激活。出现少数具有清晰边缘的小的NCC样簇。第二阶段是在确定的小分子培养基中培养表观遗传激活的细胞。大部分簇在第二阶段中扩增并且逐渐生长。
为获得ciNCC，我们进行FACS以收集Wnt1-tdTomato +细胞。已形成的ciNCC在含有N2、B27、bFGF以及EGF的常规NCC扩增培养基中连续繁殖。形态上，处于P3的M6诱导的细胞在单层培养中维持典型NCC特征(图9A)。传代之后，ciNCC变得形态均匀。为进一步表征M6诱导的Wnt1-tdTomato +细胞，我们试图检查其基因表达。我们的结果展示ciNCC表达多种NCC标记，包括P75、HNK1、AP2α以及巢蛋白(图9B)。此外，我们测试ciNCC是否具有朝向外周神经元、雪旺细胞和其它等等的分化潜能。对于ciNCC的分化，将这些细胞在不同谱系分化培养基中培养。在培养2-4周之后，通过由免疫染色评定标记表达，以检查分化细胞。值得注意的是，ciNCC还可产生表达神经元(包括Tuj1和外周蛋白)特异标记的细胞(图9C)。对于黑色素细胞分化，我们在诱导2-3周之后观察到黑色素细胞(图9C)。我们的免疫染色结果展示ciNCC可分化为雪旺细胞。诱导的雪旺细胞为GFAP +和S100β +细胞(图9D)。这些ciNCC的进一步体外分化产生间充质谱系，得到典型间充质细胞形态。我们的结果展示这些ciNCC源间充质细胞可产生成骨细胞、成脂细胞以及软骨细胞(图9E)。共同地，这些数据表明我们的ciNCC可被诱导以朝向周围神经系统谱系和间充质谱系分化。
移植到动物模型中展现ciCEC功能
为评估ciCEC是否具有体内再生角膜内皮的能力，我们将其移植到良好确立的兔模型中，该模型通过从德斯密氏膜机械刮擦角膜内皮而具有大泡性角膜病变。先前研究发现注射补充有ROCK抑制剂的人pCEC恢复了内皮功能。根据这一研究，将ciCEC与ROCK 抑制剂(Y27636)组合注射到眼的前房中(图14A)。每个接受者接受1×10 6个ciCEC。对侧眼(正常)和未处理的眼(PBS注射)用作实验对照。与未处理的眼相比，在ciCEC移植之后，角膜水肿减少早得多。与展示角膜透明度未改变的未处理的眼相比，我们注意到经移植眼的角膜的透明度在移植之后逐渐提高(图14B、图19A)。7天之后，经移植眼的角膜变得透明，而未处理的眼中角膜混浊和基质水肿仍然不良。裂隙灯检查结果展示注射之后经移植眼的角膜的透明度也显著改良，并且可清楚观察到瞳孔和虹膜纹理(图14C、图19A)。
接下来，我们研究经移植眼中的ciCEC存活。术后第28天剜出眼球以评估移植的ciCEC细胞。荧光显微法检查证实tdTomato-标记的移植细胞的存在。免疫组织化学展现ZO-1表达，表明移植的ciCEC细胞的泵功能(图14D、图19C)。对眼前段的Visante光学相干断层扫描(OCT)也展示在ciCEC注射之后角膜厚度减小(图14E)。共聚焦显微镜证实ciCEC移植的疾病模型中的德斯密氏膜上的多角形内皮细胞的全覆盖，而这在未处理的模型中由于其严重角膜混浊而无法检测到(图14G)。在放大下，在第28天时，移植的ciCEC以单层紧密粘附于角膜后表面，而未处理的模型中的德斯密氏膜裸露并且不具有可检测的CEC。
ciCEC注射之后4周内存在角膜厚度的快速减小，接着为在接下来2周内更渐进的减小(图14F、图19D)。在未处理的组中，在整个42天观察过程中平均角膜厚度是大致1200μm。相比之下，其在移植的组中快速减小，显著小于未处理的组中的平均角膜厚度。我们观察到在手术后第14天(P<0.01)、第21、28、35和42天(P<0.001)时，ciCEC移植的组中的平均角膜厚度显著小于对照组中的平均角膜厚度，表明角膜水肿明显逆转。这些结果强有力地表明ciCEC移植在角膜后表面再填充且自组织，并且具有使角膜内皮再生的能力。
小分子促进ciNCC诱导成ciCEC
为从ciNCC生成小鼠CEC样细胞，我们还基于小分子在体外CEC器官发生和维持中的重要性寻求一组小分子作为候选物。在初步筛选中，我们发现SB431542和CKI-7能够从ciNCC诱导CEC样细胞。这两种化合物其后包括于分化条件中(图10A)。为确定ciNCC是否可进一步分化成成熟CEC，我们用含有这两种小分子的分化培养基处理ciNCC。在这一分化培养基中培养7-15天之后，簇外或簇内的小群体显示具有均匀且多角形形态的典型紧密聚集体(图10B)。我们还观察到这些集落快速扩增，并且到第12-15天时，小簇合并 到较大的簇中(图10B)。这些CEC样细胞快速生长并且具有强增殖能力。ciNCC诱导的CEC样细胞形成六角形和五角形细胞的单层。为证实CEC样细胞衍生自ciNCC，我们用含有5μM SB431542和5μM CKI-7的CEC分化培养基分化tdTomato +ciNCC。这些tdTomato +ciNCC还可随后经诱导以朝向CEC样细胞分化(图10C)。Na +/K +-ATP酶、AQP1、波形蛋白、ZO-1以及N-钙粘蛋白表达进一步通过免疫荧光染色验证(图10D)。在这一研究中，我们通过Dil标记的乙酰化低密度脂蛋白(Dil-Ac-LDL)摄入鉴别CEC样细胞的功能(图10E)。通过RNA测序进行的全局基因表达分析展示CEC样细胞与原代CEC(pCEC)共享一类似基因表达谱，但这一谱不同于初始MEF的谱(图10F)。通过TEM观察到CEC样细胞中存在紧密连接(图10G)。为进一步监测重编程过程，我们通过使用qRT-PCR进一步证实一组NCC和CEC标记在不同时间时的表达。到第12天时，在细胞中检测到NCC基因的稳健表达，包括Hnk1、P75、Sox10、Sox9、Pax3以及Ap2(图11A)。另外，还通过qRT-PCR检测到CEC基因(如Slc4a1c、Col8a1、Na +/K +-ATP酶、Aqp1以及N-钙粘蛋白)的基因激活的类似动力学(图11B)。已知的在pCEC中富集的基因在ciCEC中极大地上调。为验证从成纤维细胞到CEC的转化过程，我们通过使用RNA测序分析转录组(图11C)。ciCEC中16种CEC特征基因的表达显著上调，与pCEC一致。然而成纤维细胞特征基因明显下调。值得注意的是，在诱导过程期间，一组NCC标记基因最初上调并且随后下调。主成分分析展现M6处理的细胞不同于初始MEF，这表明化学重编程引起显著转录变化(图11D)。这些结果表明CEC样细胞获得CEC属性。共同地，这些数据表明SB431542与CKI-7的组合有效促进ciNCC培养中10-15天内CEC的生成。那些成纤维细胞起源的CEC样细胞其后被称作化学诱导的CEC样细胞(ciCEC)。
用以证实从成纤维细胞诱导ciCEC的谱系追踪
为证实用于基于小分子的重编程的初始成纤维细胞的起源，我们寻求纯化成纤维细胞特异性蛋白1(Fsp1)-tdTomato阳性成纤维细胞的遗传谱系追踪策略(图12A)。Fsp1-Cre作为用于谱系追踪的特异性成纤维细胞标记已被证实；因此，使Fsp1-Cre小鼠与ROSA26tdTomato小鼠杂交。MEF从处于E13.5的转基因小鼠(Fsp1-Cre/ROSA26tdTomato)分离，并且成纤维细胞特异性表达tdTomato；这些细胞在下文中命名为tdMEF(图16A)。为避免可能出现的NCC祖细胞对MEF的污染，我们进行FACS以收集tdTomato +/p75 -群体(图12B)。这些tdMEF对所有NCC标记(包括P75、HNK1、Sox10以及AP2α)呈阴性(图12C)。
随后，将这些tdMEF用上述M6培养基诱导。在ciNCC诱导期间观察到表达tdTomato的上皮簇(图12D、图16B)。我们使Fsp1-tdTomato +ciNCC传代并且将其在NCC扩增培养基中培养以供进一步实验(2周诱导之后)。免疫荧光分析证实这些Fsp1-tdTomato +ciNCC对NC标记P75、HNK1、SOX10以及AP2α呈阳性，这表明Fsp1-tdTomato +ciNCC的集落已朝向CEC样细胞分化(图16C)。此外，Fsp1-tdTomato +ciNCC可分化成tdTomato +ciCEC(图16D)。通过免疫染色，我们发现分化的CEC样细胞共表达Na +/K +-ATP酶、AQP1、层粘连蛋白、ZO-1、Na +/K +-ATP酶以及tdTomato(图12E)。值得注意的是，所有这些ciCEC还表达tdTomato，这展现了从成纤维细胞的转化。这些结果清楚地证实ciNCC和ciCEC通过两步谱系重编程从成纤维细胞转化。
通过化学品生成的ciCEC绕过诱导多能干细胞(iPSC)阶段
因为我们的谱系重编程的机制可能类似于以化学方式重编程的iPSC，所以我们试图评定ciCEC是否经历iPSC阶段。我们进行从MEF进行化学iPSC重编程与ciCEC诱导之间的比较，该MEF衍生自携带Oct4启动子驱动GFP(OG2)报导子。我们观察到M6处理的这些MEF形态上经历了特征性的间充质向上皮的转化(MET)，并且临近第6天时小的细胞集落逐渐出现(图17A)。这些细胞集落表达NCC标记Sox10(图17B)。相比之下，我们基于我们的方法在从MEF到ciCEC的整个过程期间未观察到任何Oct4-GFP阳性细胞(图17C，3D)。此外，在体外十次连续传代期间，ciCEC维持正常核型(图17E)。为评估肿瘤发生的潜在风险，将总共5×10 6个ciCEC和2×10 6个小鼠胚胎干细胞(ESC)皮下移植到NOD/SCID小鼠中。值得注意的是，在用ciCEC移植之后，6个月内无肿瘤形成，而在用ESC移植的小鼠中在4-8周之后出现大畸胎瘤(数据未展示)。这一结果表明ciCEC不具有致瘤潜能。为更好地理解其体内分化，我们将ciCEC移植到NOD/SCID小鼠的眼的前房中。在4-8周之后，移植之后6个月内，移植的ciCEC不形成肿瘤。这些结果展现我们的方法可直接将MEF重编程为ciNCC并且最终重编程为ciCEC，同时绕过iPSC阶段。
ciCEC体外长期扩增能力
已证明的是，体外维持培养的CEC的形态和正常生理功能具有挑战性。我们旨在测试是否可从成纤维细胞生成大量功能性ciCEC，以使得能够大规模施加ciCEC。基于我们的观察结果，即在含SB431542(5μM)和CKI-7(5μM)的培养基中培养的ciCEC是小的六角形细胞(无上皮向间充质转化样细胞)(图18A)，我们假设SB431542和CKI-7将促进体外ciCEC生长。我们通过以1:6比率连续传代ciCEC来评估ciCEC体外长期扩增能力， 并且发现表型在P3与P30之间是类似的(图13A、B)。这一结果展示SB431542和CKI-7强有力地促进ciCEC扩增。在基于小分子的培养基中，这些ciCEC保持自身为具有六角形形态的同质细胞群至少30代(P30)。另外，我们成功地克隆源性地培养这些ciCEC到10代并且展现一致形态。我们的免疫染色结果展示处于P3的ciCEC中的Ki67阳性细胞比率高于处于P3的pCEC(图18B)。ciCEC具有高度增殖性，因为处于P1、P3和P6的这些细胞的24.6％、37.8％、48.1％展示了EdU并入(图18C)。利用碘化丙啶(PI)染色的FACS分析的结果展示细胞周期分布(G0/G1、S以及G2/M期)对于处于P3的ciCEC为46.30％、45.11％以及8.59％，并且对于处于P3的pCEC为67.30％、21.50％以及11.20％(图18D)。其快速扩增成大的同质集落，群体倍增时间为22.3±3.7小时(图13C)。引起关注的是，在处于P2到P10的ciCEC的表面上发现大“液泡样”结构(图13D)。当细胞连续繁殖时，在P20时这些液泡样结构消失。值得注意的是，处于P30的ciCEC还表达典型CEC标记，包括Na+/K+-ATP酶、AQP1以及ZO-1(图13E)。当通过成像分析迁移到由划痕产生的间隙中的能力时，相比于pCEC，在含SB431542和CKI-7的培养基中培养的处于不同传代数的ciCEC展示更强的增殖和迁移能力(图18E、F)。共同地，这些结果展现SB431542和CKI-7对于ciCEC体外长期扩增具有稳健且普遍的效果。

Claims (46)

  1. 一种用于将第一类型的细胞重编程成第二类型的细胞的方法，其包含在第一组重编程因子存在下培养所述第一类型的细胞，其中所述第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子-β(TGFβ)抑制剂以及环状AMP诱导剂。
  2. 根据权利要求1所述的方法，其中所述第一组重编程因子进一步包含碱性成纤维细胞生长因子(bFGF)、DNA甲基转移酶抑制剂、DOT1L抑制剂、组蛋白脱乙酰基酶抑制剂、BMP4或其组合。
  3. 根据权利要求1所述的方法，其中所述第一组重编程因子由以下组成：(a)GSK3抑制剂、TGFβ抑制剂以及环状AMP诱导剂，(b)GSK3抑制剂、TGFβ抑制剂、环状AMP诱导剂以及bFGF，或(c)GSK3抑制剂、TGFβ抑制剂、环状AMP诱导剂、DNA甲基转移酶抑制剂、DOT1L抑制剂以及组蛋白脱乙酰基酶抑制剂。
  4. 根据权利要求1所述的方法，其中所述GSK3抑制剂选自由以下组成的组：CHIR99021、LiCl、Li 2CO 3、BIO((2’Z,3’E)-6-溴靛玉红-3’-肟)、TD114-2、坎帕罗酮、TWS119、CBM1078、SB216763、3F8(TOCRIS)、AR-A014418、FRATide、靛玉红3’-肟以及L803。
  5. 根据权利要求1所述的方法，其中所述TGFβ抑制剂选自由以下组成的组：SB431542、Repsox、616452、LDN193189、A8301、GW788388、SD208、SB525334、LY364947、D4476、SB505124以及曲尼司特。
  6. 根据权利要求1所述的方法，其中所述环状AMP诱导剂是毛喉素、IBMX、咯利普兰、8BrcAMP、前列腺素E2(PGE2)、NKH477、二丁酰一环腺苷酸(DBcAMP)、Sp-8-Br-cAMPs。
  7. 根据权利要求2所述的方法，其中所述DNA甲基转移酶抑制剂选自由以下组成的组：5-氮杂-dC、5-氮杂胞苷以及RG108。
  8. 根据权利要求2所述的方法，其中所述DOT1L抑制剂是EPZ004777。
  9. 根据权利要求2所述的方法，其中所述组蛋白脱乙酰基酶抑制剂选自由以下组成的组：丙戊酸(VPA)、曲古霉素A(TSA)、伏立诺他、缩酚酸肽、Trapoxin、Depudecin、FR901228以及丁酸盐。
  10. 根据权利要求1所述的方法，其中所述第一类型的细胞是体细胞。
  11. 根据权利要求10所述的方法，其中所述体细胞衍生自中胚层、外胚层或内胚层。
  12. 根据权利要求10所述的方法，其中所述体细胞是成纤维细胞。
  13. 根据权利要求12所述的方法，其中所述成纤维细胞选自由以下组成的组：小鼠胚胎成纤维细胞(MEF)、小鼠尾尖成纤维细胞(TTF)、人胚胎成纤维细胞(HEF)、人新生成纤维细胞(HNF)、成人成纤维细胞(HAF)、人包皮成纤维细胞(HFF)以及其混合物。
  14. 根据权利要求10所述的方法，其中所述体细胞是人脱落肾上皮细胞。
  15. 根据权利要求1所述的方法，其中所述第一类型的细胞是干细胞。
  16. 根据权利要求15所述的方法，其中所述干细胞选自由以下组成的组：人脐带间充质干细胞、人胚胎干细胞以及诱导多能干细胞(iPSC)。
  17. 根据权利要求1所述的方法，其中所述第二类型的细胞是干细胞。
  18. 根据权利要求17所述的方法，其中所述干细胞是神经嵴细胞样细胞(NCC样细胞)。
  19. 根据权利要求18所述的方法，其中所述NCC样细胞呈P75、Hnk1、AP2α以及Sox10阳性。
  20. 根据权利要求1所述的方法，其中所述第一类型的细胞在所述第一组重编程因子存在下培养(a)至少1、2、3、4、5、6、7、8、9、10、11或12天或(b)不超过20、19、18、17、16、15、14、13或12天。
  21. 一种用于将第二类型的细胞重编程成第三类型的细胞的方法，其包含在第二组重编程因子存在下培养所述第二类型的细胞，其中所述第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
  22. 根据权利要求21所述的方法，其中所述第二组重编程因子进一步包含BMP4和/或DNA甲基转移酶抑制剂。
  23. 根据权利要求21所述的方法，其中所述酪蛋白激酶1抑制剂是CKI-7。
  24. 根据权利要求22所述的方法，其中所述DNA甲基转移酶抑制剂选自由以下组成的组：5-氮杂-dC、5-氮杂胞苷以及RG108。
  25. 根据权利要求21所述的方法，其中所述第三类型的细胞是体细胞。
  26. 根据权利要求25所述的方法，其中所述体细胞是角膜内皮细胞(CEC)样细胞。
  27. 根据权利要求26所述的方法，其中所述CEC样细胞呈ZO-1和Na +/K +-ATP酶阳性。
  28. 根据权利要求21所述的方法，其中所述第二类型的细胞在所述第二组重编程因子存在下培养(a)至少1、2、3、4、5、6、7、8、9、10、11或12天或(b)不超过20、19、18、17、16、15、14、13或12天。
  29. 一种将第一类型的细胞重编程成第三类型的细胞的方法，其包含步骤(a)在第一组重编程因子存在下培养所述第一类型细胞，其中所述第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子(TGFβ)抑制剂以及环状AMP诱导剂，和步骤(b)在第二组重编程因子存在下培养从步骤(a)获得的细胞，其中所述第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
  30. 根据权利要求29所述的方法，其中所述第一组重编程因子进一步包含DNA甲基转移酶抑制剂、DOT1L抑制剂以及组蛋白脱乙酰基酶抑制剂。
  31. 根据权利要求29所述的方法，所述方法进一步包含在开始步骤(b)之前洗涤从步骤(a)获得的细胞。
  32. 根据权利要求29所述的方法，其中步骤(a)与步骤(b)之间不存在洗涤步骤。
  33. 一种将第一类型的细胞重编程成第三类型的细胞的方法，其包含在第一组重编程因子和第二组重编程因子存在下培养所述第一类型的细胞，其中所述第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、转化生长因子(TGFβ)抑制剂以及环状AMP诱导剂，并且所述第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
  34. 根据权利要求33所述的方法，其中所述第一组重编程因子进一步包含DNA甲基转移酶抑制剂、DOT1L抑制剂以及组蛋白脱乙酰基酶抑制剂。
  35. NCC样细胞群，其根据如权利要求1至20中任一项所述的方法产生。
  36. 角膜内皮细胞样细胞(CEC样细胞)群，其根据如权利要求21至34中任一项所述的方法产生。
  37. 一种组合物，其包含根据权利要求35所述的NCC样细胞或根据权利要求36所述的CEC样细胞。
  38. 一种治疗与功能失调或受损角膜内皮细胞相关的疾病或病状的方法，其包含向有需要的受试者施用有效量的根据权利要求36所述的CEC样细胞或根据权利要求37所述的组合物。
  39. 根据权利要求38所述的方法，其中所述受试者是人。
  40. 根据权利要求38所述的方法，其中所述疾病或病状选自由以下组成的组：富克氏营养不良、虹膜角膜内皮综合征、后部多态性营养不良、先天性遗传性角膜内皮营养不良、年龄相关性黄斑变性(AMD)、视网膜色素变性、青光眼、角膜营养不良、隐形眼镜使用、白内障手术以及角膜移植中的晚期内皮衰竭。
  41. 一种用于将第一类型的细胞重编程成第二类型的细胞的试剂盒，其中所述试剂盒包含第一组重编程因子，并且所述第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、TGFβ抑制剂以及环状AMP诱导剂。
  42. 根据权利要求41所述的试剂盒，其中所述第一组重编程因子进一步包含DNA甲基转移酶抑制剂、DOT1L抑制剂以及组蛋白脱乙酰基酶抑制剂。
  43. 一种用于将第二类型的细胞重编程成第三类型的细胞的试剂盒，其中所述试剂盒包含第二组重编程因子，并且所述第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
  44. 一种用于将第一类型的细胞重编程成第三类型的细胞的试剂盒，其中所述试剂盒包含第一组重编程因子和第二组重编程因子，其中所述第一组重编程因子包含糖原合酶激酶3(GSK3)抑制剂、TGFβ抑制剂以及环状AMP诱导剂，并且所述第二组重编程因子包含TGFβ抑制剂和酪蛋白激酶1抑制剂。
  45. 根据权利要求44所述的试剂盒，其中所述第一组重编程因子进一步包含DNA甲基转移酶抑制剂、DOT1L抑制剂以及组蛋白脱乙酰基酶抑制剂。
  46. 一种鉴别药物的方法，其包含向根据权利要求35所述的NCC样细胞或根据权利要求36所述的CEC样细胞施用药物候选物，并且检测所述细胞对所述药物候选物的反应，从而鉴别所述药物。
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