Human functional corneal endothelial cell and application thereof

Abstract

To provide a human functional corneal endothelial cell and its application.SOLUTION: The present invention completes a technique for treating a corneal disorder or disease by injection into the anterior chamber of a human eye. Specifically, the present invention has found that cultured human corneal endothelial cells are composed of multiple subpopulations, most of which are not suitable for infusion into patients. The problem is solved by providing, as a medicine, a cell of functionally high-grade quality which is a specific subpopulation and has a function of a mature differentiated human corneal endothelial cell characterized by biochemical and functional phenotypes. The present invention provides this functional mature differentiated corneal endothelial cell, a medicament comprising the same, a production method, quality control, and techniques related thereto.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to human functional corneal endothelial cells that can induce human corneal endothelial functional properties when injected into the anterior chamber of the human eye, a pharmaceutical containing the cells, a method for producing the same, and applications such as quality control of the produced cells and the production process. [Background technology]

[0002] The only current treatment for corneal endothelial disorders, such as bullous keratopathy, is keratoplasty using donor corneas, but the long-term clinical results of this surgery are unsatisfactory. Furthermore, visual acuity after corneal transplantation is poor due to the induction of irregular corneal astigmatism. Approximately 60% of corneal transplant patients suffer from corneal endothelial dysfunction (bullous keratopathy). The main causes of bullous keratopathy are corneal endothelial damage caused by ophthalmic surgery such as cataract surgery, glaucoma surgery, vitreoretinal surgery, or laser iridotomy, corneal trauma, pseudoexfoliation syndrome, and Fuchs' endothelial dystrophy. The potential prevalence of a genetic predisposition to Fuchs' endothelial corneal dystrophy in Europe and the United States is approximately 5%. The above has been reported. Corneal transplantation requires one donor cornea to treat one diseased eye, and therefore does not solve the ongoing shortage of donors. Given the large number of potential patients, the provision of innovative medical treatments that are more versatile than corneal transplantation techniques and can be applied at a wider range of medical institutions is an urgent issue and is strongly desired worldwide. In addition, cell injection therapy results in the normal shape of the cornea without distortion, resulting in the recovery of good visual function. Summary of the Invention [Means for solving the problem]

[0003] The present inventors were the first in the world to discover that cultured human corneal endothelial cells are composed of multiple subpopulations due to cell phase transitions during culture (fibrosis, epithelial-mesenchymal transition, senescence, dedifferentiation, etc.), and by devising a technique for selectively proliferating subpopulations during culture, they confirmed that a specific subpopulation, namely functional cells (also called effector cells) that fully possess the functions of mature, differentiated human corneal endothelial cells, form small, hexagonal, paving stone-like shapes that are ideal for cell injection therapy and that utilize an energy metabolic system primarily based on mitochondrial function, and thus completed the revolutionary present invention.

[0004] We have succeeded in developing an in vitro culture technique for functional human corneal endothelial cells, which had long been thought to be impossible using conventional technology, and have established a method for injecting high-quality functional cultured human corneal endothelial cells produced using this technique into the anterior chamber of the human eye. The concept of regenerating corneal endothelium by intracameral injection is (1) minimally invasive, (2) does not use artificial materials as a base, and (3) makes it possible to use high-quality functional cultured human corneal endothelial cells derived from young individuals that have little aging as master cells.

[0005] Thus, the present invention provides the following: (Cell invention) In another embodiment, the present invention also provides the following: (Item 1) Human functional corneal endothelial cells that can induce human corneal endothelial functional properties when injected into the anterior chamber of the human eye. (Item 2) The cells described in Item 1, which express cell surface antigens including a CD166-positive and CD133-negative phenotype. (Item 3) The cells described in Item 2, wherein the cell surface antigens include a CD166-positive, CD133-negative, and CD44-negative to intermediate-positive phenotype. (Item 4) The cells described in Item 2, wherein the cell surface antigens include a CD166-positive, CD133-negative, and CD44-negative to CD44-weakly-positive phenotype. (Item 5) The cells described in Item 2, wherein the cell surface antigens include a CD166-positive, CD133-negative, and CD200-negative phenotype. (Item 6) CD90 negative to weakly positive, CD105 negative to weakly positive, CD24 negative, CD26 negative, LGR5 negative, SSEA3 negative, MHC1 weakly positive, MHC2 negative, PDL1 positive, ZO1 positive, Na + / K + ATPase positive and the table below [Table A] 6. The cell according to any one of items 2 to 5, further comprising at least one expression characteristic selected from the group consisting of the cell surface antigens described in (Item 7) The cell according to any one of Items 1 to 6, wherein the cell has at least one characteristic selected from the group consisting of high PDGF-BB production, low IL-8 production, low MCP-1 production, high TNF-α production, high IFN-γ production, and high IL-1R antagonist production. (Item 8) The cell according to any one of Items 1 to 7, wherein the cell has at least one miRNA having the cell characteristic of a5 mature differentiated corneal endothelial functional cells, wherein the cell surface antigen characteristics of the a5 are CD44 negative to weakly positive and CD24 negative and CD26 negative. (Item 9) The characteristics of the miRNA are as follows: (A) Functional mature differentiated corneal endothelial cells (a5): Moderately differentiated corneal endothelial cells (a1): Non-functional corneal endothelial cells (a2) = High expression: High expression: Low expression: (Intracellular) miR23a-3p, miR23b-3p, miR23c, miR27a-3p, miR27b-3p, miR181a-5p, miR181b-5p, miR181c-5p, miR181d-5p (cell-secreted) miR24-3p, miR1273e; (B) a5:a1:a2 = High expression: Medium expression: Low expression: (Intracellular) miR30a-3p, miR30a-5p, miR30b-5p, miR30c-5p, miR30e-3p, miR30e-5p, miR130a-3p, miR13 0b-3p, miR378a-3p, miR378c, miR378d, miR378e, miR378f, miR378h, miR378i, miR184, miR148a-3p (cell-secreted) miR184; (C) a5:a1:a2 = High expression: Low expression: Low expression: (Intracellular) miR34a-5p, miR34b-5p (Cell-secreted) miR4419b, miR371b-5p, miR135a-3p, miR3131, miR296-3p, miR920, miR6501-3p; (D) a5:a1:a2 = Low expression: Low expression: Medium to high expression: (Intracellular) miR29a-3p, miR29b-3p, miR199a-3p, miR199a-5p, miR199b-5p, miR143-3p (cell-secreted) miR1915-3p, miR3130-3p, miR92a-2-5p, miR1260a; (E) a5:a1:a2 = Low expression: Medium expression: High expression: (Intracellular) miR31-3p, miR31-5p, miR193a-3p, miR193b-3p, miR138-5p (F) a5:a1:a2 = High expression: Low expression: High expression: (cell secreted type) miR92b-5p (G) a5:a1:a2 = Low expression: High expression: Low expression: (Cell secreted type) miR1246, miR4732-5p, miR23b-3p, miR23a-3p, miR1285-3p, miR5096 9. The cell according to item 8, comprising at least one miRNA selected from the group consisting of: (Item 10) The cell described in Item 9, wherein the miRNA marker includes at least one selected from (B) or (C). (Item 11) The average cell area of ​​the cells is 250 μm 2 The cell according to any one of items 1 to 10, wherein the cell is: (Item 12) A cell described in any one of items 1 to 11, having cellular functional characteristics homologous to a5 in at least one cellular indicator selected from the group consisting of cell surface markers; proteinaceous products or biological substances related to said products; SASP-related proteins; miRNA; exosomes; cellular metabolic products including amino acids and biological substances related to said products; cell size; cell density, and the presence of autoantibody-reactive cells. (Item 13) The cell according to any one of Items 1 to 12, wherein the cell has no karyotype or higher. (Item 14) A cell population comprising the cells according to any one of Items 1 to 13. (Item 15) The average cell density of the cell population at saturation cell culture (confluent culture) is at least 1500 cells / mm 2 The cell population according to Item 14, wherein (Item 16) The average cell density of the cell population at saturation cell culture (confluent culture) is at least 2000 cells / mm 2 The cell population according to item 14 or 15, wherein (Item 17) The average cell density of the cells engrafted on the surface of the human corneal endothelium after injection of the cell population is at least 1000 cells / mm 2 The cell population according to any one of items 14 to 16. (Item 18) The average cell density of the cells engrafted on the surface of the human corneal endothelium after injection of the cell population is at least 2000 cells / mm 2 The cell population according to any one of items 14 to 17. (Item 19) The cell population according to any one of Items A14 to A18, wherein at least 70% of the cells in the cell population have the characteristic described in Item A2 or A3. (Item 20) At least 90% of the cells in the cell population are selected from the group consisting of the cells described in items A2 and A3. A cell population according to any one of items A14 to A19, having the characteristics described above. (Item 21) The cell population according to any one of Items 14 to 20, wherein at least 40% of the cells in the cell population have the characteristic described in Item 4. (Item 22) The cell population according to any one of Items 14 to 21, wherein at least 70% of the cells in the cell population have the characteristic described in Item 4. (Item 23) The cell population according to any one of Items 14 to 22, wherein at least 80% of the cells in the cell population have the characteristic described in Item 4. (Item 24) The cells according to any one of Items 1 to 13 or the cell population according to any one of Items 14 to 23, which do not induce an allogeneic (allogenic) rejection reaction when injected into the anterior chamber. (Item 25) The cells according to any one of Items 1 to 13 or the cell population according to any one of Items 14 to 24, which, after administration to a living body, do not substantially induce an extraneous biological response unrelated to human corneal endothelial tissue reconstruction, such as an increase in serum inflammatory cytokines in the serum cytokine profile. (Item 26) A product comprising the cells according to any one of Items 1 to 13 or the cell population according to any one of Items 14 to 25. (Item 27) A method for preserving a cell or a cell population, for maintaining and preserving the cell according to any one of Items 1 to 13 or the cell population according to any one of Items 14 to 25. (Item 28) A method for delivering the cells according to any one of Items 1 to 13 or the cell population according to any one of Items 14 to 25, comprising a step of carrying out the method for preserving the cells or cell population. (Pharmaceuticals) (Item A1) A pharmaceutical comprising human functional corneal endothelial cells that can induce human corneal functional characteristics when injected into the anterior chamber of a human eye. (Item A2) The pharmaceutical according to Item A1, wherein the pharmaceutical is for treating corneal endothelial dysfunction or disease. (Item A3) The corneal endothelial dysfunction or disease includes at least one selected from the group consisting of Grade 3 corneal endothelial disorder and Grade 4 corneal endothelial disorder (bullous keratopathy) (e.g., Fuchs endothelial corneal dystrophy, PEX-BK (pseudoexfoliation bullous keratopathy; bullous keratopathy associated with pseudoexfoliation syndrome), bullous keratopathy after laser iridotomy, bullous keratopathy after cataract surgery (pseudophakic or aphakic bullous keratopathy), bullous keratopathy after glaucoma surgery, bullous keratopathy after trauma, bullous keratopathy after multiple surgeries of unknown cause, graft failure after corneal transplantation, congenital hereditary corneal endothelial dystrophy, and congenital anterior chamber angle hypoplasia syndrome). The grading system used herein is based on the severity classification of corneal endothelial disease based on the Japanese Journal of Ophthalmology 118: 81-83, 2014. (Item A4) The pharmaceutical agent according to any one of Items A1 to A3, wherein the cells are administered into the anterior chamber. (Item A5) The pharmaceutical composition according to any one of Items A1 to A4, wherein the cells are administered together with an additional drug. (Item A6) The pharmaceutical according to Item A5, wherein the additional drug comprises at least one drug selected from the group consisting of steroids, antibacterial agents, and NSAIDs. (Item A7) The pharmaceutical agent according to Item A5 or A6, wherein the additional drug comprises a ROCK inhibitor. (Item A8) The pharmaceutical composition according to any one of Items A5 to A7, wherein the additional drug is contained in the pharmaceutical composition. (Item A9) The medicine is 5 × 10 4 cells / 300μL~2×10 6 The pharmaceutical composition according to any one of items A1 to A8, comprising the cells at a density of cells / 300 μL. (Item A10) The pharmaceutical according to any one of Items A1 to A9, further comprising a cell infusion vehicle. (Item A11) The pharmaceutical described in Item A10, wherein the cell infusion vehicle further contains at least one of a ROCK inhibitor, albumin, ascorbic acid, and lactic acid. (Item A12) The pharmaceutical according to Item A10 or A11, wherein the cell infusion vehicle further comprises albumin, ascorbic acid, and lactic acid. (Item A13) The pharmaceutical according to any one of Items A10 to A12, wherein the cell infusion vehicle further contains all of a ROCK inhibitor, albumin, ascorbic acid, and lactic acid. (Item A14) The pharmaceutical according to any one of Items A10 to A13, wherein the cell injection vehicle comprises OPEGUARD-MA (registered trademark). (Item A15) The human functional corneal endothelial cells capable of inducing human corneal endothelial functional characteristics when injected into the anterior chamber of the human eye are selected from the following (A15-2) to (A15-13): (A15-2) expressing cell surface antigens including CD166-positive and CD133-negative phenotype; (A15-3) The cell surface antigen comprises a CD166-positive, CD133-negative, and CD44-negative to intermediate-positive phenotype; (A15-4) The cell surface antigens include a CD166-positive, CD133-negative, and CD44-negative to CD44-weakly-positive phenotype; (A15-5) the cell surface antigen comprises a CD166-positive, CD133-negative, and CD200-negative phenotype; (A15-6) The cell surface antigen is one of the following: CD90 negative to weak positive, CD105 negative to weak positive, CD24 negative, CD26 negative, LGR5 negative, SSEA3 negative, MHC1 weak positive, MHC2 negative, PDL1 positive, ZO1 positive, Na + / K + ATPase positive and the table below [Table B] further comprising at least one expression characteristic selected from the group consisting of the cell surface antigens described in (A15-7) The cells have at least one characteristic selected from the group consisting of high PDGF-BB production, low IL-8 production, low MCP-1 production, high TNF-α production, high IFN-γ production, and high IL-1R antagonist production; (A15-8) The cells have at least one miRNA that has the cell characteristics of mature, differentiated, functional corneal endothelial cells a5, and the cell surface antigen characteristics of the a5 are CD44 negative to weakly positive, CD24 negative, and CD26 negative; (A15-9) The miRNA has the following characteristics: (A) Functional mature differentiated corneal endothelial cells (a5): Moderately differentiated corneal endothelial cells (a1): Non-functional corneal endothelial cells (a2) = High expression: High expression: Low expression: (Intracellular) miR23a-3p, miR23b-3p, miR23c, miR27a-3p, miR27b-3p, miR181a-5p, miR181b-5p, miR181c-5p, miR181d-5p (cell-secreted) miR24-3p, miR1273e; (B) a5:a1:a2 = High expression: Medium expression: Low expression: (Intracellular) miR30a-3p, miR30a-5p, miR30b-5p, miR30c-5p, miR30e-3p, miR30e-5p, miR130a-3p, miR13 0b-3p, miR378a-3p, miR378c, miR378d, miR378e, miR378f, miR378h, miR378i, miR184, miR148a-3p (cell-secreted) miR184; (C) a5:a1:a2 = High expression: Low expression: Low expression: (Intracellular) miR34a-5p, miR34b-5p (Cell-secreted) miR4419b, miR371b-5p, miR135a-3p, miR3131, miR296-3p, miR920, miR6501-3p; (D) a5:a1:a2 = Low expression: Low expression: Medium to high expression: (Intracellular) miR29a-3p, miR29b-3p, miR199a-3p, miR199a-5p, miR199b-5p, miR143-3p (cell-secreted) miR1915-3p, miR3130-3p, miR92a-2-5p, miR1260a; (E) a5:a1:a2 = Low expression: Medium expression: High expression: (Intracellular) miR31-3p, miR31-5p, miR193a-3p, miR193b-3p, miR138-5p (F) a5:a1:a2 = High expression: Low expression: High expression: (cell secreted type) miR92b-5p (G) a5:a1:a2 = Low expression: High expression: Low expression: (Cell secreted type) miR1246, miR4732-5p, miR23b-3p, miR23a-3p, miR1285-3p, miR5096 The cell according to item 8, comprising at least one miRNA selected from the group consisting of: CD44 positive, CD24 negative, CD26 negative The expression of the cell surface antigen of a2 is CD44 strongly positive, CD24 negative, and CD26 positive; (A15-10) The miRNA marker includes at least one selected from (B) or (C); (A15-11) The average cell area of ​​the cells is 250 μm 2 Below is; (A15-12) Cells homologous to a5 in at least one cellular indicator selected from the group consisting of cell surface markers; proteinaceous products and related biological substances of said products; SASP-related proteins; miRNA; exosomes; cellular metabolic products including amino acids and related biological substances of said products; cell size; cell density; and the presence of autoantibody-reactive cells. Possessing cytotoxic properties; (A15-13) The cells do not have karyotypic abnormalities; or a cell population having one or more characteristics of (A15-14) (A15-2) to (A15-13), wherein the cell population comprises the cells described in any one of (A15-14) to (A15-13); (A15-15) The average cell density of the cell population at saturation cell culture (confluent culture) is at least 1500 cells / mm 2 the cell population according to (A15-14), (A15-16) The average cell density of the cell population at saturation cell culture (confluent culture) is at least 2000 cells / mm 2 the cell population according to (A15-14) to (A15-15); (A15-17) After transplantation of the cell population, the average cell density of the cells engrafted on the surface of the human corneal endothelium is at least 1000 cells / mm 2 The cell population according to any one of (A15-14) to (A15-16), (A15-18) After transplantation of the cell population, the average cell density of the cells engrafted on the surface of the human corneal endothelium is at least 2000 cells / mm 2 The cell population according to any one of (A15-14) to (A15-17) above; (A15-19) A cell population according to any one of (A15-14) to (A15-18), wherein at least 70% of the cells in the cell population have the characteristic described in (A15-2) or (A15-3); (A15-20) A cell population according to any one of (A15-14) to (A15-19), wherein at least 90% of the cells in the cell population have the characteristic described in (A15-2) or (A15-3); (A15-21) A cell population according to any one of (A15-14) to (A15-20), wherein at least 40% of the cells in the cell population have the characteristic described in (A15-4); (A15-22) A cell population according to any one of (A15-14) to (A15-21), wherein at least 70% of the cells in the cell population have the characteristic described in (A15-4); (A15-23) The cell population according to any one of (A15-14) to (A15-22), wherein at least 80% of the cells in the cell population have the characteristic according to (A15-4). (A15-24) The cells according to any one of (A15-2) to (A15-13) or the cell population according to any one of (A15-14) to (A15-22), which do not cause allogeneic rejection when injected into the anterior chamber; (A15-25) The cells according to any one of (A15-2) to (A15-13) or the cell population according to any one of (A15-14) to (A15-24), wherein the cells or cell population do not substantially induce an extraneous biological response unrelated to human corneal endothelial tissue reconstruction, such as an increase in serum inflammatory cytokines, after administration to a living body. A medicine characterized by being: (Manufacturing method) The present invention also provides the following: (Item B1) A method for producing human functional corneal endothelial cells that can induce human corneal functional properties when injected into the anterior chamber of the human eye, including a step of directly or indirectly via a dedifferentiation step, maturing and differentiating corneal endothelial tissue-derived cells or corneal endothelial progenitor cells. (Item B2) A method for producing human functional corneal endothelial cells that can induce human corneal functional properties when injected into the anterior chamber of the human eye, comprising a step of culturing corneal endothelial tissue-derived cells or corneal endothelial precursor cells through a step including actin depolymerization, and allowing them to mature and differentiate. (Item B3) A manufacturing method described in Item B1 or B2, wherein the actin depolymerization is achieved by one or more agents selected from the group consisting of ROCK inhibitors, HDAC inhibitors, actin depolymerization inhibitors, PPARγ inhibitors, MMP2 inhibitors, p53 activators and miRNAs. (Item B4) The method for producing according to Item B3, wherein the ROCK inhibitor is Y-27632. (Item B5) The method according to Item B3, wherein the actin depolymerization inhibitor is selected from the group consisting of latrunculin A and swinholide A. (Item B6) The method according to any one of Items B1 to B5, further comprising the step of culturing the corneal endothelial tissue-derived cells or corneal endothelial precursor cells under conditions that result in transition to epithelial-mesenchymal transition-like transformation, proliferation, maturation, and differentiation. (Item B7) The manufacturing method described in Item B6, wherein the conditions for proliferation, maturation, and differentiation include culturing in the absence of a transforming growth factor β (TGF-β) signaling inhibitor. (Item B8) The method according to any one of Items B1 to B7, further comprising a step of culturing the corneal endothelial tissue-derived cells or corneal endothelial precursor cells under conditions in which cellular senescence is suppressed. (Item B9) The production method according to Item B8, wherein the conditions in which cellular senescence is suppressed include culturing in the presence of a p38 MAP kinase inhibitor. (Item B10) The method for producing according to Item B9, wherein the p38 MAP kinase inhibitor comprises SB203580. (Item B11) The method according to any one of Items B1 to B10, wherein the corneal endothelial tissue-derived cells or corneal endothelial precursor cells are collected from a living body or differentiated from stem cells or precursor cells. (Item B12) The culture is performed at a density of 100 to 1000 cells / mm 2 The method for producing the oocytes according to any one of items B1 to B11, wherein the seeding density is 1000 μg / ml or more. (Item B13) The method according to any one of Items B1 to B12, which comprises a step of further culturing the cultured cells after the cell density reaches saturation density to allow the cells to mature functionally. (Item B14) The production method described in Item B13, wherein after the cultured cells reach saturation cell density and thereafter differentiation and maturation of the cultured cells are completed by sufficient formation of tight junctions, the cultured cells are further cultured for one week or more with only medium replacement for preservation. (Item B15) Any one of Items B1 to B14, further comprising a step of assaying the cell function after the culture using at least one cell indicator that identifies the human functional corneal endothelial cells. The manufacturing method described in paragraph . (Item B16) The manufacturing method according to Item B15, further comprising a step of selectively growing in culture a fraction determined to be the corneal endothelial functional effector cells after the assay. (Item B17) The production method according to any one of Items B1 to B16, further comprising the step of monitoring the composition of cell subpopulations during the culture. (Item B18) The monitors include mitochondrial function, oxygen consumption and pH of the culture medium, amino acid composition, protein products, soluble miRNA, cell density by non-invasive engineering techniques, cell The manufacturing method according to item B17, comprising tracking at least one item selected from the group consisting of cell size, and cell uniformity. (Item B19) The method according to any one of Items B1 to B18, wherein the culturing step includes a subculturing step. (Item B20) The manufacturing method according to any one of Items B1 to B19, wherein the culture step includes a step of adding one or more drugs selected from the group consisting of a ROCK inhibitor, an HDAC inhibitor, an actin depolymerization inhibitor, a PPARγ inhibitor and an MMP2 inhibitor, a p53 activator, and miRNA during subculture. (Item B21) The method according to any one of Items B1 to B20, which comprises culturing in the presence of a serum-free medium. (Item B22) The manufacturing method according to any one of Items B1 to B21, wherein the corneal endothelial tissue-derived cells or corneal endothelial precursor cells are selected from the group consisting of pluripotent stem cells, mesenchymal stem cells, corneal endothelial precursor cells collected from corneal endothelium, cells collected from corneal endothelium, and corneal endothelial precursor cells and corneal endothelial-like cells prepared by a direct programming method. (Item B23) A method for preserving mature, differentiated, functional corneal endothelial cells, comprising the step of continuing to culture the mature, differentiated, functional corneal endothelial cells according to any one of Items B1 to B22 after production. (Quality control) (Item C1) A method for quality control or process control of cultured human functional corneal endothelial cells capable of eliciting human corneal endothelial functional properties upon injection into the anterior chamber of a human eye, comprising a step of measuring at least one cellular indicator selected from the group consisting of cell surface markers; proteinaceous products and biological substances associated with said products; SASP-related proteins; miRNA; exosomes; cellular metabolic products including amino acids and biological substances associated with said metabolic products; cell size; cell density, and the presence of autoantibody-reactive cells. (Item C2) The method described in Item C1, wherein at least three cell indicators are used. (Item C3) The method described in Item C1 or C2, wherein the cell indicator includes cell size, cell density, or a combination thereof. (Item C4) The method according to any one of Items C1 to C3, wherein the cellular indicator comprises a combination of at least one of a cell surface marker, a proteinaceous product and a biological substance associated with the product, at least one miRNA, and at least one cellular metabolite and a biological substance associated with the metabolite. (Item C5) The method according to any one of Items C1 to C4, further comprising identifying a subpopulation of the cultured functional corneal endothelial cells based on a corneal functional characteristic. (Item C6) The method described in Item C5, wherein the corneal functional characteristic is expression of cell surface antigens including CD166 positive and CD133 negative on the cell surface. (Item C7) The method described in Item C5 or C6, characterized in that the cell surface antigens include CD166 positive, CD133 negative, and CD44 negative to intermediate positive. (Item C8) The method according to any one of Items C5 to C7, wherein the cell surface antigens include CD166 positive, CD133 negative, and CD44 negative to CD44 weakly positive. (Item C9) The method according to any one of Items C5 to C8, wherein the cell surface antigens include CD166 positive, CD133 negative, CD44 negative to CD44 weakly positive, and CD90 negative to weakly positive. (Item C10) The method according to any one of items C5 to C9, characterized in that the cell surface antigens include CD166 positive, CD133 negative, and CD200 negative. (Item C11) A method according to any one of items C5 to C10, characterized in that multiple indicators are selected from each of proteinaceous products and biological substances related to the products; secreted miRNA; cellular metabolites including amino acids and biological substances related to the metabolites; and fluctuations in the profile of each indicator are confirmed to determine the homogeneity of cells having cell indicators including CD166 positive, CD133 negative, CD44 negative to CD44 weakly positive, and CD90 negative to weakly positive. (Item C12) The proteinaceous product and the related biological material of the product are: (A) Expression is increased in human functional corneal endothelial cells, which can induce human corneal functional characteristics upon injection into the anterior chamber of the human eye: COL4A1, COL4A2, COL8A1, COL8A2, CDH2, and TGF-β2; and (B) Expression is reduced in human functional corneal endothelial cells, which can induce human corneal functional characteristics when injected into the anterior chamber of the human eye. The method according to any one of items C1 to C11, wherein the IL-13 is selected from the group consisting of MMP1, MMP2, TIMP1, BMP2, IL13RA2, TGF-β1, CD44, COL3A1, IL6, IL8, HGF, THBS2, and IGFBP3. (Item C13) The miRNA has the following characteristics: (A) Mature differentiated functional corneal endothelial cells (a5): Mature differentiated corneal endothelial progenitor cells (a1): Non-functional corneal endothelial cells (a2) = High expression: High expression: Low expression: (Intracellular) miR23a-3p, miR23b-3p, miR23c, miR27a-3p, miR27b-3p, miR181a-5p, miR181b-5p, miR181c-5p, miR181d-5p (cell-secreted) miR24-3p, miR1273e; (B) a5:a1:a2 = High expression: Medium expression: Low expression: (Intracellular) miR30a-3p, miR30a-5p, miR30b-5p, miR30c-5p, miR30e-3p, miR30e-5p, miR130a-3p, miR13 0b-3p, miR378a-3p, miR378c, miR378d, miR378e, miR378f, miR378h, miR378i, miR184, miR148a-3p (cell-secreted) miR184; (C) a5:a1:a2 = High expression: Low expression: Low expression: (Intracellular) miR34a-5p, miR34b-5p (Cell-secreted) miR4419b, miR371b-5p, miR135a-3p, miR3131, miR296-3p, miR920, miR6501-3p; (D) a5:a1:a2 = Low expression: Low expression: Medium to high expression: (Intracellular) miR29a-3p, miR29b-3p, miR199a-3p, miR199a-5p, miR199b-5p, miR143-3p (cell-secreted) miR1915-3p, miR3130-3p, miR92a-2-5p, miR1260a; (E) a5:a1:a2 = Low expression: Medium expression: High expression: (Intracellular) miR31-3p, miR31-5p, miR193a-3p, miR193b-3p, miR138-5p (F) a5:a1:a2 = High expression: Low expression: High expression: (cell secreted type) miR92b-5p (G) a5:a1:a2 = Low expression: High expression: Low expression: (Cell secreted type) miR1246, miR4732-5p, miR23b-3p, miR23a-3p, miR1285-3p, miR5096 and the expression level is 3. It is the relative intensity between cells of a species, and is defined as the order of high expression > medium expression > low expression, with the expression intensity decreasing. The cell surface antigen characteristics of the a5 are CD44 negative to weakly positive, CD24 negative and CD26 negative, The expression of the cell surface antigen of a1 is CD44 positive, CD24 negative, and CD26 negative. The expression of the cell surface antigen of the a2 is CD44 strongly positive, CD24 negative, and CD26 positive. The method according to any one of items C1 to C12. (Item C14) The exosomes are the following cellular indicators: (A) Expression of a compound that is decreased in human functional corneal endothelial cells that can induce human corneal functional characteristics when injected into the anterior chamber of the human eye: The method according to any one of items C1 to C13, comprising at least one indicator selected from the group consisting of CD63, CD9, CD81 and HSP70. (Item C15) The cellular metabolites and the related biological substances of the metabolites are as follows: at least one selected from the group consisting of succinic acid, Pro, Gly, glycerol 3-phosphate, Glu, lactic acid, argininosuccinic acid, xanthine, N-carbamoyl aspartic acid, isocitrate, cis-aconitic acid, citric acid, Ala, 3-phosphoglyceric acid, hydroxyproline, malic acid, uric acid, betaine, folic acid, Gln, 2-oxoisovaleric acid, pyruvate, Ser, hypoxanthine, Asn, Trp, Lys, choline, Tyr, urea, Phe, Met, carnosine, Asp, ornithine, Arg, creatine, 2-hydroxyglutamic acid, β-Ala, citrulline, Thr, Ile, Leu, Val, creatinine, His, N,N-dimethylglycine, or combinations or relative ratios thereof; The method according to any one of items C1 to C14. (Item C16) The cell metabolites and the related biological substances of the metabolites are serine in the culture supernatant. , alanine, proline, glutamine or citrate / lactate ratio. (Item C17) The size of the cells is an average cell area of ​​250 μm 2 The method according to any one of items C1 to C16, wherein: (Item C18) The average cell density of the cells when cultured to saturation is at least 2000 cells / mm 2 The method according to any one of items C1 to C17, which is as described above. (Item C19) A method for detecting non-functional corneal endothelial cells present in cultured human corneal endothelial cells, comprising a step of measuring at least one cell indicator selected from the group consisting of cell size, cell density, and the presence of autoantibody-reactive cells. (Item C20) A quality evaluation agent for functional, mature, differentiated corneal endothelial cells, a process control agent, or an agent for detecting non-functional corneal endothelial cells, comprising a reagent or means for measuring the cell indicator according to any one of Items C1 to C19. (Item C21) The quality evaluation agent, process control agent or detection agent according to Item C20, wherein the measuring means is labeled. (Item C22) A) providing a sample that may contain human functional corneal endothelial cells that can induce human corneal functional characteristics when injected into the anterior chamber of a human eye; B) a step of determining whether the sample contains human functional corneal endothelial cells capable of inducing human corneal functional characteristics when injected into the anterior chamber of the human eye, using a quality evaluation agent, process control agent, or non-functional corneal endothelial cell detection agent described in item C20 or C21, wherein, when the evaluation result using the quality evaluation agent, process control agent, or non-functional corneal endothelial cell detection agent indicates that the cells are human functional corneal endothelial cells capable of inducing human corneal functional characteristics when injected into the anterior chamber of the human eye, determining that the sample contains human functional corneal endothelial cells capable of inducing human corneal functional characteristics when injected into the anterior chamber of the human eye; C) Selectively expanding in culture cells determined to be human functional corneal endothelial cells capable of eliciting human corneal functional properties upon injection into the anterior chamber of a human eye. A method for selectively growing human functional corneal endothelial cells, comprising: (Item C23) A) a step of obtaining information about the cell indicators of functional corneal endothelial cells of cells provided as human functional corneal endothelial cells capable of inducing human corneal functional properties when injected into the anterior chamber of a human eye, using the quality evaluation agent, process control agent, or non-functional corneal endothelial cell detection agent described in either Item C20 or C21; and B) determining, based on the information, that the provided cells are human functional corneal endothelial cells capable of inducing human corneal functional properties when injected into the anterior chamber of a human eye; A method for assaying the quality of human functional corneal endothelial cells, comprising: (Item C24) A method for controlling the quality of a preparation of human functional corneal endothelial cells capable of inducing human corneal functional properties when injected into the anterior chamber of a human eye, comprising: A) a step of obtaining information regarding cell indicators of mature, differentiated, functional corneal endothelial cells of the cells obtained in the preparation using the quality evaluation agent, process control agent, or corneal endothelial non-functional cell detection agent described in item C20 or C21; and B) determining, based on the information, that the preparation is suitable for preparing human functional corneal endothelial cells capable of inducing human corneal functional properties when injected into the anterior chamber of a human eye. (Item C25) A method for assaying the purity of human functional corneal endothelial cells capable of inducing human corneal functional characteristics when injected into the anterior chamber of a human eye, comprising: A) providing a sample that may contain human functional corneal endothelial cells that can induce human corneal functional characteristics when injected into the anterior chamber of the human eye; B) A step of obtaining information regarding the cell indicators of functional corneal endothelial cells of the cells using the quality evaluation agent, process control agent, or non-functional corneal endothelial cell detection agent described in item C20 or C21; and C) calculating the purity of human functional corneal endothelial cells in the sample that are capable of inducing human corneal functional properties when injected into the anterior chamber of the human eye based on the information; The method includes: (Item C26) A) a step of culturing in a culture medium cells provided as functional mature differentiated corneal endothelial cells capable of inducing human corneal functional properties when injected into the anterior chamber of a human eye, and obtaining information about the cell indicators of the functional corneal endothelial cells of the cells using the quality evaluation agent, process control agent, or non-functional corneal endothelial cell detection agent described in either Item C20 or C21; and B) determining, based on the information, that the medium is suitable for producing human functional corneal endothelial cells capable of inducing human corneal functional properties when injected into the anterior chamber of the human eye; A method for testing the quality of a culture medium for human functional corneal endothelial cells, comprising: (Item C27) A) a step of culturing cells provided as human functional corneal endothelial cells capable of inducing human corneal functional properties when injected into the anterior chamber of a human eye in a cell injection vehicle, and obtaining information about the cell indicators of the functional corneal endothelial cells of the cells using the quality evaluation agent, process control agent, or non-functional corneal endothelial cell detection agent described in either Item C20 or C21; and B) determining, based on the information, that the cell injection vehicle is suitable for cell injection therapy. (Item C28) Below: (1) Purity test of culture supernatant by ELISA TIMP-1: 500ng / mL or less IL-8: 500pg / mL or less PDGF-BB: 30pg / mL or more MCP-1: 3000pg / mL or less (2) Purity test by cell FACS CD166=95% or more CD133=5% or less CD105 low positive = 95% or more CD44 low positive = 70% or more CD44 high positivity = 15% or less CD24=10% or less CD26 positive = 5% or less CD200=5% or less (3) Barrier function (ZO-1) positive (4) Pump function (Na+ / K+ATPase) positive (5) Cell viability Trypan blue staining: 70% or more (6)Cell morphology No transformed cells detected in visual examination (7)Claudin10 positive (8) Effector cell (E-ratio) > 50% (9) Non-target cells Non-target cells A (strongly CD44-positive cells) <15%, non-target cells B (CD26-positive cells) <5%, non-target cells C (CD24-positive cells) <10% (10) Negative karyotype abnormality A method for quality control or process control of cultured human functional corneal endothelial cells that can induce human corneal functional properties when injected into the anterior chamber of the human eye, or a method for detecting non-functional corneal endothelial cells mixed in with cultured human corneal endothelial cells, comprising a step of confirming one or more of the following: (Item C29) The method according to Item C28, wherein the confirmation is carried out 3 weeks or more immediately before cell injection therapy or during conservative culture with only medium exchange. (Item C30) The confirmation includes being performed about 7 days before to immediately before cell injection therapy. The method according to item C28 or C29. (Item C31) The method according to any one of Items C22 to C27, characterized by one or more of the features according to Items C28 to C30. (Item C32) A method for quality control or process control of human functional corneal endothelial cells that can induce human corneal functional properties when injected into the anterior chamber of the human eye, comprising steps of determining one or more characteristics of the target cells: (1) retention of endothelial pump and barrier function, (2) adhesion and binding to specific laminins, (3) secreted cytokine profile, (4) produced metabolic product profile, (5) saturation cell density during in vitro culture, (6) spatial size and distribution of cells obtained during culture, and (8) cell maintenance when injected into a mouse cornea after liquid nitrogen freeze injury. (Item C33) The method described in Item C32, wherein the maintenance of the endothelial pump and barrier function is determined using a pump function measurement method or barrier function measurement method commonly used for corneal endothelium. (Item C34) The determination of the adhesiveness and binding properties to the specific laminin is based on the adhesiveness and / or the affinity to laminin 511 (a complex of α5 chain, β1 chain, and γ chain 1), laminin 521 (a complex of α5 chain, β2 chain, and γ chain 1) or a functional fragment thereof. The method according to item C32 or C33, wherein the disease is determined using an increase in expression of glioma as an index. (Item C35) The method according to any one of Items C32 to C34, wherein the determination of the secreted cytokine profile comprises measuring the production level of the cytokine profile in serum or aqueous humor. (Item C36) The method according to any one of Items C32 to C35, wherein determining the profile of the produced metabolites comprises measuring the production level of the metabolites of the cells. (Item C37) The method according to any one of Items C32 to C36, wherein determining the produced microRNA (miRNA) profile comprises obtaining total RNA and obtaining its microRNA expression profile. (Item C38) The method according to any one of Items C32 to C37, wherein determining the saturation cell density during in vitro culture comprises counting cells in an image of the cells obtained using an image acquisition system. (Item C39) The method according to any one of Items C32 to C38, wherein determining the spatial size and distribution of the cells obtained during the culture comprises counting the cells in an image of the cells obtained using an image acquisition system. (Item C40) The method according to any one of Items C32 to C39, in which the determination of cell maintenance when cells are injected after liquid nitrogen freeze injury to the mouse cornea includes injecting the cells to be determined into the anterior chamber of a model prepared by pretreating the central region of the mouse cornea with cryoinjury and removing endothelial cells, clinically observing the characteristics of the cornea, evaluating the thickness of the cornea using a pachymeter, and histopathologically examining the adhesion of HCECs using human nuclear staining, to confirm whether the cells are functional. Alternative Embodiments Thus, the present invention provides the following: (cell) (Item X1) Human functional corneal endothelial cells that can induce human corneal endothelial functional properties when transplanted into the anterior chamber of the human eye. (Item X2) The cells described in Item X1, characterized in that they express cell surface antigens including CD166 positive and CD133 negative. (Item X3) The cells described in Item X2, characterized in that the cell surface antigens include a CD166-positive, CD133-negative, and CD44-negative to intermediate-positive phenotype. (Item X4) The cells according to item X2 or X3, characterized in that the cell surface antigens include CD166 positive, CD133 negative, and CD44 negative to CD44 weakly positive. (Item X4A) Cells according to Item X1, which express cell surface antigens including a CD44 negative to CD44 weakly positive phenotype. (Item X4B) The cell according to X1, which expresses cell surface antigens including a CD44-negative phenotype. (Item X5) The cell according to any one of items X2 to X4, X4A and X4B, wherein the cell surface antigens include a CD166-positive, CD133-negative and CD200-negative phenotype. Cells on the plate. (Item X6) The cells described in any one of items X2 to X4, X4A, X4B and X5, wherein the cell surface antigens include a CD166 positive, CD133 negative, CD44 negative to intermediate positive and CD90 negative phenotype. (Item X7) The cell surface antigen is one of the following: CD90 negative to weakly positive, CD105 negative to weakly positive, CD24 negative, CD26 negative, LGR5 negative, SSEA3 negative, MHC1 weakly positive, MHC2 negative, PDL1 positive, ZO-1 positive, and Na + / K + The cell according to any one of items X2 to X4, X4A, X4B and X5 to X6, further comprising at least one surface antigen expression characteristic selected from the group consisting of ATPase positive. (Item X8) The cells according to any one of Items X1 to X4, X4A, X4B, and X5 to X7, have at least one characteristic selected from the group consisting of high PDGF-BB production, low IL-8 production, low MCP-1 production, high TNF-α production, high IFN-γ production, and high IL-1R antagonist production. (Item X9) The cells are described in any one of Items X1 to X4, X4A, X4B and X5 to X8, wherein at least one miRNA has the cell characteristics of mature differentiated corneal endothelial functional cells a5, and the cell surface antigen characteristics of the a5 are CD44 negative to weakly positive and CD24 negative and CD26 negative. (Item X10) The characteristics of the miRNA are as follows: (A) miR23a-3p, miR23b-3p, miR23c, miR27a-3p, miR27b-3p, miR181a-5p, miR181b-5p, miR181c-5p, miR181d-5p, miR24-3p, miR1273e; (B) miR30a-3p, miR30a-5p, miR30b-5p, miR30c-5p, miR30e-3p, miR30e-5p, miR130a-3p, miR130b-3p , miR378a-3p, miR378c, miR378d, miR378e, miR378f, miR378h, miR378i, miR184, miR148a-3p, miR184; (C) miR34a-5p, miR34b-5p, miR4419b, miR371b-5p, miR135a-3p, miR3131, miR296-3p, miR920, miR6501-3p; (D) miR29a-3p, miR29b-3p, miR199a-3p, miR199a-5p, miR199b-5p, miR143-3p, miR1915-3p, miR3130-3p, miR92a-2-5p, miR1260a; (E) miR31-3p, miR31-5p, miR193a-3p, miR193b-3p, miR138-5p; (F) miR92b-5p; and (G) miR1246, miR4732-5p, miR23b-3p, miR23a-3p, miR1285-3p, miR5096 Item X9, comprising at least one miRNA selected from the group consisting of: (Item X11) The cell described in Item X10, wherein the miRNA marker includes at least one selected from (B) or (C). (Item X12) The average cell area of ​​the cells is 250 μm 2 The cell according to any one of items X1 to X4, X4A, X4B, and X5 to X11, which is: (Item X13) A cell described in any one of items X1 to X4, X4A, X4B and X5 to X12, which has cell functional characteristics homologous to a5 in at least one cell indicator selected from the group consisting of cell surface markers; proteinaceous products and biological substances related to said products; SASP-related proteins; intracellular and secreted miRNA; exosomes; cellular metabolic products including amino acids and biological substances related to said metabolic products; cell size; cell density and the presence of autoantibody-reactive cells. (Item X14) The cells have no karyotypic abnormalities, Items X1 to X4, X4A, X4B and The cell according to any one of items X5 to X13. (Item X15) A cell population comprising the cells according to any one of items X1 to X4, X4A, X4B, and X5 to X14. (Item X16) The average cell density of the cell population at saturation cell culture (confluent culture) is at least 1500 cells / mm 2 The cell population described in item X15. (Item X17) The average cell density of the cell population at saturation cell culture (confluent culture) is at least 2000 cells / mm 2 (Item X18) The cell population according to Item X15 or X16, wherein the average cell density of the cells engrafted on the surface of human corneal endothelium after transplantation of the cell population is at least 1000 cells / mm 2 The cell population according to any one of items X15 to X17. (Item X19) After transplantation of the cell population, the average cell density of the cells engrafted on the surface of the human corneal endothelium is at least 2000 cells / mm 2 The cell population according to any one of items X15 to X18. (Item X20) A cell population according to any one of Items X15 to X19, wherein at least 70% of the cells in the cell population have the characteristic described in any one of Items X2 to X4, X4A, X4B, and X5 to X6. (Item X21) A cell population according to any one of items X15 to X20, wherein at least 90% of the cells in the cell population have the characteristic described in any one of items X2 to X4, X4A, X4B, and X5 to X6. (Item X22) A cell population according to any one of Items X15 to X21, wherein at least 40% of the cells in the cell population have the characteristic described in Item X4. (Item X23) A cell population according to any one of Items X15 to X22, wherein at least 70% of the cells in the cell population have the characteristic described in Item X4. (Item X24) A cell population according to any one of Items X15 to X23, wherein at least 80% of the cells in the cell population have the characteristic described in Item X4. (Item X25) The cells according to any one of Items X1 to X4, X4A, X4B, and X5 to X14 or the cell population according to any one of Items X15 to X24, which do not cause allogeneic rejection when transplanted into the anterior chamber. (Item X26) The cells or cell population according to any one of Items X1 to X4, X4A, X4B, and X5 to X14 or the cell population according to any one of Items X15 to X25, wherein the cells or cell population do not substantially induce an extraneous biological response unrelated to human corneal endothelial tissue reconstruction, such as an increase in serum inflammatory cytokines, after administration to a living body. (Item X27) A product comprising a cell according to any one of items X1 to X4, X4A, X4B, and X5 to X14, or a cell population according to any one of items X15 to X26. (Item X28) A method for preserving the cells or cell population according to any one of Items X1 to X4, X4A, X4B, and X5 to X14 or the cell population according to any one of Items X15 to X26, by replacing the medium to maintain and preserve the cell functional characteristics. (Item X29) A method for delivering the cells according to any one of Items X1 to X4, X4A, X4B, and X5 to X14 or the cell population according to any one of Items X15 to X26, comprising a step of carrying out a method for preserving the cells or cell population. (Pharmaceuticals) (Item XA1) A pharmaceutical comprising functional corneal endothelial cells that can induce human corneal functional characteristics when transplanted into the anterior chamber of a human eye. (Item XA2) The pharmaceutical according to Item XA1, wherein the pharmaceutical is for treating corneal endothelial dysfunction or disease. (Item XA3) The corneal endothelial dysfunction or disease is corneal endothelial disorder Grade 3 and corneal endothelial disorder Grade 4 (bullous keratopathy) (e.g., Fuchs corneal endothelial dystrophy, PEX-BK (pseudoexfoliation bullous keratopathy; bullous keratopathy associated with pseudoexfoliation syndrome), post-laser iridotomy bullous keratopathy, post-cataract surgery bullous keratopathy (pseudophakic or aphakic bullous keratopathy) The pharmaceutical composition according to Item XA2, which comprises at least one selected from the group consisting of: post-glaucoma surgery bullous keratopathy, post-traumatic bullous keratopathy, post-multiple surgery bullous keratopathy of unknown cause, graft failure after corneal transplantation, congenital hereditary corneal endothelial dystrophy, and congenital anterior chamber angle hypoplasia syndrome. The grading system used herein is based on the severity classification of corneal endothelial diseases according to the Japanese Journal of Ophthalmology 118: 81-83, 2014. (Item XA4) The pharmaceutical according to any one of Items XA1 to XA3, wherein the cells are administered into the anterior chamber. (Item XA5) The pharmaceutical composition according to any one of Items XA1 to XA4, wherein the cells are administered together with an additional drug. (Item XA6) The pharmaceutical described in Item XA5, wherein the additional drug comprises at least one drug selected from the group consisting of steroids, antibacterial agents, and NSAIDs. (Item XA7) The pharmaceutical described in Item XA5 or XA6, wherein the additional drug comprises a ROCK inhibitor. (Item XA8) The pharmaceutical composition according to any one of Items XA5 to XA7, wherein the additional drug is contained in the pharmaceutical composition. (Item XA9) The medicine is 5 × 10 4 cells / 300μL~2×10 6 The pharmaceutical according to any one of items XA1 to XA8, comprising the cells at a density of cells / 300 μL. (Item XA10) The pharmaceutical according to any one of Items XA1 to XA9, further comprising a cell transfer solution. (Item XA11) The pharmaceutical described in Item XA10, wherein the cell infusion vehicle further comprises at least one of a ROCK inhibitor, albumin, ascorbic acid, and lactic acid. (Item XA12) The pharmaceutical described in Item XA10 or XA11, wherein the cell infusion vehicle further comprises albumin, ascorbic acid, and lactic acid. (Item XA13) The pharmaceutical according to any one of Items XA10 to XA12, wherein the cell infusion vehicle further contains all of a ROCK inhibitor, albumin, ascorbic acid, and lactic acid. (Item XA14) The pharmaceutical according to any one of items XA10 to XA13 above, wherein the cell infusion vehicle comprises OPEGUARD-MA (registered trademark). (Item XA15) The pharmaceutical according to any one of Items XA1 to XA14, wherein the human functional corneal endothelial cells are cells according to any one of Items X1 to X4, X4A, X4B, and X5 to X14, or a cell population according to any one of Items X15 to X26. (Manufacturing method) (Item XB1) A method for producing human functional corneal endothelial cells that can induce human corneal functional properties when transplanted into the anterior chamber of the human eye, comprising a step of directly or indirectly via a dedifferentiation step, proliferating, maturing, and differentiating human corneal endothelial tissue-derived cells or corneal endothelial progenitor cells. (Item XB2) A method for producing human functional corneal endothelial cells that can induce human corneal endothelial functional properties when transplanted into the anterior chamber of the human eye, comprising a step of culturing corneal endothelial tissue-derived cells or corneal endothelial precursor cells through a step including actin depolymerization, and allowing them to mature and differentiate. (Item XB3) A manufacturing method described in Item XB1 or XB2, wherein the actin depolymerization is achieved by one or more drugs selected from the group consisting of ROCK inhibitors, HDAC inhibitors, actin depolymerization inhibitors, PPARγ inhibitors, MMP2 inhibitors, p53 activators and miRNAs. (Item XB4) The manufacturing method described in Item XB3, wherein the ROCK inhibitor is Y-27632. (Item XB5) The method for producing according to item XB3 or XB4, wherein the actin depolymerization inhibitor is selected from the group consisting of latrunculin A and swinholide A. (Item XB6) The method according to any one of Items XB1 to XB5, further comprising a step of culturing the corneal endothelial tissue-derived cells or corneal endothelial precursor cells under conditions in which they undergo epithelial-mesenchymal transition-like transformation, proliferation, maturation, and differentiation. (Item XB7) The conditions for proliferation, maturation and differentiation include transforming growth factor beta The method of production according to paragraph XB6, comprising culturing in the absence of a (TGF-β) signaling inhibitor. (Item XB8) The method according to any one of Items XB1 to XB7, further comprising the step of culturing the corneal endothelial tissue-derived cells or corneal endothelial precursor cells under conditions that suppress cellular senescence. (Item XB9) A manufacturing method described in Item XB8, wherein the conditions under which cellular senescence is suppressed include culturing in the presence of a p38 MAP kinase inhibitor. (Item XB10) The manufacturing method described in Item XB9, wherein the p38 MAP kinase inhibitor includes SB203580. (Item XB11) A manufacturing method according to any one of Items XB1 to XB10, wherein the corneal endothelial tissue-derived cells or corneal endothelial precursor cells are collected from a living body or differentiated from stem cells or precursor cells. (Item XB12) The culture is performed at a density of 100 to 1000 cells / mm 2 The production method according to any one of items XB1 to XB11, wherein the seeding density is (Item XB13) A method for producing the cells according to any one of Items XB1 to XB12, which comprises a step of further culturing the cells for functional maturation after the cell density of the cultured cells has reached saturation density. (Item XB14) A manufacturing method described in Item XB13, in which after the cultured cells reach saturation cell density and thereafter differentiation and maturation of the cultured cells are completed by sufficient formation of tight junctions, the cultured cells are cultured for an additional week or more with only medium changes to preserve the cultured cells. (Item XB15) A manufacturing method described in any one of Items XB1 to XB14, further comprising a step of assaying the cell function after the culture using at least one cell indicator that identifies the human functional corneal endothelial cells. (Item XB16) A manufacturing method according to Item XB15, further comprising a step of selecting the fraction determined to be the human functional corneal endothelial cells after the assay. (Item XB17) A manufacturing method according to any one of items XB1 to XB16, further comprising the step of monitoring the composition of cell subpopulations during the culture. (Item XB18) The manufacturing method described in Item XB17, wherein the monitor includes tracking at least one item selected from the group consisting of mitochondrial function, oxygen consumption and culture medium pH, amino acid composition, proteinaceous products, soluble miRNA, cell density by non-invasive engineering techniques, cell size, and cell uniformity. (Item XB19) The method of any one of Items XB1 to XB18, wherein the culturing step includes a subculturing step. (Item XB20) A manufacturing method described in any one of Items XB1 to XB19, wherein the culture step includes a step of adding one or more drugs selected from the group consisting of a ROCK inhibitor, an HDAC inhibitor, an actin depolymerization inhibitor, a PPARγ inhibitor and an MMP2 inhibitor, a p53 activator, and miRNA during subculture. (Item XB21) The method according to any one of Items XB1 to XB20, which comprises culturing in the presence of a serum-free medium. (Item XB22) A manufacturing method described in any one of Items XB1 to XB21, wherein the corneal endothelial tissue-derived cells or corneal endothelial progenitor cells are selected from the group consisting of pluripotent stem cells, mesenchymal stem cells, corneal endothelial progenitor cells collected from corneal endothelium, cells collected from corneal endothelium, and corneal endothelial progenitor cells and corneal endothelial-like cells prepared by a direct programming method. (Item XB23) A method for preserving mature, differentiated, functional human corneal endothelial cells, comprising the step of continuing to culture the mature, differentiated, functional human corneal endothelial cells according to any one of Items XB1 to XB22 after production. (Item XB24) The method described in any one of Items XB1 to XB23, wherein the human functional corneal endothelial cells are cells described in any one of Items X1 to X4, X4A, X4B, X5 to X14, and X25 to X26, or a cell population described in any one of Items X15 to X26. (Quality control) (Item XC1) A method for quality control or process control of cultured human functional corneal endothelial cells capable of eliciting human corneal endothelial functional characteristics when transplanted into the anterior chamber of a human eye, comprising a step of measuring at least one cell function indicator selected from the group consisting of cell surface markers; proteinaceous products and biological substances related to said products; SASP-related proteins; intracellular and secreted miRNA; exosomes; cellular metabolic products including amino acids and biological substances related to said metabolic products; cell size; cell density, and the presence of autoantibody-reactive cells. (Item XC2) The method described in Item XC1, wherein at least three cell indicators are used. (Item XC3) The method described in Item XC1 or XC2, wherein the cell indicator includes cell size, cell density, or a combination thereof. (Item XC4) The method described in any one of Items XC1 to XC3, wherein the cellular indicator includes a combination of at least one of a cell surface marker, a proteinaceous product and a biological substance associated with the product, at least one miRNA, and at least one cellular metabolite and a biological substance associated with the metabolite. (Item XC5) The method according to any one of Items XC1 to XC4, further comprising identifying a subpopulation of the human functional cultured corneal endothelial cells based on a corneal functional characteristic. (Item XC6) The method according to any one of Items XC1 to XC4, further comprising a step of identifying a subpopulation of the cultured functional corneal endothelial cells based on the corneal functional characteristic according to any one of Items X1 to X4, X4A, X4B, X5 to X14, and X25 to X26 and / or Items X15 to X26. (Item XC7) A method according to any one of items XC5 to XC6, characterized in that multiple indicators are selected from each of the following: proteinaceous products and biological substances related to said products; secreted miRNA; cellular metabolites including amino acids and biological substances related to said metabolites; and fluctuations in the profile of each indicator are confirmed, thereby determining the homogeneity of cells having cell indicators including CD166 positive, CD133 negative, CD44 negative to CD44 weakly positive, and CD90 negative to weakly positive. (Item XC8) The proteinaceous product and its associated biological material are: (A) Expression is elevated in human functional corneal endothelial cells, which can induce human corneal functional characteristics when transplanted into the anterior chamber of the human eye. COL4A1, COL4A2, COL8A1, COL8A2, CDH2, and TGF-β2; and (B) Expression is reduced in human functional corneal endothelial cells, which can induce human corneal functional characteristics when transplanted into the anterior chamber of the human eye. The method according to any one of items XC1 to XC7, wherein the IL-13A1 is selected from the group consisting of MMP1, MMP2, TIMP1, BMP2, IL13RA2, TGF-β1, CD44, COL3A1, IL6, IL8, HGF, THBS2, and IGFBP3. (Item XC9) The exosomes are the following cellular indicators: (A) Substances whose expression is decreased in human functional corneal endothelial cells that can induce human corneal functional characteristics when transplanted into the anterior chamber of the human eye: The method according to any one of items XC1 to XC8, comprising at least one indicator selected from the group consisting of CD63, CD9, CD81 and HSP70. (Item XC10) The cellular metabolites and the associated biological substances of the metabolites are as follows: at least one selected from the group consisting of succinic acid, Pro, Gly, glycerol 3-phosphate, Glu, lactic acid, argininosuccinic acid, xanthine, N-carbamoyl aspartic acid, isocitrate, cis-aconitic acid, citric acid, Ala, 3-phosphoglyceric acid, hydroxyproline, malic acid, uric acid, betaine, folic acid, Gln, 2-oxoisovaleric acid, pyruvate, Ser, hypoxanthine, Asn, Trp, Lys, choline, Tyr, urea, Phe, Met, carnosine, Asp, ornithine, Arg, creatine, 2-hydroxyglutamic acid, β-Ala, citrulline, Thr, Ile, Leu, Val, creatinine, His, N,N-dimethylglycine, or combinations or relative ratios thereof; The method according to any one of items XC1 to XC9. (Item XC11) The method described in Item XC10, wherein the cellular metabolites and the related biological substances of the metabolites include an increase in serine, alanine, proline, glutamine or citrate / lactate ratio in the culture supernatant. (Item XC12) A method for detecting non-functional corneal endothelial cells present in cultured human corneal endothelial cells, comprising a step of measuring at least one cell function indicator selected from the group consisting of cell surface markers; proteinaceous products and biological substances related to said products; SASP-related proteins; intracellular and secreted miRNA; exosomes; cellular metabolic products including amino acids and biological substances related to said metabolic products; cell size; cell density, and the presence of autoantibody-reactive cells. (Item XC13) A quality evaluation agent, process control agent, or corneal endothelial non-functional cell detection agent for mature differentiated corneal endothelial functional cells, comprising a reagent or means for measuring the cell indicator described in any one of Items XC1 to XC12. (Item XC14) The quality assessment agent, process control agent or detection agent according to Item XC13, wherein the measuring means is labeled. (Item XC15) A) providing a sample that may contain human functional corneal endothelial cells that can induce human corneal functional characteristics when transplanted into the anterior chamber of a human eye; B) a step of determining whether the sample contains human functional corneal endothelial cells capable of inducing human corneal functional characteristics when transplanted into the anterior chamber of the eye using a quality evaluation agent, process control agent, or non-functional corneal endothelial cell detection agent described in item XC13 or XC14, wherein, when the evaluation result using the quality evaluation agent, process control agent, or non-functional corneal endothelial cell detection agent indicates that the cells are human functional corneal endothelial cells capable of inducing human corneal functional characteristics when transplanted into the anterior chamber of the eye, a step of determining that the sample contains human functional corneal endothelial cells capable of inducing human corneal functional characteristics when transplanted into the anterior chamber of the eye; C) A step of selecting cells determined to be human functional corneal endothelial cells capable of inducing human corneal functional properties when transplanted into the anterior chamber of the eye. A method for selecting human functional corneal endothelial cells, comprising: (Item XC16) A) a step of obtaining information about the cell indicators of human functional corneal endothelial cells of cells provided as human functional corneal endothelial cells capable of inducing human corneal functional properties when transplanted into the anterior chamber of the eye, using the quality evaluation agent, process control agent, or non-functional corneal endothelial cell detection agent described in either Item XC13 or XC14; and B) determining, based on the information, that the provided cells are human functional corneal endothelial cells capable of inducing human corneal functional properties when transplanted into the anterior chamber of the eye; A method for assaying the quality of human functional corneal endothelial cells, comprising: (Item XC17) A method for controlling the quality of a preparation of human functional corneal endothelial cells capable of inducing human corneal functional properties when transplanted into the anterior chamber of the eye, comprising: A) a step of obtaining information regarding the cell indicators of mature, differentiated, functional corneal endothelial cells of the cells obtained in the preparation using the quality evaluation agent, process control agent, or corneal endothelial non-functional cell detection agent described in item XC13 or XC14; and B) determining, based on the information, that the preparation is suitable for preparing human functional corneal endothelial cells capable of inducing human corneal functional properties when transplanted into the anterior chamber of the eye. (Item XC18) A method for assaying the purity of human functional corneal endothelial cells that can induce human corneal functional characteristics when transplanted into the anterior chamber of the eye, comprising: A) providing a sample that may contain human functional corneal endothelial cells that can induce human corneal functional characteristics when transplanted into the anterior chamber of the eye; B) A step of obtaining information regarding the cell indicators of human functional corneal endothelial cells of the cells using the quality assessment agent, process control agent, or non-functional corneal endothelial cell detection agent described in item XC13 or XC14; and C) calculating the purity of human functional corneal endothelial cells in the sample that can induce human corneal functional properties when transplanted into the anterior chamber of the eye based on the information; The method includes: (Item XC19) A) a step of culturing in a culture medium cells provided as mature differentiated human functional corneal endothelial cells capable of inducing human corneal functional properties when transplanted into the anterior chamber of the eye, and obtaining information about the cell indicators of the human functional corneal endothelial cells of the cells using the quality assessment agent, process control agent, or non-functional corneal endothelial cell detection agent described in either Item XC13 or XC14; and B) determining, based on the information, that the culture medium is suitable for producing human functional corneal endothelial cells capable of inducing human corneal functional properties when transplanted into the anterior chamber of the eye; A method for testing the quality of a culture medium for human functional corneal endothelial cells, comprising: (Item XC20) A) a step of culturing cells provided as human functional corneal endothelial cells capable of inducing human corneal functional properties when transplanted into the anterior chamber of the eye in a cell injection vehicle, and obtaining information about the cell indicators of the human functional corneal endothelial cells of the cells using the quality assessment agent, process control agent, or non-functional corneal endothelial cell detection agent described in either Item XC13 or XC14; and B) determining, based on the information, that the cell injection vehicle is suitable for cell transfer therapy. (Item XC21) Below: (1) Purity test of culture supernatant by ELISA TIMP-1: 500ng / mL or less IL-8: 500pg / mL or less PDGF-BB: 30pg / mL or more MCP-1: 3000pg / mL or less (2) Purity test by cell FACS CD166=95% or more CD133=5% or less CD105 negative ~ low positive = 95% or more CD44 negative ~ low positive = 70% or more CD44 moderate to high positivity = 15% or less CD24=5% or less CD26 positive = 5% or less CD200=5% or less (3) Barrier function (ZO-1) positive (4) Pump function (Na+ / K+ATPase) positive (5) Cell viability Trypan blue staining: 70% or more (6)Cell morphology No transformed cells detected in visual examination (7)Claudin10 positive (8) Effector cell (E-ratio) > 50% (9) Non-target cells Non-target cells A (strongly CD44-positive cells) <15%, non-target cells B (CD26-positive cells) <5%, non-target cells C (CD24-positive cells) <5% (10) Negative karyotype abnormality A method for quality control or process control of cultured human functional corneal endothelial cells that can induce human corneal functional properties when transplanted into the anterior chamber of the human eye, or a method for detecting non-functional corneal endothelial cells mixed in with cultured human corneal endothelial cells, comprising a step of confirming one or more of the following: (Item XC22) The method described in Item XC21, wherein the confirmation is performed 3 weeks or more immediately before cell injection therapy or during conservative culture with only medium change. (Item XC23) The confirmation includes being performed approximately 7 days before or immediately before cell infusion therapy. , the method described in item XC21 or XC22. (Item XC24) Characterized by one or more of the features described in Items XC19 to XC21 The method according to any one of items XC15 to XC22. (Item XC25) A method for quality control or process control of cultured human functional corneal endothelial cells that can induce human corneal functional characteristics when transplanted into the anterior chamber of the human eye, comprising steps of determining one or more characteristics of the target cells: (1) retention of endothelial pump and barrier function, (2) adhesion and binding to specific laminins, (3) cytokine profile produced, (4) metabolic product profile produced, (5) saturation cell density during in vitro culture, (6) spatial size and distribution of cells obtained during culture, and (8) cell maintenance when transferred to a mouse cornea after liquid nitrogen freeze injury. (Item XC26) The method described in Item XC25, wherein the maintenance of the endothelial pump and barrier function is determined using a pump function measurement method or barrier function measurement method commonly used for corneal endothelium. (Item XC27) The determination of adhesion and binding properties to the specific laminin is based on the adhesion and / or binding properties to laminin 511 (a complex of α5 chain, β1 chain, and γ chain 1), laminin 521 (a complex of α5 chain, β2 chain, and γ chain 1) or functional fragments thereof. The method according to Item XC25 or XC26, wherein the determination is made using an increase in tegrin expression as an index. (Item XC28) The method according to Item XC23, wherein the determination of the cytokine profile produced comprises measuring the production level of the cytokine profile in serum or aqueous humor. (Item XC29) The method according to any one of Items XC25 to XC28, wherein determining the metabolic product profile produced comprises measuring the metabolic product production level of the cells. (Item XC30) The method according to any one of Items XC25 to XC28, wherein determining the produced microRNA (miRNA) profile comprises obtaining total RNA and obtaining its microRNA expression profile. (Item XC31) A method according to any one of items XC25 to XC30, wherein determining the saturation cell density during in vitro culture comprises counting cells in an image of the cells obtained using an image acquisition system. (Item XC32) A method described in any one of items XC25 to XC31, wherein determining the spatial size and distribution of the cells obtained during the culture comprises counting the cells in an image of the cells obtained using an image acquisition system. (Item XC33) The method for determining cell maintenance when cells are transferred after liquid nitrogen freeze damage to the mouse cornea includes injecting the cells to be determined into the anterior chamber of a model prepared by pretreating the central region of the mouse cornea with cryodamage and removing endothelial cells, clinically observing the characteristics of the cornea, evaluating the thickness of the cornea using a pachymeter, and histopathologically examining the adhesion of HCECs using human nuclear staining, and confirming whether the cells are functional.

[0006] It is contemplated that one or more of the above-described features may be provided in combinations other than those explicitly stated. Still further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0007] This treatment represents a paradigm shift in corneal endothelial regenerative medicine, and as a versatile medical treatment that can be deployed internationally, it has the potential to be applied to more than one million patients worldwide. do. In the brief description of the figures below, with regard to the expression intensity of cell surface markers, -, +, ++, +++ indicate negative, weakly positive, moderately positive, and strongly positive, respectively. ± is included in - (negative) in this specification. Neg, low, med, and high indicate negative, weakly positive, moderately positive, and strongly positive, respectively. Weakly positive (also refers to low in this specification) and moderately positive (also refers to moderate in this specification) are also used. Strong positivity (also referred to herein as high positivity) was determined as follows: PE-Cy7-labeled anti-human CD44 antibody (BD Biosciences) was used to detect seroconversion by FACS Canto. When the area scaling factor for the blue laser in step II was set to 0.75 and the voltage for PE-Cy7 was set to 495, the weak fluorescence intensity range was approximately less than 3800, the medium fluorescence intensity range was approximately 3800 to less than 27500, and the strong fluorescence intensity range was approximately 27500 or more. A negative control (isotype control) with the same staining intensity pattern was considered negative, while a slight shift was considered positive. The mean fluorescence intensity of the negative control (isotype control) under these settings was approximately 50 (55 ± 25 range). The other fluorescent dyes were set as follows: area scaling factors: FSC = 0.5, blue laser = 0.75, red laser = 0.8; voltages: FSC = 270, SSC = 400, FITC = 290, PE = 290, PerCP-Cy 5.5 = 410, PE-Cy 7 = 495, APC = 430. Spillover of each fluorescence into other fluorescence was corrected using BD comp Beads (BD Biosciences) and FACS DiVa software. The mean fluorescence intensities of the negative controls (isotype controls) were as follows: FITC, approximately 130; PE, approximately 120; PerCP-Cy5.5, approximately 120; PE-Cy7, approximately 50; and APC, approximately 110. When using Lyoplate experiments (Examples, Table 2), an Alexa Fluor 647-labeled secondary antibody was used for detection. (Included in the kit) In this case, the median fluorescence intensity of each marker / median fluorescence intensity of the negative control (stained with isotype control antibody) is defined as -, +, ++, and +++ if the value is less than 5, 5 to less than 10, 10 to less than 30, or 30 or more, respectively. [Brief explanation of the drawings]

[0008] [Figure 1-A]Figure 1-A shows the change in subpopulation (SP) composition depending on the passage number. The results of FACS analysis and phase-contrast micrographs are shown for primary cultures of HCEC #82 and passages 1 to 3. The vertical axis of the graph represents the percentage of cells selectively expanded in each gate relative to the total cell count. The gate conditions were as follows: Gate 1: CD24-CD44- to CD105-CD166+, Gate 2: CD24-CD44++CD105+CD166+, Gate 3: CD24-CD44+++CD105++CD166+, Gate 4: CD24+CD44+ to CD105+CD166+, Gate 5: CD24+CD44+++CD105++CD166+. [Figure 1-B] Figure 1-B shows the change in subpopulation (SP) composition depending on the passage number. The results of FACS analysis and phase-contrast micrographs of primary cultures and the first to third passages of HCEC #88 are shown. The vertical axis of the graph indicates the percentage of cells selectively expanded in the culture at each gate relative to the total cell count. The gate conditions were the same as in Figure 1-A. [Figure 1-C] Figure 1-C shows the change in subpopulation (SP) composition depending on the passage number. The results of FACS analysis and phase-contrast micrographs of primary cultures and 1st to 3rd passages of HCEC #83 are shown. The vertical axis of the graph indicates the percentage of cells selectively expanded in the culture at each gate relative to the total cell count. The gate conditions were the same as in Figure 1-A. [Figure 1-D] Figure 1-D shows the change in subpopulation (SP) composition depending on the passage number. The results of FACS analysis and phase-contrast micrographs of primary cultures and 1st to 3rd passages of HCEC #84 are shown. The vertical axis of the graph indicates the percentage of cells selectively expanded in the culture at each gate relative to the total cell count. The gate conditions were the same as in Figure 1-A. [Figure 2-A] Figure 2-A shows representative FACS analyses demonstrating changes in subpopulation composition depending on the passage number. The results of FACS analysis of CD44, CD166, CD24, and CD105 expression were obtained for primary cultures and the first to third passages of HCEC #82. [Figure 2-B]Figure 2-B shows representative FACS analysis results demonstrating the changes in subpopulation composition depending on the passage number. The results show the expression of CD44, CD166, CD24, and CD105 in primary culture and the first to third passages of HCEC #83. [Figure 3] Figure 3 shows the marker expression and morphology of subpopulations characterized by CD44 and CD24 expression. Nuclei were stained with hematoxylin. From top to bottom, bright-field images for Na+ / K+ ATPase (developed with 3,3'-diaminobenzidine [DAB], brown), bright-field images for ZO-1 (developed with 3,3'-diaminobenzidine [DAB], brown), and phase-contrast microscopy images are shown. From left to right, C19 (passage 2) is CD24-CD44-~+, C16 (passage 3) is CD24-CD44++, C17 (passage 3) is CD24+CD44+++, and C18 (passage 2) is CD24+CD44+++. [Figure 4] Figure 4 shows FACS analysis of CD200 and CD44 expression in each culture. In the dot plots, the horizontal axis shows the logarithmic expression intensity of human CD44 for each culture, and the vertical axis shows the logarithmic expression intensity of CD200. Also shown are dot plots showing the logarithmic expression intensity of mouse IgG as a control on both the vertical and horizontal axes. In the histograms, the horizontal axis shows the logarithmic expression intensity of CD200 or CD44 together with the expression intensity of mouse IgG (control, gray) for each culture, and the vertical axis shows the corresponding cell number. [Figure 5]Figure 5 shows the results of FACS analysis, demonstrating that the expression of surface HLA class I antigens decreased along with the decrease in CD26 and CD44 expression in each culture. We investigated which subpopulations were suitable for patient infusion based on the expression of immune rejection-related molecules. In the dot plots, the horizontal axis represents the logarithmic expression intensity of human CD44 for each culture, and the vertical axis represents the logarithmic expression intensity of CD26. In the histograms, the horizontal axis represents the logarithmic expression intensity of HLA class I antigen for each culture, and the vertical axis represents the corresponding cell number. The colors correspond to the cells shown in the CD26 and CD44 dot plots. The numbers in the histograms represent the mean fluorescence intensity (MFI) of each histogram. The arrowheads indicate lower expression of CD44neg~low compared to CD44high. [Figure 6-A] FIG. 6-A shows the results of FACS analysis of the expression of CD44, CD166, CD24, and CD105 in corneal tissue (from a 71-year-old donor) immediately after tissue excision and preparation of a single cell suspension. [Figure 6-B] Figure 6-B shows the results of immunohistochemical staining showing the expression of markers in corneal tissue (derived from a 65-year-old donor) treated in the same manner as in Figure 6-B. Figure 6-B shows staining for LGR5, CD24, and CD26 (top row, from left), and CD166, CD44, and control (isotype control) (bottom row, from left), superimposed with DAPI staining. The scale bar is 100 μm. [Figure 7]Figure 7 shows that cHCECs derived from subpopulations with different CD44 intensities exhibit distinct morphologies. The upper left shows a phase-contrast micrograph of the cultured cells before gating by FACS. The upper right shows FACS analysis of the cultured cells for CD44 and CD24. Gate A contained 12.8% of the cells, while gate B contained 61.1% of the cells. The lower panels show phase-contrast and fluorescent micrographs of the cultured cells from each gate. The upper panel shows cultured cells with moderate to high CD44 expression obtained from gate A, and the lower panel shows cultured cells with low CD44 expression obtained from gate B. From the left, phase-contrast and bright-field images stained with DAPI and anti-Na+ / K+ ATPase antibody are shown on days 3, 10, and 17. [Figure 8] Figure 8 shows a comparison of gene expression in two different subpopulations of cultured endothelial cells that underwent effector cell differentiation or CST. Gene signatures were compared using hierarchical clustering and displayed as a heatmap. Red indicates relatively high expression, and green indicates relatively low expression. [Figure 9] Figure 9 shows some of the results of FACS analysis of cell surface marker expression in different cell subpopulations among cHCECs. Figure 9 shows histograms representing the expression intensity of each marker for two FACS-gated subpopulations (CD166+CD105-CD24-CD44-~+ (blue) and CD166+CD105-CD24+CD44+++ (red)) of #154 (first passage, top) and #127D (sixth passage, bottom). The horizontal axis shows the expression intensity of CD73, CD13, CD147, or CD200 in logarithmic scale, along with the expression intensity of the negative control (labeled with isotype control antibody, gray). The vertical axis shows the corresponding cell number. [Figure 10-A]Figure 10-A shows the correlation between the effector cell percentage (E ratio) and other donor parameters. In each graph, each plot represents a different tissue donor, and the vertical axis represents the E ratio in the first passage culture. In the upper panel, the horizontal axis represents the donor's endothelial cell density, and the correlation coefficient between the E ratio and the donor's endothelial cell density is 0.4107. In the middle panel, the horizontal axis represents the donor's age, and the correlation coefficient between the E ratio and the donor's age is 0.7333. In the lower panel, the horizontal axis represents the cell death during storage, and the correlation coefficient between the E ratio and the cell death during storage is 0.0015. [Figure 10-B] Figure 10-B shows the results of FACS analysis of five different lots of cultured HCE cells for three types of immune rejection-related molecules. Figure 10-B examines which subpopulations are suitable for infusion into patients based on the expression of immune-related molecules. The horizontal axis represents the expression intensity of each immune rejection-related molecule, and the vertical axis represents the corresponding cell count. Figure 10-B shows, from left to right, cultured human corneal endothelial cells C18, C19, C16, C17, and #118. The red histograms from top to bottom show the expression of HLAI, HLAII, and PDL1, and the MFI represents the mean fluorescence intensity for these molecules. The gray histogram represents the control. HLAI and PDL1 are positive in all cultured human corneal endothelial cells, but HLAII is nearly negative. [Figure 10-C] Figure 10-C shows fluorescence and phase-contrast microscopy images of cultured cells derived from human corneal endothelial tissue containing cells that have undergone cell phase transition. The upper left shows a fluorescence microscopy image of cells reacted with serum from a healthy donor labeled with anti-human IgG antibody, the upper center shows a fluorescence microscopy image of cells labeled with anti-human IgM antibody, and the upper right shows a fluorescence microscopy image of cells labeled with DAPI. The lower left shows a superposition of the three fluorescence microscopy images in the upper row, and the lower right shows a phase-contrast microscopy image. IgG and IgM are partially bound to cultured cells derived from human corneal endothelial tissue that have undergone phase transition, indicating the presence of natural antibodies in human serum against cells that have undergone cell phase transition and are unsuitable for injection therapy. [Figure 11-A]Figure 11-A shows that CD44 expression gradually decreases with prolonged primary culture, showing, from the left, FACS analysis of CD166, CD24, CD105, and CD44 in cultures harvested at weeks 1, 2, and 3 of culture. [Figure 11-B] Figure 11-B shows the effects of the addition of [(R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate] (Y-27632) on CD166, CD24, CD105, and CD44 by FACS analysis and phase-contrast microscopy. Y(+) indicates the addition of Y-27632, and Y(-) indicates the absence of Y-27632. The scale bar indicates 200 μm. [Figure 12-A] Figure 12-A shows that the addition of Y-27632 during culture enriches a cell subpopulation with a small cell area. The left image shows the case without Y-27632, and the right image shows the case with Y-27632. The top image shows a phase-contrast photograph of the culture on day 47 after washing with PBS, and the bottom image shows the cell area identified using the BZ-H3C Hybrid cell counting software. [Figure 12-B] Figure 12-B is a histogram showing that the addition of Y-27632 during culture enriched the cell subpopulation with a small cell area. -Y indicates the case without Y-27632, and +Y indicates the case with Y-27632. The vertical axis indicates the cell number, and the horizontal axis indicates the cell area. [Figure 13]Figure 13 shows an example of karyotypic aneuploidy. The upper left image shows a normal karyotype. The upper right image shows loss of a Y chromosome. The lower image shows trisomy of chromosome 20. Examining the slide in the upper left image reveals that 30 of the 30 cells counted had 46 chromosomes. Detailed karyotyping revealed that 20 of the cells had 46 chromosomes, XX. Examining the slide in the upper right image reveals that 50 of the 50 cells counted had 45 chromosomes. Detailed karyotyping revealed that 20 of the cells had 45 chromosomes, X, -Y. Examining the slide in the lower image reveals that 33 of the 50 cells counted had 46 chromosomes, and 17 of the 50 cells had 45 chromosomes. Detailed karyotyping revealed that 8 of the 20 cells had 47 chromosomes, XX, and 12 of the 20 cells had 46 chromosomes, XX. [Figure 14] Figure 14 shows a typical example of karyotype classification. Phase-contrast microscope images and detailed karyotypes of each cultured cell are shown. a) is a third-passage cHCEC derived from a 58-year-old woman; b) is a second-passage cHCEC derived from a 23-year-old man; c) is a second-passage cHCEC derived from a 23-year-old man; and d) is a third-passage cHCEC derived from a 15-year-old woman. [Figure 15] Figure 15 shows phase contrast microscopy images of different cHCECs. The top row shows the second passage from a 29-year-old male donor, where the cells exhibit hexagonal morphology and no signs of CST. The middle row shows the third passage from a 22-year-old female donor, where the cells exhibit abnormal CST-like morphology. The bottom row shows the fifth passage from a 9-year-old male donor, where the cells exhibit abnormal CST-like morphology. ECD refers to the cell density (cells / µm2) in the culture. [Figure 16] Figure 16 shows FACS analysis of different cHCECs. The FACS analysis results in a, b, and c correspond to the cultured cells shown in the phase-contrast microscopy images in Figure 15a, b, and c, respectively. The top row shows a culture containing a subpopulation enriched in CD44-. The middle row shows a subpopulation that is mostly CD44+++, representing a culture containing cells that strongly express CD24. The bottom row shows a culture containing cells that strongly express CD26. [Figure 17] Figure 17 shows phase-contrast microscopy and chromosome images demonstrating karyotypic aneuploidy observed when specific subpopulations were cultured. (A) The CD44+++, CD166+, CD24-, CD26+ subpopulation exhibited loss of sex chromosomes. (B) The CD44+++, CD166+, CD24+, CD26-(G3) subpopulation exhibited frequent trisomies on chromosomes 6, 7, and 8. (C) The CD44-, CD166+, CD105-, CD24-, CD26-(G1) subpopulation did not exhibit aneuploidy. [Figure 18] FIG. 18 shows phase contrast microscopy images of four types of cHCECs (C01, #87, C03, and #C04) that characterize cHCECs derived from different donors. [Figure 19] FIG. 19 shows the results of FACS analysis of CD44, CD166, CD24, CD26, and CD105 of two types of cHCECs (#2 from a 57-year-old donor and #4 from a 58-year-old donor) that characterize cHCECs derived from different donors. [Figure 20] Figure 20 shows bright-field immunostained images (DAB development) of two types of cHCECs (#2 from a 57-year-old donor and #4 from a 58-year-old donor) characterizing cHCECs derived from different donors. The images show antibody staining for Na+ / K+ ATPase, ZO1, Claudin 10, and CD26. Nuclei were stained with hematoxylin. [Figure 21] FIG. 21 shows the subpopulation distribution when cultured in the presence and absence of Y-27632. [Figure 22-A] Figure 22-A shows that culturing in the absence of the TGF-β inhibitor SB431542 did not induce morphological changes in cultured cells (CST). Figure 22-A shows phase-contrast microscopic images and FACS analysis results for CD44, CD166, CD24, CD26, and CD105 with and without the addition of SB431542. SB4(+) indicates the addition of SB431542, and SB4(-) indicates the absence of SB431542. [Figure 22-B]Figure 22-B shows phase-contrast microscopic images and FACS gating results of cHCECs derived from two different donors (both 22 years old). For each cHCEC, the phase-contrast microscopic image is shown on the left, and the FACS gating results are shown on the right. The cHCECs were cultured in the presence of Y-27632. The scale bar indicates 100 μm. [Figure 22-C] Figure 22-C shows the results of culturing cells collected from a 71-year-old subject with continuous addition of Y-27632 throughout the culture period, as in Figure 22-B. Photographs of the cells are shown on the left, and FACS gating results for CD44, CD166, CD24, CD26, and CD105 are shown on the right. The scale bar indicates 100 μm. [Figure 22-D] Figure 22-D shows representative microscopic images of cHEHCs treated with two drugs. In the upper panel, the upper panel shows fluorescence microscopic images of cHEHCs treated with trichostatin A (TSA), and the lower panel shows fluorescence microscopic images of cHEHCs treated with Y-27632. Figure 22-D shows, from left to right, ZO-1 fluorescence, Na+ / K+ ATPase fluorescence, DAPI fluorescence, and an overlay of these images. The bar indicates 100 μm. The lower panel shows phase-contrast microscopic images of cHEHCs treated with trichostatin A (TSA) or Y-27632, respectively. [Figure 23]Figure 23a shows phenotypically transformed cells that are positive for c-Myc. The left image shows a phase-contrast image, the center image shows c-Myc fluorescence, and the right image shows DAPI fluorescence. The upper and lower images show different microscopic images. Immunocytochemical evaluation of c-Myc expression in bulk-cultured cHCECs revealed c-Myc expression in the morphologically transformed cell-like areas under a phase-contrast microscope. Figure 23b shows glucose uptake in bulk-cultured cHCECs analyzed by flow cytometry. Detached cHCECs were incubated with 600 μM 2NBGD for 5, 10, and 30 minutes at 37°C. The culture medium was then replaced with fresh glucose-depleted medium for 15 minutes. After washing twice with cold FACS buffer (PBS containing 1% BSA), the cells were resuspended in ice-cold FACS buffer, and subjected to flow cytometry. Samples were analyzed using a BD FACS Canto II (BD Biosciences) in the FITC range (excitation 490 nm, emission 525 nm bandpass filter). The mean fluorescence intensity of the different groups was analyzed using BD FACS Diva software and corrected for autofluorescence from unlabeled cells. All incubation times showed a single peak of 2-NBGD uptake. From left to right, the unincubated group, the 5-minute incubation group, the 10-minute incubation group, and the 30-minute incubation group are shown. [Figure 24-A] Figure 24-A shows morphological changes detected by phase contrast microscopy in culture of cHCECs in glucose-starved DMEM without FBS in the presence of lactate. After passage to P3 under normal culture conditions, the cultured cells were incubated in DMEM containing 10 mM lactate but without glucose for 72 hours, and the resulting cHCECs were then further cultured under normal conditions for 4 weeks at three different dilution passages (1:3, 1:9, 1:30). [Figure 24-B]Figure 24-B shows immunohistochemical staining of Na+ / K+-ATPase before and after glucose starvation to assess the partial loss of a subpopulation of cHCECs in bulk culture. Na+ / K+-ATPase was used as a functional marker of HCECs. The efficiency of recovery from the loss effect clearly depended on the concentration of added lactate. [Figure 25-A] Figure 25-A is a photograph showing the change in morphology of cHCEC (#55) before and after Lac treatment. Left: before Lac treatment. Right: after Lac treatment. [Figure 25-B] Figure 25-B shows the results of quantitative real-time PCR analysis of gene expression related to CST, including EMT, cellular senescence, and fibrosis, in cHCECs (#55) before and after Lac treatment. The expression intensity is shown relative to the expression intensity of each gene before treatment, which is set to 1. [Figure 25-C] Figure 25-C shows the results of quantitative real-time PCR analysis of gene expression related to CST, including EMT, cellular senescence, and fibrosis, in cHCECs (#55) before and after Lac treatment. The expression intensity is shown relative to the expression intensity of each gene before treatment, which is set to 1. [Figure 25-D] Figure 25-D shows changes in the morphology of cHCEC (#72) before and after Lac treatment. Left: before Lac treatment. Right: after Lac treatment. [Figure 25-E] Figure 25-E shows the results of quantitative real-time PCR analysis of the expression of genes related to CST, such as EMT, cellular senescence, and fibrosis, in cHCECs (#72) before and after Lac treatment. The expression intensities are shown relative to the expression intensity of each gene before treatment, which is set to 1. [Figure 25-F] Figure 25-F shows the results of quantitative real-time PCR analysis of the expression of genes related to CST, such as EMT, cellular senescence, and fibrosis, in cHCECs (#72) before and after Lac treatment. The expression intensities are shown relative to the expression intensity of each gene before treatment, which is set to 1. [Figure 25-G]Figure 25-G shows the results of quantitative real-time PCR analysis of the expression of genes related to CST, such as EMT, cellular senescence, and fibrosis, in cHCECs (#72) before and after Lac treatment. The expression intensities are shown relative to the expression intensity of each gene before treatment, which is set to 1. [Figure 25-H] Figure 25-H shows the results of quantitative real-time PCR analysis of gene expression related to CST, including EMT, cellular senescence, and fibrosis, in cHCECs (#72) before and after Lac treatment. The expression intensity is shown relative to the expression intensity of each gene before treatment, which is set to 1. [Figure 26-A] FIG. 26-A shows the relative intracellular metabolite signal positioning in three lots of cHCEC (164P1, C16P6 and C21P3, which are effector cells, cell phase transition cells 1 and cell phase transition cells 2, respectively) as a PCA analysis. [Figure 26-B] Figure 26-B shows typical metabolites in the PC1 component of intracellular metabolite signals in three lots of cHCECs (164P1, C16P6 and C21P3, which are effector cells, cell phase transition cells 1 and cell phase transition cells 2, respectively). [Figure 26-C] Figure 26-C shows typical metabolites in the PC2 component of intracellular metabolite signals in three lots of cHCECs (164P1, C16P6 and C21P3, which are effector cells, cell phase transition cells 1 and cell phase transition cells 2, respectively). [Figure 26-D] Figure 26-D shows hierarchical clustering (HCA) using the normalized intensities of each metabolite in duplicate samples from each lot. High normalized intensities are shown in red, and low normalized intensities are shown in green. [Figure 26-E] Figures 26E, 26F, and 26G show the morphological differences of the three lots of cHCECs, along with flow cytometry analysis. The bottom panels show FACS analysis of each cHCEC, and the subpopulation composition of each lot, classified by the same criteria as in Figure 1, based on the expression of cell surface antigens. [Figure 26-F] Figures 26E, 26F, and 26G show the morphological differences of the three lots of cHCECs, along with flow cytometry analysis. The bottom panels show FACS analysis of each cHCEC, and the subpopulation composition of each lot, classified by the same criteria as in Figure 1, based on the expression of cell surface antigens. [Figure 26-G] Figures 26E, 26F, and 26G show the morphological differences of the three lots of cHCECs, along with flow cytometry analysis. The bottom panels show FACS analysis of each cHCEC, and the subpopulation composition of each lot, classified by the same criteria as in Figure 1, based on the expression of cell surface antigens. [Figure 26-H] Figure 26-H shows the lactate / pyruvate ratio and the amount of metabolites controlling intracellular physiological functions in each cHCEC lot. Markers include GSH / GSSG, total glutathione, NADP+, NADPH, and NADPH / NADP+. [Figure 27-A] Figure 27-A shows the characterization of metabolite changes in different lots of cHCECs cultured in the conditioned medium. Hierarchical clustering of the metabolomic profiles is shown, and clusters of metabolites correlated with the presence of CSTs are identified. The intensity of each metabolite is indicated by red (high) and green (low). The cluster was divided into at least four metabolite subclusters: from top to bottom: metabolites that increased in all cHCECs (#66, #72, #55); metabolites that increased mainly in #72 and #55 but not #66; metabolites that decreased most significantly in #66; and metabolites that were roughly present in all three groups. [Figure 27-B] FIG. 27-B is a graph showing that the lactate / pyruvate ratio is higher in cells at cell phase transitions 1 and 2 than in effector cells. [Figure 27-C] FIG. 27C is a microscopic image showing the morphology of #66, #72, and #55. [Figure 28-A]Figures 28-A and 28-B show the results of FACS analysis of four distinct cHCECs with different subpopulation compositions produced under GMP conditions, i.e., cHCECs without CD44+++ cells, CD24+ cells, or CD26+ cells. The reference values ​​for the expression intensities of CD166, CD24, CD26, CD44, and CD105 were as described above. Figure 28-A shows the composition of effector cells, moderately differentiated cells, and non-target cells in each lot, respectively, based on surface CD expression. [Figure 28-B] Figures 28-A and 28-B show the results of FACS analysis of four distinct cHCECs with different subpopulation compositions produced under GMP conditions, i.e., cHCECs without CD44+++ cells, CD24+ cells, or CD26+ cells. The reference values ​​for the expression intensities of CD166, CD24, CD26, CD44, and CD105 were as described above. Figure 28-B shows the composition of effector cells, moderately differentiated cells, and non-target cells in each lot, respectively, based on surface CD expression. [Figure 28-C] Figures 28-C and 28-D show the results of FACS analysis of four distinct cHCECs with different subpopulation compositions produced under GMP conditions, i.e., cHCECs without CD44+++ cells, CD24+ cells, or CD26+ cells. The reference values ​​for the expression intensities of CD166, CD24, CD26, CD44, and CD105 were as described above. Figure 28C shows the composition of effector cells, moderately differentiated cells, and non-target cells in each lot, respectively, based on surface CD expression. [Figure 28-D] Figures 28-C and 28-D show the results of FACS analysis of four distinct cHCECs with different subpopulation compositions produced under GMP conditions, i.e., cHCECs without CD44+++ cells, CD24+ cells, or CD26+ cells. The reference values ​​for the expression intensities of CD166, CD24, CD26, CD44, and CD105 were as described above. Figure 28-D shows the composition of effector cells, quasi-passive cells, and non-target cells in each lot, respectively, based on surface CD expression. [Figure 28-E]Figure 28-E shows hierarchical clustering of four cHCECs and conditioned media with different subpopulation compositions. The intensity of each metabolite is indicated by red for high and green for low. The cluster was divided into four metabolite subclusters. [Figure 28-F] FIG. 28-F shows PCA analysis of four cHCECs and conditioned media with different compositions of the subpopulations. [Figure 28-G] FIG. 28-G shows the main metabolites correlated with PC1 and PC2 in the PCA analysis of four cHCECs with different subpopulation compositions. [Figure 28-H] Figure 28-H is a graph showing, in the upper row, the difference in citrate / lactate ratio between C21, C22, C23, and C24, which differ only in the proportion of CD44- to CD44+ to CD44++, and, in the lower row, the difference in citrate / lactate ratio between #66, #55, and #72, which differ in the content of the CD44+++ subpopulation. [Figure 29-A] Figure 29-A shows microscopic images of 665C (effector cells), 3411 (a culture lot whose constituent cultured cell subpopulations are unknown), 675A (a culture lot composed of a cultured cell subpopulation in which cell phase transition is morphologically recognized), and C1121 (phase-transition cells containing island-like clusters (presumed to be senescent cells)). [Figure 29-B] Figure 29-B is a scatter plot of the relative amounts of various intracellular miRs, comparing the intracellular miR profiles detected using 3D gene (Toray) between effector cells (#66 P5) and cHCECs (2911, 3411, and 3511) whose constituent cell subpopulations are unknown. The horizontal axis represents the relative amount of miR in effector cells, and the vertical axis represents the relative amount of miR in culture lots whose constituent subpopulations are unknown. [Figure 29-C]Figure 29-C is a scatter plot of the relative amounts of various intracellular miR profiles detected using 3D gene (Toray) between effector cells (#66 P5) and cHCECs (675A1-A3) composed of a culture cell subpopulation in which cell phase transition is morphologically observed. The horizontal axis represents the relative amount of miR in the effector cells, and the vertical axis represents the relative amount of miR in the culture lot composed of a cHCEC subpopulation in which cell phase transition is morphologically observed. [Figure 29-D] Figure 29-D shows a scatter plot of the relative abundance of various intracellular miR profiles detected using 3D gene (Toray) between effector cells (#66 P5) and phase transition cells (C1121 and C1122) containing island-like clusters. The horizontal axis represents the relative abundance of miR in effector cells, and the vertical axis represents the relative abundance of miR in phase transition cells containing island-like clusters. [Figure 30-A] Figure 30-A shows the subpopulation composition of a5 subjected to 3D gene analysis based on CD44-CD24-CD26- subpopulations. The upper image shows the morphology of a5, and the lower image shows CD44-CD26- subpopulations measured by FACS. The reference values ​​for the expression intensities of CD166, CD24, CD26, CD44, and CD105 were as described above. a5 contains a subpopulation mainly composed of CD44-CD24-CD26- subpopulations. [Figure 30-B] Figure 30-B shows the subpopulation composition of a1 subjected to 3D gene analysis based on CD44+ / -CD24- / -CD26- expression measured by FACS. The upper image shows the morphology of a1, and the lower image shows CD44+ / -CD26- expression measured by FACS. The reference values ​​for the expression intensities of CD166, CD24, CD26, CD44, and CD105 were as described above. a1 contains a subpopulation mainly composed of CD44+ / -CD24- / -CD26-. [Figure 30-C]Figure 30-C shows the subpopulation composition of a2 subjected to 3D gene analysis based on CD44+++CD24-CD26++ subpopulation. The upper image shows the morphology of a2, and the lower image shows CD44 expression measured by FACS. The reference values ​​for the expression intensities of CD166, CD24, CD26, CD44, and CD105 were as described above. a2 contains a subpopulation mainly composed of the CD44+++CD24-CD26++ subpopulation. [Figure 31-A] FIG. 31-A is a table summarizing the relative expression intensities of various intracellular miRs of the miR378 family in a5, a1, and a2 cells. [Figure 31-B] Figure 31-B shows the classification of intracellular miRNAs into five classes based on the variation in expression intensity between cells. The expression of each intracellular miR is classified as shown below the graph. [Figure 32-A] Figure 32-A is a photograph showing the morphology of #66 P4 (effector cells, passage 4), #66 P5 (effector cells, passage 5), C11 P2 (passage 2), C09 P2 (passage 2), #55 P5 (passage 5), and #73 P2 (passage 2). [Figure 32-B] Figure 32-B shows the relative expression levels of miRs in the culture supernatants of cells #66 P4 (effector cells, passage 4), #66 P5 (effector cells, passage 5), C11 P2 (passage 2), C09 P2 (passage 2), #55 P5 (passage 5), and #73 P2 (passage 2). Secreted miRs exhibit characteristic patterns of expression change for each cell type. Relative expression intensities are expressed relative to the expression level in #66 P4 (effector cells, passage 4). [Figure 33-A] Figures 33-A to 33-B show the patterns of several secreted miRs, which have clear trends in expression per cell. Figure 33-A shows an overview of the changes in secreted miR expression patterns. [Figure 33-B] Figures 33-A to 33-B show several secreted miR patterns with clear trends in cell-specific expression. Figure 33-B shows a graph showing the specific expression intensities of secreted miRs belonging to each pattern. [Figure 34-A] Figure 34-A shows a comparison of miR profiles in fresh corneal endothelial tissues. Figure 34-A is a scatter plot showing a comparison of miR profiles between tissues with guttata and a medium ECD level and tissues with guttata and a low ECD level (ECD378). [Figure 34-B] Figure 34-B shows a comparison of miR profiles in fresh corneal endothelial tissue. Figure 34-B is a scatter plot showing a comparison of miR profiles between tissue with guttata and low ECD levels (ECD378) and normal tissue. [Figure 34-C] Figure 34-C is a graph showing the expression of miR-378a-5p in corneal epithelial tissue, corneal endothelial tissue from newborns, adolescents, and adults, and in corneal endothelial tissue from adults with different ECD levels and guttata. The 378 family of miRs, which were upregulated in corneal endothelial tissue compared to epithelial tissue, were dramatically reduced in endothelial tissue with lower ECD levels and guttata. [Figure 34-D] Figure 34-D is a graph showing the expression of miR-378f in corneal epithelial tissue, corneal endothelial tissue from newborns, adolescents, and adults, and in corneal endothelium from adults with different ECD levels and guttata. The 378 family of miRs, which were upregulated in corneal endothelial tissue compared to epithelial tissue, was dramatically reduced in endothelial tissue from tissues with lower ECD and guttata. [Figure 34-E] Figure 34-E is a graph showing the expression of miR-146b-5p in corneal epithelial tissue, corneal endothelium from newborns, adolescents, and adults, and in corneal endothelial tissue from adults with different ECD levels and guttata. [Figure 34-F] Figure 34-F is a graph showing the expression of miR-146b-3p in corneal epithelial tissue, corneal endothelial tissue from newborns, adolescents, and adults, and in corneal endothelial tissue from adults with different ECD levels and guttata. [Figure 34-G]The left column of Figure 34-G shows images of the morphology of cells with ECD378, ECD1552, and ECD2457. The right column of Figure 34-G shows the results of Q-RT-PCR for the miR378 family (a-3p, e, and f) in normal tissues and tissues with ECD795 and 1410. The expression intensity is shown as a relative expression level, with the expression intensity in normal tissues set to 1. [Figure 35-A] FIG. 35-A is a photograph showing the morphology of 66P5 (effector cells, passage 5) and 67P5 (cells in which CST is clearly observed, passage 5). [Figure 35-B] FIG. 35-B is a scatter plot showing the difference in expression intensity of various genes between 66P5 (effector cells, passage 5) and 67P5 (cells in which CST is clearly observed, passage 5). [Figure 35-C] Figure 35-C shows the results of pre-transfection of miR378a-3p or 5f mimics into a CD44+++ cHCEC subpopulation in which miR378a-3p or 5f expression had not been detected. Heat maps of gene signatures after transfection of the two miR mimics, as assayed by PCR array for senescence, EMT, fibrosis, p53, and EMA, are shown. Transfected cells showed upregulation of multiple gene signatures, including collagen, ITG and MMP families, and CD44. For each cell and each gene, red indicates relatively high expression, and green indicates relatively low expression. [Figure 35-D] Figure 35-D shows the classification of the expression patterns of secreted miRs for each subpopulation (effector, moderately differentiated, CD44+++). As shown in Figure 35-D, miRs in the culture supernatants for each subpopulation were classified into six patterns. [Figure 35-E]The upper panel of Figure 35-E shows the contents and morphology of subpopulations with different expression levels of CD markers in different culture lots of cHCECs, a1, a2, and a5. The lower panel of Figure 35-E is a volcano plot comparing the miR expression profiles among the culture supernatants of cultures a1, a2, and a3 shown in A. [Figure 36] Figure 36 shows a schematic diagram of the centrifugal cell adhesion assay for testing the binding ability of HCECs. Cultured HCECs were added to a U-bottom 96-well culture plate pre-coated with collagen, laminin, or proteoglycan, and the plate was then centrifuged. Adherent cells were evaluated under a phase-contrast microscope. [Figure 37] Figure 37 shows the binding of cultured HCECs to laminin in a centrifugation assay. The top row shows the results of binding to plates coated with 2 nM of laminin, and the bottom row shows the results of binding to plates coated with 5 nM of laminin. From left to right, laminin-521, laminin-411, laminin-332, and BSA are shown. [Figure 38] Figure 38 shows cultured HCECs bound to laminin-521 and laminin-511 in a concentration-dependent manner in a centrifugation assay. The top row shows cultured HCECs bound to laminin-521, and the bottom row shows cultured HCECs bound to laminin-511. From left to right, the laminin coating concentrations are 500 pM, 100 pM, 20 pM, 4 pM, 0.8 pM, and 0 pM. [Figure 39] Figure 39 shows cultured HCECs binding to type IV collagen in a concentration-dependent manner in a centrifugation assay. From left to right, the coating concentrations of type IV collagen are 4000 ng / mL, 1000 ng / mL, 250 ng / mL, 62.5 ng / mL, and 0 ng / mL. [Figure 40] Figure 40 shows the binding of cultured HCECs to various proteoglycans and glycoproteins in a centrifugation assay. From left, coating with agrin, nidogen-1, fibulin 5, TSP-1, perlecan, and BSA (all coated at a concentration of 400 nM) is shown. [Figure 41]Figure 41 shows the binding of laminin-411 to HCECs cultured in different media. From the left, Opti-MEM, Opeguard-MA, and BSS are shown. Laminin-411 concentrations of 5 nM, 1.25 nM, and 0 nM were used. [Figure 42] Figure 42 shows the change in binding affinity of cultured HCECs to laminin-511 with or without the addition of human serum albumin (HSA), ascorbic acid, or lactic acid (components of aqueous humor). Laminin-511 concentrations of 0.8 nM, 0.2 nM, and 0.05 nM were used. [Figure 43] Figure 43 shows the change in binding affinity of cultured HCECs to laminin-411 with or without the addition of human serum albumin (HSA), ascorbic acid, or lactic acid (components of aqueous humor). Laminin-411 was added at concentrations of 5 nM, 1.25 nM, and 0 nM. [Figure 44] Figure 44 shows a cHCEC subpopulation with a hexagonal shape that shows no signs of CST, prepared by magnetic bead cell sorting (MACS). The left panel shows a phase-contrast micrograph of the cHCEC subpopulation that shows no signs of CST; the upper scale bar indicates 500 μm, and the lower scale bar indicates 100 μm. The right panel shows the results of flow cytometry to measure the purity of the cHCEC subpopulation provided for analysis. [Figure 45] Figure 45 shows representative fluorescence microscopy images of two types of subpopulations. The upper row shows the subpopulations shown in Figure 44, and the lower row shows the subpopulations shown in Figure 46. From the left, the images show ZO-1 fluorescence, Na+ / K+ATPase fluorescence, DAPI fluorescence, and a merged image of these. The bar indicates 50 μm. [Figure 46] Figure 46 shows a subpopulation with an EMT phenotype prepared by magnetic bead cell sorting (MACS). The left panel shows a phase-contrast microscopic image of the subpopulation with an EMT phenotype, with the upper scale bar representing 500 μm and the lower scale bar representing 100 μm. The right panel shows the results of flow cytometry to measure the purity of the subpopulation provided for analysis. [Figure 47]Figure 47 shows a comparison of the binding ability of HCEC subpopulations to components of Descemet's membrane. Upper panel: Each subpopulation used was prepared under controlled culture conditions or by magnetic cell separation and confirmed by staining with cell surface markers. Then, a centrifugal cell adhesion assay was performed. A mature HCEC subpopulation and an EMT phenotype subpopulation were used. Lower panel: The binding ability of HCEC subpopulations to laminin and type IV collagen was compared by centrifugal cell adhesion assay. The binding index was calculated as follows: Binding index = (Δbasal (laminin or collagen) - ΔBSA) / ΔBSA, where Δ indicates area. [Figure 48] Figure 48 shows the expression of integrin α subunits in HCEC subpopulations. The upper row shows the expression of integrin α2, the middle row shows the expression of integrin α3, and the lower row shows the expression of integrin α6. The left column shows the mature phenotype, and the right column shows the EMT phenotype. [Figure 49-A] Figure 49-A shows a phase-contrast microscope image and the expression of cell surface markers of the cHCECs used in Example 7. FACS analysis was performed as follows: Cells were detached from the culture dish and analyzed by FACS for the expression of CD166, CD24, CD44, CD105, and CD26. [Figure 49-B] Figure 49-B shows a phase-contrast microscope image and the expression of cell surface markers of the cHCECs used in Example 7. FACS analysis was performed as follows: Cells were detached from the culture dish and analyzed by FACS for the expression of CD166, CD24, CD44, CD105, and CD26. [Figure 50]Figure 50 shows the endothelial nuclei at the endothelial surface, corneal transparency, and central corneal thickness after endothelial cryoinjury. Horizontally mounted corneas were stained with DAPI to observe the loss and recovery of mouse endothelial cells 24 to 72 hours after cryoinjury. (A) The white dotted lines indicate the area of ​​endothelial defect (a, d, g). The dotted and solid boxes (a, d, g) in the horizontally mounted tissues indicate the areas shown in Figure 50(b), (e), (h), and Figure 50(c), (f), (i), respectively. The arrows indicate the margin between normal and defective endothelium. (B) Clinical appearance after injection of 0 to 2.0 x 104 HCECs into freeze-injured eyes (24, 48, and 72 hours). (C) Corneal thickness before and after injection (24 hours, 48 ​​hours, and 72 hours) (*p < 0.05). [Figure 51] Figure 51(A) shows corneal transparency 48 hours after injection of cHCECs in different cell suspension vehicles. BALB / c eyes were cryoinjured, and 2.0 x 104 HCECs suspended in Opti-MEM (a) or Opeguard MA (b) were injected into the anterior chamber. As a control, Opeguard MA alone (c) without cells was injected into the anterior chamber (N = 3 for each). Figure 51(B) shows the same experiment as in (A), but with cells suspended in Opi-MEM (a) or Opeguard F (b). As a control, Opeguard F alone (c) without cells was injected into the anterior chamber (N = 3 for each). [Figure 52]Figure 52(A) shows corneal thickness after injection of cHCECs in different cell suspension vehicles. BALB / c eyes were cryoinjured, and 2.0 x 104 HCECs suspended in Opti-MEM (a) or Opeguard MA (b) were injected into the anterior chamber. Also, Opeguard MA alone (c) without cells was injected into the anterior chamber as a control (N = 3 for each). Figure 52(B) shows the same experiment as in Figure 52(A) except that cells were suspended in Opti-MEM (a) or Opeguard F (b). Also, Opeguard F alone (c) without cells was injected into the anterior chamber as a control (N = 3 for each). Corneal thickness was evaluated before injection, and 24 and 48 hours after injection. * indicates statistically significant difference (p < 0.05). [Figure 53] Figure 53(A) shows fluorescence microscopy images showing the adhesion of HCECs injected into the anterior chamber in different cell suspension vehicles. BALB / c eyes were cryoinjured, and 2.0 x 104 HCECs suspended in Opti-MEM (a) or Opeguard MA (b) were injected into the anterior chamber. Also, Opeguard MA alone (c) without cells was injected into the anterior chamber as a control (N = 3 for each). Figure 53(B) shows the same experiment as in Figure 53(A) except that cells were suspended in Opti-MEM (a) or Opeguard F (b). Also, Opeguard F alone (c) without cells was injected into the anterior chamber as a control (N = 3 for each). 48 hours later, corneal transparency and thickness were assessed, and immediately afterwards, these corneas were stained with anti-human nuclei antibody (to identify injected HCECs and host-derived CECs) and DAPI. The dotted circle indicates the cryoinjured area. DAPI (red), anti-human nuclei antibody (green), and overlay images show high magnification of the white boxed area in the DAPI (blue) image. [Figure 54-A(a)]Figures 54-A(a) and 54-A(b) show the results of analyzing human corneal endothelial (Endo) / epithelial (EP) tissues and cHCECs for their mRNA and miRNA signatures using 3D-Gene. The analysis was performed using Human_25K_Ver2.1 and Human_miRNA_Ver17. Figure 54-A(a) shows the correlation coefficients between the mRNA (upper left) and miRNA (upper right) signatures of six human corneal endothelial tissues and five human corneal epithelial tissues, as well as the correlation coefficients between the mRNA (lower left) and miRNA (lower right) signatures of fresh tissues from seven donors. [Figure 54-A(b)] Figures 54-A(a) to 54-A(b) show the results of analyzing the mRNA and miRNA signatures of human corneal endothelial (Endo) / epithelial (EP) tissues and cHCECs using 3D-Gene. The analysis was performed using Human_25K_Ver2.1 and Human_miRNA_Ver17. Figure 54-A(b) shows the correlation coefficients between the mRNA (left) and miRNA (right) signatures of six fresh tissues, three normal cHCECs, and three cHCECs undergoing cell phase transition. [Figure 54-B] Figure 54-B shows the results of analyzing human corneal endothelial (Endo) / epithelial (EP) tissues and cHCECs for their mRNA and miRNA signatures using 3D-Gene. The analysis was performed using Human_25K_Ver2.1 and Human_miRNA_Ver17. Scatter plots of gene and miR expression profiles of Endo, EP, and cHCECs are shown. Values ​​are the average of global normalized values. Lines representing 2-fold change and 1 / 2-fold change are shown in the scatter plots. [Figure 55-A]Figures 55-A to 55-B show a comparison of gene signatures between cHCECs without CST (#14, #18, #19), cHCECs with CST (#29, #34, #35), and fresh tissues (Endo tissues from 8 and 9 of 20Y and 12 of 12Y) using RT2 profiler PCR-Array for senescence, EMT, and fibrosis. In Figure 55-A, (1) shows phase-contrast micrographs of #14 (passage 3), #18 (passage 3), and #19 (passage 3), from the left. (2) shows phase-contrast micrographs of #29 (passage 1), #34 (passage 1), and #359 (passage 1), from the left. [Figure 55-B] Figures 55-A and 55-B show a comparison of gene signatures between cHCECs (#14, #18, #19) without CST, cHCECs (#29, #34, #35) with CST, and fresh tissues (8 and 9 of 20 years, and 12 of 12 Endo tissues) using RT2 profiler PCR-Array for senescence, EMT, and fibrosis. Figure 55-B shows microarray analysis of senescence, EMT, and fibrosis using mRNA extracted from cHCECs without CST, cHCECs with CST, and fresh Endo tissue. The gene signatures were compared using hierarchical clustering and displayed as heatmaps. Red indicates relatively high expression intensity, and green indicates relatively low expression intensity. [Figure 56-A] Figure 56-A shows the results of comparing the expression of individual mRNAs in two cHCECs (#66 passage 5 (effector cells) and #67 passage 5 with CST) by qRT-PCR. (a) is a phase-contrast microscope image of #66 passage 5 and #67 passage 5. These two cultures were morphologically different. (b) is the result of measuring and comparing the mRNA expression intensity for some of 50 candidate genes by qRT-PCR. In each bar graph, the left represents #66 passage 5, and the right represents #67 passage 5. The vertical axis represents the relative expression level of mRNA, with the expression intensity of #66 passage 5 set to 1. [Figure 56-B] Figure 56-B is a table summarizing genes whose mRNA expression intensities differed between #66 and #67. [Figure 57-A] Figure 57-A shows photographs of cultures submitted for analysis of selected genes by qRT-PCR. Figure 57-A shows phase-contrast microscopy images of each culture, along with the donor number, passage number, and score assigned for morphological classification of cHCECs. A higher score indicates higher quality cHCECs. [Figure 57-B] Figure 57B shows a graph of the expression intensity of selected genes by qRT-PCR among morphologically classified cHCECs. In Figure 57B, for each gene, the vertical axis shows the relative mRNA expression intensity, with the expression level of #66 passage 5 set to 1. The horizontal axis shows the type of culture from which mRNA was extracted. Cultures with a score of 10 are shown as light bars, and cultures with scores of 0 to 8 are shown as dark bars. [Figure 58-A] Figure 58-A shows photographs of cultures provided for analysis of selected cells by qRT-PCR among cHCECs produced at the cell processing center under GMP. (a) shows phase-contrast microscopy images of #66 (passage 5), C09 (derived from a 16-year-old donor) (passage 3), and C11 (derived from a 26-year-old donor) (passage 3). miR expression levels were evaluated by qRT-PCR. For each gene, in each bar graph, the bars represent, from left to right, #66 (passage 5) culture well A, #66 (passage 5) culture well C, C09 (derived from a 16-year-old donor) (passage 3), and C11 (derived from a 26-year-old donor) (passage 3). The vertical axis represents the relative expression intensity of mRNA, with the expression intensity of #66 (passage 5) culture well A set to 1. [Figure 58-B] Figure 58B is a table summarizing genes that were highly expressed in each culture as a result of analysis of cHCECs by qRT-PCR among cHCECs produced at the cell processing center under GMP. [Figure 59-A]Figure 59-A shows the results of Bio-Plex analysis of cytokine levels in the supernatants of cultured HCEC #82 (P0-P3, 72-year-old donor, ECD = 3192 / 3409), #84 (P0-P3, 75-year-old donor, ECD = 2598), and #88 (P0-P3, 10-year-old donor, ECD = 3879). A shows the quantitative results for IL-6, B shows IFN-γ, C shows MCP-1, D shows PDGF-bb, and E shows MIP-1b, respectively. [Figure 59-B] Figures 59-B to 59-C show ELISA assays of culture supernatants for quality assessment of cHCECs. Quantitation was performed in triplicate by ELISA. Graphs show the amount of IL8, PDGFbb, or MCP1 secreted in cultures C17, C18, C23, or C24. P1 indicates the first passage, P2 indicates the second passage, and P3 indicates the third passage. Quantitation was performed on cultures with various culture days. [Figure 59-C] Figures 59-B to 59-C show ELISA assays in culture supernatants for quality assessment of cHCECs. Quantification was performed in triplicate by ELISA. The amounts of IL8, PDGFbb, or MCP1 secreted in cultures C17, C18, C23, or C24 are shown. P1 indicates the first passage, P2 indicates the second passage, and P3 indicates the third passage. Quantification was performed on cultures with various culture days. [Figure 60-A] Figures 60-A and 60-B show ELISA assays of culture supernatants for quality assessment of cHCECs. Quantitation was performed in triplicate. The graphs show the amount of TIMP1, IL8, PDGFbb, or MCP1 secreted in each culture. From top to bottom, Figures 60-A and 60-B show C14 (passage 3, week 4), C15 (passage 3, week 4), C24 (passage 3, day 27), C23 (passage 2, day 31), and C32 (passage 2, day 45). [Figure 60-B]Figures 60-A and 60-B show ELISA assays of culture supernatants for quality assessment of cHCECs. Quantitation was performed in triplicate. The graphs show the amount of TIMP1, IL8, PDGFbb, or MCP1 secreted in each culture. From top to bottom, Figures 60-A and 60-B show C14 (passage 3, week 4), C15 (passage 3, week 4), C24 (passage 3, day 27), C23 (passage 2, day 31), and C32 (passage 2, day 45). [Figure 61-A] Figure 61-A shows a cytokine profile. It shows the profile of cytokine levels in the serum of a patient who was infused with low-quality cHCECs. A comparison of the profiles is shown before cHCEC infusion, two days after treatment, one week after treatment, and one month after treatment. The amount of cytokines present in the patient's serum at each time point is expressed as a relative value. This shows that low-quality cells induce unintended biological responses. [Figure 61-B] Figure 61-B is a cytokine profile diagram. It shows the profile of cytokine levels in the serum of a patient who was infused with low-quality cHCECs. This shows a different surgical example from Figure 61-A. This shows a comparison of serum cytokine profiles before cHCEC infusion, two days after surgery, one week after surgery, and one month after surgery. The amount of cytokines present in the patient's serum at each time point is expressed as a relative value. This shows that low-quality cells induce unintended biological responses. [Figure 62] Figures 62 and 63 show cytokine profile diagrams. The figures show the cytokine level profiles of the serum of patients infused with high-quality cHCECs. A comparison of the profiles is shown before cHCEC infusion, two days after treatment, and one week after treatment. The amount of cytokines present in the patient serum at each time point is expressed as a relative value. This indicates that high-quality cells are less likely to induce unintended biological responses. [Figure 63] Figure 63 shows a cytokine profile. It shows the profile of cytokine levels in the serum of a patient who was infused with high-quality cHCECs. A comparison of the profiles is shown before cHCEC infusion, two days after treatment, and one week after treatment. The amount of cytokines present in the patient's serum at each time point is expressed as a relative value. This shows that high-quality cells are less likely to induce unintended biological responses. [Figure 64-A]Figure 64-A shows the results of Western blot using anti-CD63 and anti-CD9 antibodies to detect secreted exosomes in culture supernatants from cHCECs with or without CST. [Figure 64-B] The upper panel of Figure 64-B shows phase-contrast microscopic images showing the morphology of cells #66 (passage 4), #77 (passage 2), and C11 (passage 2). The lower panel of Figure 64-B shows a graph showing the amount of exosomes detected by ExoScreen using CD9 and / or CD63 in each cell culture medium. [Figure 65] Figure 65 shows the results of FACS analysis demonstrating changes in cell population composition using magnetic bead cell sorting (MACS) with CD44 magnetic beads. C23 cHCECs at passage 2 were treated. The gated dot plots show, from the left, no MACS treatment, the unbound fraction after MACS treatment, and the bound fraction after MACS treatment. Analysis of CD105 and CD44 expression after gating on CD166 and CD24 expression is shown. The dot plots for CD26 and CD44 expression on the right show, from the top, no MACS treatment, the unbound fraction after MACS treatment, and the bound fraction after MACS treatment. [Figure 66] Figure 66 shows the results of FACS analysis demonstrating changes in cell population composition using magnetic bead cell sorting (MACS) with CD44 magnetic beads. C23 cHCECs at passage 4 were treated. The left image shows the results without MACS treatment, and the right image shows the unbound fraction after MACS treatment. Analysis of CD105 and CD44 expression, as well as analysis of CD26 and CD44 expression, are shown after gating on CD166 and CD24 expression. [Figure 67]Figure 67 shows the results of FACS analysis demonstrating changes in cell population composition using magnetic bead cell sorting (MACS) with CD44 magnetic beads. C27 cHCECs at passage 2 were treated. The left image shows the results without MACS treatment, and the right image shows the unbound fraction after MACS treatment. Analysis of CD105 and CD44 expression, as well as analysis of CD26 and CD44 expression, is shown after gating on CD166 and CD24 expression. [Figure 68] Figure 68 shows a method for estimating the effector content (E ratio) in a cultured cell population by FACS analysis. Using a PE-Cy7-labeled anti-human CD44 antibody (BD Biosciences), the FACS Canto II Blue laser was set to an area scaling factor of 0.75 and the PE-Cy7 voltage was set to 495. The gates were set as follows: First, fractions A, B, C, and D were set in a dot plot (upper left) with CD24 on the X axis and CD166 on the Y axis. Fraction A is CD24-negative and CD166-positive, fraction B is CD24-positive and CD166-positive, fraction C is CD24-negative and CD166-negative, and fraction D is CD24-positive and CD166-negative. The percentage of fraction B, relative to the analysis control cells (100%), represents the content of non-target cells C [CD24-positive cells]. For fraction B, fractions 1, 2, and 3 are defined as shown in the bottom left figure in a dot plot with CD44 on the X axis and CD105 on the Y axis. The percentage of fraction 1 is the E ratio, the sum of the percentages of fraction 1 and fraction 2 is the "effector cell + progenitor cell" content, and the percentage of fraction 3 is the content of non-target cell A [strongly CD44-positive cells]. Separately, a dot plot is created with CD44 on the X axis and CD26 on the Y axis, and fractions a', b', c', and d' are defined as shown in the top right figure below. The percentage of fraction B, when the analysis control cells are set to 100%, is the content of non-target cell B [CD26-positive cells]. [Figure 69]Figure 69 shows the relationship between postoperative corneal thickness and the quality of injected cells (E ratio). It shows the results of measuring corneal thickness before cell injection, 1 month, 3 months, and 6 months (4 weeks, 12 weeks, and 24 weeks, respectively), and 1 year and 2 years for patients A-N who received injections of cell populations with E ratios of less than 90% and patients I-O who received injections of cell populations with E ratios of 90% or higher. The horizontal axis represents time (before surgery, 1 month, 3 months, and 6 months (4 weeks, 12 weeks, and 24 weeks)), and the vertical axis represents corneal thickness. It can be seen that early thinning was achieved extremely well with functional cells with E ratios of 90% or higher. [Figure 70] Figure 70 compares clinical outcomes using cHCECs with different E ratios. The top row shows the results of cHCEC injection using C15 (passage 3), which has a very low E ratio. The middle row shows the results of cHCEC injection using C23 (passage 2) according to the present invention (E ratio <90%). The bottom row shows the results of cHCEC injection using C32 (passage 2) according to the present invention (E ratio ≥90%). In each row, the left side shows phase-contrast micrographs and FACS analysis of the injected cells based on CD24, CD26, and CD44. The right side shows specular micrographs taken after injection, showing the ECD values ​​(cells / mm2) in the endothelial tissue. When a cell population with a low E ratio (<10%) was injected, the tissue was opaque and no cells adhered to the endothelial tissue were detectable both one and three months later. Cells adhered to the endothelial tissue were detectable only six months later. When the injection surgery according to the present invention (with subpopulation selection, E-ratio < 90%) was used, cells could not be detected one month later due to turbidity, but this improved after three months, making cell detection possible. Furthermore, as shown in the lower panel, when the transplant surgery according to the present invention (with subpopulation selection, E-ratio ≥ 90%) was used, this improved after one month, making cell detection possible. It was found that cells prepared using the technology first developed in the present invention showed significantly earlier effects than conventional methods. [Figure 71]Figure 71 shows the postoperative results of the cultured endothelial cell injection of the present invention (top row), DSAEK (conventional method; middle row), and PKO (corneal transplant, conventional method; bottom row). Photographs of the eye after each procedure are shown on the left, and the corneal thickness distribution is shown in the center of the top row. Horizontal cross-sectional photographs are shown on the right. Endothelial injection of the present invention results in distortion-free corneal reconstruction and good recovery of QAV, whereas DSAEK presents distortion and a surface due to the surgical method, and PKP presents significant distortion. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments of the present invention will be described in detail. Note that similar content will be omitted where appropriate to avoid repetition. Furthermore, throughout this specification, singular expressions should be understood to include the plural concept, unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept, unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the relevant field, unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of a conflict, the present specification (including definitions) will prevail.

[0010] First, the terms and general techniques used in the present invention will be explained.

[0011] In this specification, the terms "corneal endothelium" and "human corneal endothelium" are used in the usual sense as used in this field. The cornea is one of the layered tissues that make up the eye, is transparent, and is the part closest to the outside world. In humans, the cornea is said to be made up of five layers, starting from the outside (body surface), and is composed of, from the outside, the corneal epithelium, Bowman's membrane (outer boundary line), lamina propria, Descemet's membrane (inner boundary line), and corneal endothelium. Unless otherwise specified, parts other than the epithelium and endothelium are sometimes collectively referred to as the "corneal stroma," and will be referred to as such in this specification.

[0012] In this specification, cells derived from corneal endothelial tissue are referred to as "corneal endothelial tissue-derived cells." Furthermore, cells that differentiate to become corneal endothelial cells are referred to as "corneal endothelial progenitor cells."

[0013] As used herein, "human functional corneal endothelial cells capable of inducing corneal endothelial functional characteristics when injected into the anterior chamber of a human eye" refer to cells having the functionality of corneal endothelium, which have the ability to express corneal endothelial functional characteristics (when referring to humans, these are referred to as "human corneal endothelial functional characteristics" and, without any particular limitation, are simply referred to as "corneal endothelial functional characteristics" in this specification) when injected into the anterior chamber of a human eye. When particularly abbreviated, they are also referred to as "functional cells endowed with corneal endothelial characteristics of the present invention." When referring to human cells, they are referred to as "human functional corneal endothelial cells capable of inducing human corneal endothelial functional characteristics when injected into the anterior chamber of a human eye." Because the present invention primarily relates to human corneal cells, it is understood that the term refers to human cells unless otherwise specified. In this specification, the functional cells having corneal endothelial properties of the present invention encompass "functional mature differentiated corneal endothelial cells" which have corneal endothelial functional properties in their original state, and "moderately differentiated corneal endothelial cells" which lack some of the functions but which, when used in the same way or after injection, exhibit functions equivalent to those of functional mature differentiated corneal endothelial cells.

[0014] As used herein, the term "corneal endothelial functional characteristics" refers to functional characteristics that a mature, differentiated cornea has in a normal state.

[0015] As used herein, the term "functional mature differentiated corneal endothelial cells" refers to any cells that have mature differentiated corneal endothelium and its functions (typically, the above-mentioned corneal endothelial functional characteristics), and in the case of human cells, they are referred to as functional mature differentiated human corneal endothelial cells. In particular, corneal endothelial functional characteristics are confirmed by the formation of a small, hexagonal, paving stone-like shape and the utilization of an energy metabolic system based on mitochondrial function, and can be determined by whether or not they have a therapeutic effect when injected (for example, into the anterior chamber of the human eye). However, without being limited thereto, corneal endothelial functional characteristics can also be determined using surrogate markers as indicators. Examples of such surrogate markers include (1) retention of endothelial pump and barrier function (including Claudin expression), (2) adhesion to specific laminins, and (3) the ability to differentiate between endothelial cells and corneal endothelial cells. It can also be assessed by any one or a combination of the following eight criteria: (1) binding affinity, (2) secreted cytokine profile, (3) microRNA (miRNA) profile, (4) produced microRNA (miRNA) profile, (5) produced metabolite profile, (6) saturation cell density during in vitro culture, (7) spatial size and distribution of cells obtained during culture, and (8) cell maintenance when cells are injected into mouse corneas after liquid nitrogen cryo-freezing injury.

[0016] (1) The maintenance of endothelial pump and barrier functions can be assessed using, for example, pump function and barrier function assays commonly used for corneal endothelium. Such assessments can be performed using an Ussing chamber, which is used in the case of sheet-type endothelium. Wigham C, Hodson S.: Current Eye Research, 1, 37-41,1981, Hodson S, Wigham C.: J Physiol., 342:409-419,1983, Hatou S., Yamada M., Akune Y., Mochizuki H., Shiraishi A., Joko T., Nishida T., Tsubota K.:Investigative Ophthlmology & Visual Science, 51, 3935-3942, 2010. Claudin expression can be confirmed by a method known in the art, for example, an immunological method. Claudin expression can be confirmed using any immunological method known in the art. However, since the cells of the present invention are expected to be injected in a suspension, in this case, preferably, the cells express Claudin or any one of (2) to (8) or a combination thereof. This can be applied to evaluate corneal endothelial function.

[0017] (2) To determine adhesion and binding to specific laminins, the adhesion to laminin 511 (a complex of α5 chain, β1 chain, and γ chain 1), laminin 521 (a complex of α5 chain, β2 chain, and γ chain 1) or their functional fragments (e.g., laminin 511-E8 fragment) and / or the expression of integrins (e.g., α3β1, α6β1, etc.) corresponding to these fragments can be determined. Such a technique can be carried out by the cell adhesion assay exemplified in Example 6.

[0018] Here, the laminin α chain, "α5 chain" (LAMA5), is one of the subunits of laminin, a cell adhesion molecule protein found in the extracellular matrix, and is referred to as LAMA5;KIAA1907, etc. The gene and protein sequences of human LAMA5 are registered in NCBI accession numbers NM_005560 and NP_005551, respectively, and are identified in OMIM under accession number 601033. The laminin β chain, "β1 chain" (LAMB1), is one of the subunits of laminin, a cell adhesion molecule protein found in the extracellular matrix, and is referred to as LAMB1;CLM;LIS5, etc. The gene and protein sequences of human LAMB1 are registered in NCBI accession numbers NM_002291 and NP_002282, respectively, and are identified in OMIM under accession number 150240. The "β2 chain" (LAMB2) (laminin S) is one of the subunits of the cell adhesion molecule protein (laminin) in the extracellular matrix, and is referred to as LAMB2, LAMS, NPHS5, etc. The gene and protein sequences of human LAMB2 are registered under the NCBI accession numbers NM_002292 and NM_003293, respectively. and NP_002283. OMIM identifies it under the accession number 150325. Regarding the laminin gamma chain, "gamma chain 1" (LAMC1) is one of the subunits of the cell adhesion molecule protein (laminin) in the extracellular matrix, and is also referred to as LAMC1; LAMB2, etc. The human LAMC1 gene and protein sequences are registered in NCBI under accession numbers NM_002293 and NP_002284, respectively. OMIM identifies it under the accession number 150290.

[0019] (3) The secreted cytokine profile can be determined by measuring the production level of the cytokine profile in "serum" or "aqueous humor" as described elsewhere herein. Such cytokines include, but are not limited to, RANTES, PDGF-BB, IP-10, MIP-1b, VEGF, EOTAXIN, IL-1ra, IL-6, IL-7, IL-8, IL-0, IL-10, IL-12(p70), IL-13, IL-17, FGFbasic, G-CSF, GM-CSI, IFN-γ, MCP-1, MIP-1a, and TNF-α. Specifically, cytokines can be analyzed using a cytokine measurement kit and analysis system such as Bio-Plex for integrated cytokine analysis, an example of which is described in Example 9. (4) The produced microRNA (miRNA) profile can be determined by measuring the "miRNA profile" as described elsewhere herein. For example, this can be achieved using the microRNA expression profile analysis method described in Examples 5 or 9, and can be performed using, for example, Toray's "3D-Gene" human miRNA oligochip (miRBase version 17). Total RNA obtained from both tissue and cell samples is labeled with a label such as Hy5 using a kit such as the miRCURY LNA® microRNA Power Labeling Kits (Exiqon, Vedbaek, Denmark), and total miRNA obtained from the supernatant is labeled. The labeled microRNAs are separately hybridized to the surface of the microRNA chip and incubated under appropriate conditions (e.g., 1 After incubation at 32°C for 6 hours, the microRNA chips are washed and dried in an ozone-free environment, then scanned using a scanner such as the 3D-Gene scanner 3000 (Toray Industries Inc., Tokyo, Japan) and analyzed using 3D-Gene Extraction software (Toray). (5) The resulting metabolite profile can be performed, for example, by the method described in Example 4. Metabolic extracts of intracellular metabolites are analyzed using an internal standard such as Internal Standard Solution (Human Metabolome Technologies; HMT, Inc., Tsuruoka, Japan). Prepare the cHCEC culture vessel with methanol containing reagent, replace the medium, process the cell extract (processing conditions are exemplified in Example 4), and perform CE-MS analysis to analyze the metabolites. Metabolomic analysis was performed according to the method described by Soga, et al. (Soga, D. et al., T. Soga, et al., Anal.Chem. 2002; 74: 2233-2239 Anal.Chem. 2000; 72: 1236-1241; T. Soga, et al., J. Proteome Res. 2003; 2: 488-494). Measurements can be made using appropriate automated integrated software (MasterHands, Keio University, Tsuruoka, Japan (M. Sugimoto, et al., Metabolomics, 2009; 6: 78-95) and MassHunter Quantitative Analysis B.04.00 (Agilent Technologies, Santa Clara, CA, USA). Based on the measured m / z values, peaks are annotated and normalized by hypothetical metabolites from the HMT metabolite database. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) can be performed to obtain metabolomic measurements. (6) Saturation cell density during in vitro culture can be determined by measuring cell density using appropriate culture conditions described herein. Cell density may also be measured in parallel with cell size, and can be measured using an inverted microscope system (e.g., CKX41, Olympus, Tokyo, Japan) with an image acquisition system such as a BZ X-700 microscope system (Keyence, Osaka, Japan). A phase contrast microscope image can be obtained using a microscope, and the number of cells can be quantified using cell counting software (for example, BZ-H3C Hybrid cell counting software (Keyence)). Preferred saturating cell densities are described elsewhere herein. (7) The spatial size and distribution of cells obtained during culture can be determined by taking photographs of the cells and measuring them with any software, etc., using appropriate culture conditions described herein. This can be achieved using image processing software such as BZ-H3C Hybrid cell counting software (Keyence). Preferred confluent cell densities for the present invention are described elsewhere herein. (8) Cell maintenance when cells are injected into a mouse cornea after liquid nitrogen cryoinjury can be assessed by creating a mouse model as exemplified in Example 7. Specifically, a model is created by pretreating the central region (e.g., 2 mm) of the cornea of ​​an appropriate mouse (e.g., BALB / c) with cryoinjury and removing endothelial cells. The cells to be assessed are then injected into the anterior chamber of the model. The characteristics of corneal transparency are clinically observed, the corneal thickness is evaluated using a pachymeter, and the adhesion of HCECs is examined histopathologically using human nuclear staining to confirm whether the cells are functional. These methods are exemplified in Example 8.

[0020] It is unclear whether cells that are not derived from corneal endothelium (for example, cells produced by differentiating stem cells (for example, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), etc.) into corneal endothelium) are completely identical to corneal endothelial cells present in a living body, but as long as they have corneal endothelial functional properties, they fall within the scope of the functional corneal endothelial cells or functional mature differentiated corneal endothelial cells of the present invention that can induce corneal endothelial functional properties when injected into the anterior chamber of a human eye. In the explanations and experiments shown in this specification, the human functional mature differentiated corneal endothelial cells of the present invention are also referred to as "human functional mature differentiated corneal endothelial cells," "functional mature differentiated human corneal endothelial cells," etc., and may also be referred to as "a5" cells, "target cells," "accepted cells," or simply "effector cells," but all of these are used interchangeably. Furthermore, "functional mature differentiated corneal endothelial cells" refers to cells with enhanced functionality that are referred to as "high quality" functional mature differentiated corneal endothelial cells. Such high-quality cells can be provided by selectively expanding CD44-negative cells, and although we do not wish to be bound by theory, concentrating only on negative cells is thought to result in higher quality and a higher degree of identity with cells found in mature, differentiated biological tissues.

[0021] As used herein, "moderately differentiated corneal endothelial cells" refer to cells that can exhibit the corneal endothelial functional properties of functional, mature, differentiated corneal endothelial cells (human corneal endothelial functional properties in the case of human cells), but do not possess the complete corneal endothelial functional properties of functional, mature, differentiated corneal endothelial cells, and instead exhibit at least a portion of those functions. When referring to human cells, the terms "moderately differentiated corneal endothelial cells" or "human moderately differentiated corneal endothelial cells" are used, but it should be noted that the present invention is primarily directed to humans. "Functional, mature, differentiated corneal endothelial cells" are cells that can be used in the present invention because they have the ability to function as functional, mature, differentiated corneal endothelial cells after injection into the anterior chamber of the eye. "Moderately differentiated human corneal endothelial cells" are effective, or at least do not inhibit the therapeutic effect, even if they are mixed with other substances to a certain extent and used in injection therapy. Such moderately differentiated corneal endothelial cells are capable of maturing and differentiating into functional, mature, differentiated corneal endothelial cells in vivo after injection (for example, after injection into the anterior chamber of the human eye). In the explanations and experiments presented in this specification, the cells are also referred to as "a1" cells, or simply as "corneal endothelial semi-functional cells," "semi-functional cells," "moderately differentiated effector cells," or "quasi-target cells," all of which have the same meaning.

[0022] As used herein, "non-functional corneal endothelial cells" refer to cells other than the functional cells having corneal endothelial properties of the present invention (i.e., "functional mature differentiated corneal endothelial cells" and "moderately differentiated corneal endothelial cells"), and may also be referred to as "non-target cells," "rejected cells," "non-target cells," "non-functional cells," etc. Such cells include the "a2" fraction.

[0023] As used herein, the term "cell indicator" refers to any indicator that indicates that a certain cell is a functional cell having corneal endothelial characteristics of the present invention (for example, a functional mature differentiated corneal endothelial cell or a moderately differentiated corneal endothelial cell), and is also referred to as a "functional cell indicator" because it is a characteristic possessed by any cell that has mature differentiated human corneal endothelium and its functions. This specific characteristic is also referred to as a "cell functional characteristic." For example, when a target cell is said to have cell functional characteristics homologous to a5 cells, this means that the target cell has values ​​corresponding to the range of values ​​of each of the cell indicators indicated by a5.

[0024] As used herein, "transformation" refers to a change in the phenotype of a cell to an abnormal state, and includes the meaning of normal cells dividing indefinitely, i.e., canceration, or particularly dynamic metaplasia (dedifferentiation of stem cells or changes beyond the boundaries of the tissue's basic form). Examples of transformation include cell state transitions (CST) such as EMT, fibrosis, epithelial-mesenchymal transition, senescence, and dedifferentiation. Corneal endothelial cells are These cells often undergo transformation such as epithelial-mesenchymal phase transition, and are no longer functional mature differentiated corneal endothelial cells. The production methods of the present invention include production methods that can convert even cells that have undergone such epithelial-mesenchymal transition into functional mature differentiated corneal endothelial cells by dedifferentiating them and then causing them to mature and differentiate.

[0025] As used herein, "epithelial-mesenchymal transition" (EMT) refers to the process by which epithelial cells lose their cell polarity and cell adhesion functions with surrounding cells, and acquire the ability to migrate and invade, thereby transforming into mesenchymal-like cells.

[0026] Here, the starting cells in the various samples and production methods that can be used in the present specification may be any sample that is considered to contain functional mature differentiated corneal endothelial cells, or cells of interest, or substances derived therefrom that enable gene expression. For example, Cells directly isolated from corneal endothelial tissue (also called corneal endothelial tissue-derived cells) or cells that have been differentiated to have corneal endothelial-like functions can be used. Corneal endothelial tissue-derived cells can be obtained by known methods (Koizumi N, Okumura N, Kinoshita S., Experimental Eye Research. 2012;95:60-7.). Preferably, cells obtained from a corneal endothelium donor are used. Cells obtained by inducing differentiation into functional cells having corneal endothelial characteristics of the present invention or functional mature differentiated corneal endothelial cells can be used as cell samples. In addition, cultured cells containing functional cells having corneal endothelial characteristics of the present invention or functional mature differentiated corneal endothelial cells that have been induced to differentiate in vitro can be used as samples. In vitro differentiation into functional cells having corneal endothelial characteristics of the present invention or functional mature differentiated corneal endothelial cells can be performed by using known cells such as ES cells, iPS cells, bone marrow stromal cells, etc. as starting materials and differentiating them by known methods, for example, the AMED method, etc. <Ueno M, Matsumura M, Watanabe K, Nakamura T, Osakada F, Takahashi M, Kawasaki H, Kinoshita S, Sasai Y:, Proc Natl Acad Sci USA. 103(25): 9554-9559, 2006.> This can be achieved by carrying out a process.

[0027] In the present invention, "miRNA" is an abbreviation of microRNA, which refers to RNA encoded on the genome and generated through a multi-step production process. Various types of miRNA have been discovered. They are generally thought to be 20 to 25 bases long, but are not particularly limited to this, and known types are registered in the microRNA database, miRBase (hGp: / / www.mirbase.org / ), etc. The precursor of miRNA is called "mir." It is common for miRNAs to be labeled "miR" in the case of mature miRNAs, and "miR" in the case of mature miRNAs. Furthermore, a registration number is added after miR (mir), but in cases of similarity, a lowercase letter is added. When defining the origin of a gene, the 5'-terminal strand is designated as 5p, and the 3'-terminal strand is designated as 3p. To distinguish between biological species, hsa is added for humans. These are connected with a hyphen, e.g., "hsa-miR-15a-5p." Since this specification primarily targets humans, it is understood that humans are intended even when the hsa is not added. In particular, when used separately in the present invention, "intracellular" miRNA and "secreted" miRNA can be distinguished. One of the features of the present invention is the world's first discovery that miRNAs can be used to identify cell subpopulations. Among these, one particularly distinctive feature is the discovery that "secreted" miRNAs secreted into the cell supernatant can identify cell types or subpopulations and can be used for quality control in cell infusion therapy.

[0028] As used herein, "intracellular" miRNA refers to any miRNA present within a cell.

[0029] As used herein, "secreted" miRNA refers to any miRNA that is secreted and can be detected in the culture supernatant. In the art, miRNAs are sometimes referred to as "cell-secreted" miRNA, "culture supernatant" miRNA, "cell supernatant" miRNA, or "cell supernatant-culture" miRNA, but all of these terms refer to the same thing. Secreted miRNAs can be detected without destroying cells.

[0030] As used herein, the terms "high expression," "moderate expression," and "low expression" of miRNAs, etc., are used to relatively express the expression intensity of miRNAs, and refer to the relative intensity compared to a standard. "High expression," "moderate expression," and "low expression" indicate high expression > medium expression > low expression in terms of expression intensity. As a representative example, the miR expression level (expression intensity) can be determined by determining the genes whose fluorescence intensity is measured, and then correcting the value so that the median expression intensity of all detected genes is the same, assuming that the total gene copy number between samples does not differ significantly. "High expression" and "low expression" refer to a statistically significant difference in expression intensity (relative ratio of 2 or more, P-value 0.05 or less). Moderate expression can be included as necessary. Moderate expression may also be included in the evaluation when a third expression intensity other than the strongest and weakest is observed in three or more groups of cells. Furthermore, "high expression" and "moderate expression" There may also be a statistically significant difference between "low expression" and "moderate expression."

[0031] As used herein, typically, when the expression characteristics of a5 cells are specified as CD44 negative to weakly positive, CD24 negative, and CD26 negative, the expression characteristics of a1 cells are specified as CD44 intermediately positive, CD24 negative, and CD26 negative, and the expression characteristics of a2 cells are specified as CD44 strongly positive, CD24 negative, and CD26 positive, the terms "high expression," "medium expression," and "low expression" of miRNAs are used to express the relative expression intensities of a5 cells, a1 cells, and a2 cells. Note that there may be no "medium expression" and in such cases, the respective cells can be distinguished by "high expression" and "low expression."

[0032] As used herein, "cell size" is one of the cell indices of the functional cells with corneal endothelial characteristics of the present invention, and is measured by a technique commonly used in the art. Cell size is expressed, for example, by cell area. As used herein, "cell area" is one of the cell indices of the functional cells with corneal endothelial characteristics of the present invention, and can be measured by taking a photograph of the cells and using any software or the like. Examples of such measurement techniques include a method that uses image processing software such as BZ-H3C Hybrid cell counting software (Keyence). The average value is called the "average cell area." The arithmetic mean is usually used.

[0033] As used herein, "cell density" and "(average) cell density" are cellular indicators expressed as the number of cells present in a certain area, and are measured by any technique commonly used in the art. The average density of a cell population is one cellular indicator of functional cells with corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention. The arithmetic mean is usually used as the average. It may also be measured in parallel with cell size, and may be measured using an inverted microscope system (e.g., CKX41, Olympus, Tokyo, Japan) or a microscope such as a BZ X-700 microscope system (Keyence, Osaka, Japan). Phase-contrast microscopic images are taken using an image acquisition system, and quantification is performed using cell counting software (e.g., BZ-H3C Hybrid cell counting software (Keyence)). The cell density at the time of saturated cell culture (also referred to as (culture) confluence; in this specification, saturated cell culture and (culture) confluence are used interchangeably) can be used as an indicator, and the density at the time of seeding can also be used as a guide in the production method of the present invention. Furthermore, the cell density can also be used as an indicator of the therapeutic outcome after injection.

[0034] As used herein, the term "karyotypic abnormality" refers to any abnormal karyotype, and in humans, it can be measured according to the standard International Code of Nomenclature for Human Chromosomes (ISCN) (1995) and its definitions. Specific analytical methods are described in the Examples.

[0035] As used herein, the term "immune properties", when used with respect to certain cells, refers to the immunological responsiveness that the cells exhibit when transplanted relative to host-derived cells, and is one cellular indicator of functional cells having corneal endothelial properties or functional mature differentiated corneal endothelial cells of the present invention, and an example of this is the absence of an immune rejection response despite being an allogeneic injection.

[0036] As used herein, the term "genetic characteristic", when referring to a certain cell, refers to a characteristic such as the expression of a gene related to that cell, and is one cellular indicator of functional cells having corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention.

[0037] As used herein, the term "serum cytokine profile" refers to a profile that indicates the amount, level, etc. of at least one cytokine in serum, which is a cellular indicator of functional cells having corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention. Typically, the profile can be displayed by the centroid method exemplified herein.

[0038] As used herein, the term "cell surface marker" refers to any biological substance expressed on the cell surface. Also called cell surface antigens, surface antigens, or surface markers, these can be identified as antigens that bind to monoclonal antibodies, and include those also known in the art as CD markers or CD antigens. The traits expressed by cell surface markers are also referred to as cell surface traits, and these terms are sometimes used interchangeably herein.

[0039] As used herein, the term "proteinaceous product" refers to any proteinaceous product produced by a cell, and the term "biological substance related to the proteinaceous product (said product)" refers to any biological substance related to the cellular proteinaceous product (for example, a gene (DNA), mRNA, protein precursor, etc. that encodes the proteinaceous product), and is one cellular indicator of functional cells having corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention. Representative examples of such genes are genes and their products. In the present invention, examples include (A) genes whose expression increases in functional cells having corneal endothelial characteristics of the present invention (including functional, mature, differentiated human corneal endothelial cells) (COL4A1, COL4A2, COL8A1, COL8A2, CDH2, TGF-β2, etc.) and (B) genes whose expression levels decrease in functional cells having corneal endothelial characteristics of the present invention (including functional, mature, differentiated human corneal endothelial cells) (MMP1, MMP2, TIMP1, BMP2, IL13RA2, TGF-β1, CD44, COL3A1, IL6, IL8, HGF, THBS2, IGFBP3, etc.).

[0040] As used herein, the terms "SASP-related protein," "SASP factor," and "SASP mediator" are used interchangeably and refer to any protein related to SASP (an abbreviation for Sensence Associated Secretory Phenotype), and are one cellular indicator of functional cells with corneal endothelial characteristics, functional mature differentiated corneal endothelial cells, or non-target cells of the present invention. They are also referred to as cellular senescence-associated secretion. SASP is a phenomenon associated with cellular senescence, characterized by the high expression of various secretory proteins that have the effect of inducing inflammatory responses and carcinogenesis. Examples of such SASP-related proteins include inflammatory cytokines (IL-6), inflammatory chemokines (IL-8, MCP-1, etc.), proteases (MMPs, etc.), PAI-1, GRO-α, and VEGF.

[0041] As used herein, the term "exosome" is also referred to as an exosome complex, and is a cell indicator of functional cells having corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention, and examples thereof include CD63, CD9, CD81, and HSP70.

[0042] As used herein, the term "cellular metabolite" refers to any metabolic product produced by a cell, and the term "biological substance related to the cellular metabolite (said product)" refers to any biological substance related to the cellular metabolite (for example, an enzyme that synthesizes the metabolite, an enzyme that metabolizes the metabolite, a protein related to a signal transduction pathway, etc.), and is one of the cellular indicators of functional cells having corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention. Examples of metabolites include any products related to the mitochondrial energy metabolism system, glutathione metabolism system products, methionine metabolism cycle products, lipid metabolism products, pentose phosphate pathway products, tricarboxylic acid (TCA) cycle metabolites, glycolysis metabolism products, etc., with TCA cycle metabolites and glycolysis metabolism products being particularly important. Examples of cellular metabolic products and related biological substances include succinic acid, Pro, Gly, glycerol 3-phosphate, Glu, lactic acid, argininosuccinic acid, xanthine, N-carbamoyl aspartic acid, isocitrate, cis-aconitic acid, Ala citrate, 3-phosphoglyceric acid, hydroxyproline, malic acid, uric acid, betaine, folic acid, Gln, 2-oxoisovaleric acid, pyruvate, Ser, hypoxanthine, Asn, Trp, Lys, choline, Tyr, urea, Phe, Met, carnosine, Asp, ornithine, Arg, creatine, 2-hydroxyglutamic acid, β-Ala, citrulline, Thr, Ile, Leu, Val, creatinine, His, and N,N-dimethylglycine. Some examples include:

[0043] As used herein, "autoantibody-reactive cells" refers to any cells that react to an autoantibody, and is one cell indicator for selecting functional cells having corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention. Autoantibody-reactive cells can be detected by any technique known in the art. Since non-target cell subpopulations are often reactive to autoantibodies, reactivity to autoantibodies can be evaluated in order to clarify target cells.

[0044] Detection, identification, quality control, etc. of the cells of the present invention can be achieved by using substances that bind to marker substances or interactive molecules. In the context of the present invention, a "substance that binds" or an "interactive molecule" to a marker substance refers to a molecule or substance that at least transiently binds to a molecule such as a marker substance (e.g., CD44) and preferably indicates that it has bound (e.g., is labeled or can be labeled). Substances that bind to molecules such as CD44 can be ligands of molecules such as CD44, examples of which include antibodies, antisense oligonucleotides, siRNAs, low molecular weight molecules (LMWs), binding peptides, aptamers, ribozymes, and peptidomimetics, including binding proteins or peptides directed against molecules such as CD44, as well as nucleic acids directed against the genes of molecules such as CD44. As used herein, the term "binding protein" or "binding peptide" in reference to a molecule such as CD44 refers to a type of protein or peptide that binds to a molecule such as CD44, and includes, but is not limited to, polyclonal or monoclonal antibodies, antibody fragments, and protein scaffolds directed against a molecule such as CD44.

[0045] As used herein, the terms "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably to refer to a polymer of amino acids of any length. The polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified, but are natural when targeted to intracellular contents.

[0046] As used herein, the terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer to a polymer of nucleotides of any length. The terms also include "oligonucleotide derivative" or "polynucleotide derivative." An "oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide that contains a derivative of a nucleotide or has an unusual bond between nucleotides, and are used interchangeably. As used herein, "nucleic acid" is also used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide. As used herein, "nucleotides" may be natural or non-natural, but those present in cells are natural. When nucleic acids or nucleotides are used as detection tools, they are considered artificial.

[0047] As used herein, "gene" refers to a factor that determines a genetic trait. It is usually arranged in a specific order on a chromosome. A gene that determines the primary structure of a protein is called a structural gene, and a gene that controls its expression is called a regulatory gene. As used herein, "gene" can refer to "polynucleotide," "oligonucleotide," and "nucleic acid." "Gene product" refers to a substance produced based on a gene, such as a protein or mRNA.

[0048] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. In this specification, comparison of similarity, identity, and homology of amino acid sequences and nucleotide sequences is calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed using, for example, NCBI's BLAST 2.2.28 (published April 2, 2013). The identity values ​​in this specification are usually calculated using the above-mentioned BLAST under default conditions. The similarity value is the value obtained by lining up the sequence. However, if a higher value is obtained by changing the parameters, the highest value is used as the identity value. If identity is evaluated in multiple regions, the highest value among them is used as the identity value. Similarity is a numerical value that takes into account not only identity but also similar amino acids.

[0049] As used herein, an "isolated" substance or biological factor (e.g., nucleic acid, protein, etc.) refers to a state in which coexisting factors in the natural state are not present. On the other hand, as used herein, a "purified" substance or biological factor (e.g., nucleic acid, protein, etc.) refers to a biological factor from which at least a portion of the factors naturally associated with the biological factor have been removed. Therefore, the purity of the biological factor in a purified biological factor is usually higher (i.e., it is concentrated) than in the state in which the biological factor normally exists. As used herein, the terms "isolated" and "purified" mean that the same type of biological factor is present in an amount of preferably at least about 75% by weight, more preferably at least about 85% by weight, even more preferably at least about 95% by weight, and most preferably at least about 98% by weight. The substance used in the present invention is preferably an "isolated" or "purified" substance.

[0050] As used herein, the term "marker (substance, protein, or gene (nucleic acid))" refers to a substance that serves as an indicator for tracking whether a certain state (e.g., functionality, transformed state, disease state, disorder state, or the level or presence or absence of proliferation potential or differentiation state) is present or at risk. Examples of such markers include genes (nucleic acid = DNA level), gene products (mRNA, protein, etc.), metabolites, enzymes, etc. In the present invention, detection, diagnosis, preliminary detection, prediction, or pre-diagnosis of a certain state (e.g., disease such as differentiation disorder) can be achieved using a drug, agent, factor, or means specific to a marker associated with that state, or a composition, kit, system, etc. containing them. As used herein, the term "gene product" refers to a protein or mRNA encoded by a gene. It has been discovered herein that gene products (i.e., molecules such as CD44) that have not been shown to be associated with ocular cells, particularly corneal endothelial cells, can be used as an indicator of whether corneal endothelial cells are functional (transformed).

[0051] As used herein, "detection" or "quantification" of polynucleotide or polypeptide expression can be achieved using any suitable method, including, for example, measurement of mRNA and immunological assays, including binding or interaction with a detection agent, testing agent, or diagnostic agent. Examples of molecular biological assays include Northern blotting, dot blotting, and PCR. Immunological measurement methods include, for example, ELISA using a microtiter plate, RIA, fluorescent antibody method, luminescence immunoassay (LIA), immunoprecipitation (IP), immunoassay, etc. Examples include the diffusion method (SRID), immunoturbidimetry (TIA), Western blotting, and immunohistochemical staining. Examples of quantification methods include ELISA and RIA. Genetic analysis can also be performed using arrays (e.g., DNA arrays, protein arrays). DNA arrays are extensively reviewed in "DNA Microarrays and the Latest PCR Methods," a special edition of Cell Engineering, edited by Shujunsha. Protein arrays are described in detail in NatGenet. 2002 Dec; 32 Suppl: 526-532. In addition to the above, methods for analyzing gene expression include RT-PCR, RACE, These include, but are not limited to, the ELISA method, SSCP method, immunoprecipitation method, two-hybrid system, in vitro translation, FACS (fluorescence activated cell sorting), etc. Such further analytical methods are described, for example, in Genome Analysis Experimental Methods, Nakamura Yusuke Lab Manual, edited by Nakamura Yusuke, Yodosha (2002), etc., and all of the descriptions therein are incorporated by reference herein. Flow cytometry used in FACS is a method for measuring the amount of microscopic particles. This technique involves dispersing molecules in a fluid, causing the fluid to flow in a thin stream, and optically analyzing each particle. FACS (Fluorescence Activated Cell Sorting) is an application of this technique. FACS involves placing cells stained with fluorescent antibodies in a fluid stream, passing them through the focus of a laser beam, and measuring the fluorescence emitted by each cell, making it possible to quantitatively measure the amount of antigen expressed on the cell surface.

[0052] As used herein, the term "expression intensity" refers to the amount of a polypeptide or mRNA, etc., expressed in a target cell, tissue, etc. Such expression intensity can be expressed at the protein level of the polypeptide of the present invention, evaluated by any appropriate method using the antibody of the present invention, including immunological assays such as ELISA, RIA, fluorescent antibody assay, Western blotting, and immunohistochemical staining, or by molecular biology methods such as Northern blotting, dot blotting, and PCR. Examples of such markers include the expression intensity of the polypeptide used in the present invention at the mRNA level, as assessed by any appropriate method, including immunological assays. "Changes in expression intensity" refers to an increase or decrease in the expression intensity of the polypeptide used in the present invention at the protein level or mRNA level, as assessed by any appropriate method, including the above-mentioned immunological assays or molecular biological assays. By measuring the expression intensity of a certain marker, various detections or diagnoses based on the marker can be performed.

[0053] As used herein, the term "activity" refers to the "activity of an expression product (e.g., a protein, a transcript (RNA, etc.))" or ..."). "Reduction" or "inhibition" or synonyms thereof refer to a decrease in the amount, quality, or effect of a particular activity, transcript, or protein, or an activity that is reduced. When an activity, expression product, etc. is reduced to below the detection limit, it is sometimes specifically referred to as "elimination." As used herein, "elimination" is encompassed by "reduction" or "inhibition."

[0054] As used herein, the term "activity" refers to the "activity of an expression product (e.g., a protein, a transcript (RNA, etc.))" or ..."). "Increase" or "activation" or synonyms thereof refer to an increase or increasing activity in the amount, quality or effect of a particular activity, transcript or protein.

[0055] As used herein, the term "antibody" broadly includes polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotypic antibodies, as well as fragments thereof, such as Fv fragments, Fab' fragments, F(ab')2 and Fab fragments, and other recombinantly produced conjugates or functional equivalents (e.g., chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single chain antibodies, scFV, diabodies, sc(Fv)2 (single chain (Fv)2), scFv-Fc). Furthermore, such antibodies can be purified by the addition of enzymes, e.g., alkaline phosphatase. The antibodies may be covalently bound or recombinantly fused to enzymes such as phosphodiesterase, horseradish peroxidase, and α-galactosidase. The antibodies against CD44 and other proteins used in the present invention may be of any origin, type, or form, as long as they bind to proteins such as CD44. Specifically, known antibodies such as non-human animal antibodies (e.g., mouse antibodies, rat antibodies, and camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used. In the present invention, either monoclonal or polyclonal antibodies can be used, with monoclonal antibodies being preferred. It is preferred that the antibodies specifically bind to the respective proteins such as CD44.

[0056] As used herein, the term "means" refers to any tool that can achieve a certain purpose (e.g., detection, diagnosis, treatment), and in particular, as used herein, "means for selectively recognizing (detecting)" refers to a means that can recognize (detect) a certain object differently from others.

[0057] The detection agent, diagnostic agent or other pharmaceutical agent of the present invention may be in the form of a probe or primer. The probes and primers of the present invention can specifically hybridize with molecules such as CD44. As described herein, the expression of molecules such as CD44 is an indicator of whether corneal endothelial cells are normal or transformed, and is also useful as an indicator of the degree of transformation. Therefore, the probes and primers of the present invention can be used to distinguish between normal and transformed corneal endothelial cells and / or the degree of transformation. In one embodiment, the probes and primers of the present invention are capable of detecting the expression of molecules such as CD44, and refer to polymers composed of multiple bases or base pairs, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Double-stranded cDNA is also known to be useful in tissue in situ hybridization, and the probes and primers of the present invention also include such double-stranded cDNA. RNA probes (riboprobes) are particularly preferred probes and primers for detecting RNA in tissues.

[0058] As used herein, the term "(nucleic acid) primer" refers to a substance necessary for initiating a polymer synthase reaction to synthesize a polymer compound. In a nucleic acid molecule synthesis reaction, a nucleic acid molecule (e.g., DNA or RNA) complementary to a partial sequence of the polymer compound to be synthesized can be used. As used herein, a primer can be used as a means for detecting a marker.

[0059] Nucleic acid molecules typically used as primers include those having a nucleic acid sequence at least 8 contiguous nucleotides long that is complementary to the nucleic acid sequence of a gene of interest (e.g., a marker of the present invention). Such nucleic acid sequences are preferably at least 9 contiguous nucleotides long, more preferably at least about 10 contiguous nucleotides long, even more preferably at least about 11 contiguous nucleotides long, at least about 12 contiguous nucleotides long, at least about 13 contiguous nucleotides long, at least about 14 contiguous nucleotides long, at least about 15 contiguous nucleotides long, at least about 16 contiguous nucleotides long, at least about 17 contiguous nucleotides long, at least about 18 contiguous nucleotides long, at least about 19 contiguous nucleotides long, at least about 20 contiguous nucleotides long, at least about 25 contiguous nucleotides long, at least about 30 contiguous nucleotides long, at least about 40 contiguous nucleotides long, or at least about 50 contiguous nucleotides long. Nucleic acid sequences used as primers include those that are at least 70% homologous to the above-mentioned sequences, more preferably at least 80% homologous, even more preferably at least 90% homologous, and even more preferably at least 95% homologous. Suitable sequences for primers may vary depending on the nature of the sequence intended to be synthesized (amplified), but those skilled in the art can design appropriate primers depending on the intended sequence. Design of such primers is well known in the art and can be performed manually or using computer programs (e.g., LASERGENE, PrimerSelect, DNAStar).

[0060] The primers according to the present invention can also be used as a primer set consisting of two or more of the primers.

[0061] The primers and primer sets according to the present invention can be used as primers and primer sets according to conventional methods in known methods for detecting target genes using nucleic acid amplification methods such as PCR, RT-PCR, real-time PCR, in situ PCR, and LAMP.

[0062] The primer set according to the present invention can be selected so that the nucleotide sequence of a target protein, such as a molecule like CD44, can be amplified by a nucleic acid amplification method such as PCR. Nucleic acid amplification methods are well known, and the selection of primer pairs for nucleic acid amplification methods is obvious to those skilled in the art. For example, in PCR, two primers (primer pair) can be selected so that one primer pairs with the plus strand of double-stranded DNA of a molecular target protein such as CD44, the other primer pairs with the minus strand of the double-stranded DNA, and the other primer pairs with the extended strand elongated by one primer. Furthermore, in the LAMP method (WO 00 / 28082), three regions, F3c, F2c, and F1c, are defined from the 3' end of the target gene, and three regions, B1, B2, and B3, are defined from the 5' end, and four types of primers can be designed using these six regions. The primers of the present invention can be chemically synthesized based on the nucleotide sequences disclosed herein. Primer preparation is well known, and can be found, for example, in "Molecular Cloning, A Laboratory Manual 2 nd ed. (Cold Spring Harbor Press (1989)), or Current Protocols in Molecular Biology (John Wiley & Sons (1987-1997)).

[0063] As used herein, the term "probe" refers to a substance used as a search tool in biological experiments such as in vitro and / or in vivo screening, and examples include, but are not limited to, nucleic acid molecules containing a specific base sequence, peptides containing a specific amino acid sequence, specific antibodies or fragments thereof, etc. As used herein, a probe is used as a means for detecting a marker.

[0064] Nucleic acid molecules typically used as probes include those having a nucleic acid sequence at least about 8 contiguous nucleotides long that is homologous or complementary to the nucleic acid sequence of a gene of interest. Such nucleic acid sequences are preferably at least about 9 contiguous nucleotides long, more preferably at least about 10 contiguous nucleotides long, even more preferably at least about 11 contiguous nucleotides long, at least about 12 contiguous nucleotides long, at least about 13 contiguous nucleotides long, at least about 14 contiguous nucleotides long, at least about 15 contiguous nucleotides long, at least about 20 contiguous nucleotides long, at least about 25 contiguous nucleotides long, at least about 30 contiguous nucleotides long, at least about 40 contiguous nucleotides long, or at least about 50 contiguous nucleotides long. Nucleic acid sequences used as probes include nucleic acid sequences that are at least about 70% homologous, more preferably at least about 80% homologous, even more preferably at least about 90% homologous, or at least about 95% homologous to the above-mentioned sequences.

[0065] In one embodiment, the detection agent of the present invention may be labeled, or may have a tag bound thereto.

[0066] As used herein, the term "label" refers to an entity (e.g., substance, energy, electromagnetic waves, etc.) that distinguishes a target molecule or substance from others. Examples of such labeling methods include the RI (radioisotope) method, the fluorescence method, the biotin method, and the chemiluminescence method. When labeling multiple markers of the present invention or factors or means for capturing them using the fluorescence method, the labeling is carried out with fluorescent substances that have mutually different maximum fluorescence emission wavelengths. The difference in maximum fluorescence emission wavelength is preferably 10 nm or more. When labeling a ligand, any label that does not affect its function can be used. For example, labels actually used in FACS include FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, and Alexa. TM Fluor TM 488, Alexa TM Fluor 647. A typical example of immunostaining is Alexa TM Fluor 488, Alexa TM Fluor555 or Alexa TM Fluor 594, Alexa TM Can be used in combination with Fluor 647. Alexa TM Fluor is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series that corresponds to a wide range of fluorescent wavelengths. Compared to other fluorescent dyes of the corresponding wavelength, it is very stable, bright, and pH-sensitive. The combination of fluorescent dyes with a fluorescence maximum wavelength of 10 nm or more is Alexa TM 555 and Alexa TM 633 combinations, Alexa TM 488 and Alexa TM When labeling nucleic acids, any compound that can bind to the base moiety can be used, but cyanine dyes (e.g., CyDye TM series Cy3, Cy5, etc.), Rhodamine 6G reagent, N-acetoxy-N2- Acetylaminofluorene (AAF), AAIF (an iodine derivative of AAF), and the like can be used. Examples of labels that have actually been used include DAPI and Hoechst 33342. Examples of fluorescent substances with a difference in maximum fluorescence wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, and a combination of rhodamine 6G reagent and fluorescein. In the present invention, such labels can be used to modify a target object so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can carry out such methods appropriately depending on the label and the target object.

[0067] According to one embodiment of the detection method of the present invention, the probe of the present invention is hybridized with a nucleic acid sample (mRNA or its transcription product), and the hybridization complex, i.e., the nucleotide duplex, is directly or indirectly detected to detect the expression of a molecule such as CD44 or the gene for that molecule in a cell sample. For detailed procedures of the hybridization method, see "Molecular Cloning, A Laboratory Manual 2 nd ed.” (Cold Spring Harbor Press (1989), especially Sections 9.47-9.58), “Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997), especially Sections 6.3-6.4), “DNA Cloning 1:Core Techniques, A Practical Approach 2 nd ed.” (Oxford University (1995), see especially Section 2.10 for conditions).

[0068] Detection of the expression of molecules such as CD44 or the genes encoding these molecules using a hybridization method can be carried out, for example, by: (a) contacting a polynucleotide derived from a test sample with a probe according to the present invention; and (b) detecting the hybridization complex. In step (a), mRNA prepared from the test sample of interest or complementary DNA (cDNA) transcribed from the mRNA can be contacted with the probe as a polynucleotide derived from a test cell sample. In detection methods using probes, the probes can be labeled before use. Examples of labels include radioactivity (e.g., 32 P, 14 C, and 35 Examples of labels include those utilizing fluorescent dyes (e.g., Fluorescence Induced Cellular Thickness (FITC), europium), and enzymatic reactions such as chemical color development (e.g., peroxidase, alkaline phosphatase). Hybridization products can be detected using well-known methods such as Northern hybridization, Southern hybridization, and colony hybridization. Cells in which hybridization complexes are detected express molecules such as CD44, and therefore can be determined to have high proliferation potential (e.g., undifferentiated cells, progenitor cells, or stem cells) and / or high differentiation potential.

[0069] According to another embodiment of the detection method of the present invention, a nucleic acid sample (mRNA or its transcription product) is amplified by a nucleic acid amplification method using a primer or primer set according to the present invention, and the amplified product is detected, thereby detecting the expression of molecules such as CD44 or the genes of these molecules in the sample.

[0070] Detection of the expression of molecules such as CD44 or the genes encoding these molecules using nucleic acid amplification methods can be carried out, for example, by (i) performing nucleic acid amplification using a primer or primer set according to the present invention and a polynucleotide derived from a test sample as a template; and (ii) detecting the amplified product formed.

[0071] In step (i), mRNA prepared from the target test sample or complementary DNA (cDNA) transcribed from that mRNA can be used as a template. Detection of the amplification product can be carried out using a nucleic acid amplification method such as PCR, RT-PCR, real-time PCR, or LAMP. Cells from which amplification products are detected have a high tendency to be normal corneal endothelial cells for normal corneal endothelial cell markers, and a high tendency to be transformed corneal endothelial cells for transformed corneal endothelial cell markers, so it can be determined whether the cells are normal or transformed.

[0072] As immunological methods, known methods such as immunohistochemical staining, enzyme immunoassay, Western blotting, agglutination, competitive assay, and sandwich assay can be applied to cell samples that have been appropriately treated as necessary, for example, by cell separation or extraction. Immunohistochemical staining can be performed, for example, by a direct method using a labeled antibody or an indirect method using a labeled antibody against the antibody. Known labeling substances such as fluorescent substances, radioactive substances, enzymes, metals, and dyes can be used as labeling agents.

[0073] In the present invention, "Rho kinase" refers to a serine / threonine kinase that is activated upon activation of Rho. For example, ROKα (ROCK-II: Leung, T. et al. al., J.Biol.Chem., 270, 29051-29054, 1995), p160ROCK(ROKβ, ROCK-I:Ishizaki, T. et al., The EMBO J., 15(8), 1885-1893, 1996 ) and other proteins with serine / threonine kinase activity.

[0074] Rho kinase inhibitors are disclosed in the following documents: U.S. Patent No. 4,678,783, Japanese Patent No. 3,421,217, WO 95 / 28387, WO 99 / 20620, WO 99 / 61403, WO 02 / 076976, WO 02 / 076977, WO 2002 / 083175, WO 02 / 100833, WO 03 / 059913, WO 03 / 062227, WO 2004 / 009555, WO 2004 / 022541, WO 2004 / 1 08724, WO 2005 / 003101, WO 2005 / 039564, WO 2005 / 034866, WO 2005 / 037197, WO 2005 / 037198, WO 2005 / 035501, WO 2005 / 035503, WO 2005 / 035506, WO 2005 / 080394, WO 2005 / 103050, WO 2006 / 057270, WO 2007 / 026664, etc. Such compounds can be produced by the methods described in the respective publications. Specific examples include 1-(5-isoquinolinesulfonyl)homopiperazine or a salt thereof (e.g., fasudil (1-(5-isoquinolinesulfonyl)homopiperazine)), (+)-trans-4-(1-aminoethyl)-1-(4-pyridylcarbamoyl)cyclohexane ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) or a salt thereof (e.g., Y-27632 ((R)-(+)-trans-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide dihydrochloride monohydrate)), and these compounds can be preferably used as commercially available products (Wako Pure Chemical Industries, Ltd., Asahi Kasei Pharma, etc.).

[0075] As used herein, "diagnosis" refers to identifying various parameters related to a disease, disorder, condition (e.g., bullous keratopathy, Fuchs' endothelial dystrophy), etc. in a subject, and determining the current state or future of such disease, disorder, or condition. By using the methods, devices, and systems of the present invention, the internal state can be investigated, and such information can be used to select various parameters, such as the disease, disorder, condition, and the treatment or prophylactic formulation or method to be administered in the subject. In the narrow sense, "diagnosis" as used herein refers to diagnosing the current state, but in the broad sense, it can also be used to refer to "early diagnosis," "predictive diagnosis," "pre-diagnosis," etc. " and the like. The diagnostic method of the present invention is industrially useful because, in principle, it can utilize substances excreted from the body and can be carried out without the involvement of medical professionals such as doctors. In this specification, to clarify that it can be carried out without the involvement of medical professionals such as doctors, it is sometimes referred to as "assisting" "predictive diagnosis, advance diagnosis, or diagnosis."

[0076] As used herein, the term "treatment" refers to, in the case of a disease or disorder (e.g., bullous keratopathy, Fuchs' endothelial dystrophy), preventing the progression of such a disease or disorder when it has progressed, preferably maintaining the current state, more preferably alleviating, and even more preferably eliminating, and also refers to the symptom-improving effect of a patient's disease or one or more symptoms associated with the disease. It also includes the possibility of exerting a preventive effect. Proper diagnosis and appropriate treatment is called "companion therapy," and diagnostic agents for this purpose are sometimes called "companion diagnostic agents."

[0077] As used herein, the "combination" of a certain pharmaceutical component (e.g., the cellular drug of the present invention, etc.) with another pharmaceutical component (e.g., a concomitant drug such as a ROCK inhibitor, etc.) is intended to include simultaneous (concomitant) administration and sequential administration. Sequential administration is intended to encompass administration of a drug (one or more types) and a drug (one or more types) of the present invention, etc. to a subject in various orders. A drug for "combination" administration of a certain pharmaceutical component (e.g., the cellular drug of the present invention) with another pharmaceutical component (e.g., a ROCK inhibitor, etc.) may be referred to as a "combination drug."

[0078] As used herein, the term "prognosis" refers to predicting the likelihood of death or progression due to a disease such as bullous keratopathy or Fuchs' endothelial dystrophy. Prognostic factors are variables related to the natural history of a disease, and they affect the recurrence rate, etc., of patients who have developed the disease. Clinical indicators associated with worsening prognosis include, for example, any of the cellular indicators used in the present invention. Prognostic factors are often used to classify patients into subgroups with different pathological conditions.

[0079] As used herein, the term "detection agent" or "test agent" broadly refers to any agent capable of detecting or testing a target of interest.

[0080] As used herein, the term "diagnostic agent" refers in a broad sense to any agent that can diagnose a target condition (for example, a disease such as a corneal endothelial disease).

[0081] As used herein, the term "therapeutic agent" broadly refers to any drug capable of treating a target condition (e.g., a disease such as a corneal endothelial disease). In one embodiment of the present invention, the "therapeutic agent" refers to a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition may be a composition containing the active ingredient. The pharmaceutical composition may be prepared by, for example, mixing the active ingredient with the carrier by any method known in the technical field of pharmaceuticals. The therapeutic agent may be used in any form, provided that it is used for treatment, and may be the active ingredient alone or a mixture of the active ingredient with any other ingredient. The form of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution). The pharmaceutical includes drugs used for prevention (prophylactic drugs) or drugs that improve the condition of corneal endothelial disease (therapeutic drugs).

[0082] As used herein, the term "prevention" refers to preventing a certain disease or disorder (e.g., corneal endothelial disease) from occurring before that state is reached. Diagnosis can be performed using the agent of the present invention, and, if necessary, the agent of the present invention can be used to prevent, for example, a disease, or measures for prevention can be taken.

[0083] As used herein, the term "prophylactic agent" refers to a drug or agent that is used to treat a target condition (e.g., corneal endothelial disease) in a broad sense. This refers to any drug that can prevent diseases such as flu.

[0084] Generally, the compositions, medicaments, agents (therapeutic agents, prophylactic agents, etc.) of the present invention contain a therapeutically effective amount of a medicament or active ingredient and a pharmaceutically acceptable carrier or excipient. As used herein, "pharmaceutically acceptable" means approved by a government regulatory agency or listed in a pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more specifically, in humans. As used herein, "carrier" refers to a culture medium, infusion vehicle, perfusion fluid, diluent, adjuvant, excipient, or vehicle with which a medicament is administered. Because the cellular medicine of the present invention contains cells as a major component, carriers capable of supporting cells, such as culture medium, infusion vehicle, and perfusion fluid, are preferred. The present invention may be used in combination with other drugs, such as steroids, antibiotics, and ROCK inhibitors, and these drugs can be administered in the same dosage forms as conventional drugs. In this case, the carrier can be a sterile liquid, such as water and oil, including those of petroleum, animal, vegetable or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, when the medicament (composition) is administered intravenously, saline and aqueous dextrose are preferred carriers. Preferably, saline solution and aqueous dextrose and glycerol solution are used as liquid carriers for injectable solutions. When the medicament is administered orally, water is the preferred carrier. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, and the like.The compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions can also be formulated as suppositories, using traditional binders and carriers such as triglycerides. Oral formulations can also include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable carriers include E.W. Martin, Remington's Pharmaceutical. Sciences (Mark Publishing Company, Easton, USA). The compositions contain a therapeutically effective amount of the therapeutic agent, preferably in purified form, together with a suitable amount of carrier to provide a form suitable for administration to a patient. The formulation should be appropriate for the mode of administration. Other ingredients may include, for example, surfactants, excipients, colorants, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc.

[0085] When the present invention is administered as a pharmaceutical, various delivery systems are known, and such systems can be used to administer the pharmaceutical of the present invention to an appropriate site (e.g., the anterior chamber of the eye). A typical administration form for the cell pharmaceutical of the present invention is injection into the anterior chamber. In such cases, the cells can be suspended in an injection vehicle and injected into the anterior chamber using a needle (e.g., a 26G needle). For other concomitant drugs, administration forms similar to those for conventional pharmaceuticals are possible, and such systems include encapsulation in liposomes, microparticles, and microcapsules. Administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical can be administered by any suitable route, for example, by infusion, bolus injection, or by injection into epithelial or mucocutaneous linings (e.g., oral, rectal). It is also possible to administer drugs by absorption through the blood vessels (such as the intestinal mucosa and intestinal mucosa), and if necessary An inhaler or nebulizer may be used, optionally with an aerosolizing agent, and may be administered along with cells. Administration may be systemic or local.

[0086] In a preferred embodiment, the composition can be formulated according to known methods as a pharmaceutical composition adapted for administration to humans. Such compositions can be administered by injection or infusion. If the composition is to be administered by infusion, it can be dispensed using an infusion bottle containing a cell infusion solution, sterile pharmaceutical-grade water, or saline.

[0087] Low-molecular-weight or high-molecular-weight drugs such as the concomitant drugs of the present invention (e.g., antibiotics, ROCK inhibitors) can be formulated in a neutral or salt form or as other prodrugs (e.g., esters, etc.). Pharmaceutically acceptable salts include those formed with a free carboxyl group derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. These include those formed with free amine groups, as well as those derived from sodium, potassium, ammonium, calcium, and ferric hydroxides.

[0088] The amount of the pharmaceutical agent of the present invention that is effective in treating a particular disorder or condition (e.g., number of cells, frequency of administration, etc.) may vary depending on the nature of the disorder or condition, but can be determined by one of skill in the art using standard clinical techniques based on the teachings herein. It can also help identify the optimal dosage range. The exact dosage to be used in the formulation can also vary depending on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the attending physician and the circumstances of each patient. However, the dosage is not particularly limited, and can be any cell density and amount described herein, and can be within any two values, for example, 1.5 × 10 6 Examples are given below. The administration interval is not particularly limited, and may be, for example, a single administration, or administration once or twice per 1, 7, 14, 21, or 28 days, or once or twice per range of any two of these values. The therapeutic agent may be administered. The dosage, administration interval, and administration method may be appropriately selected depending on the age, weight, symptoms, target disease, etc. of the patient. The therapeutic agent preferably contains a therapeutically effective amount, or an effective amount of an active ingredient that exerts a desired effect. If a marker indicating a pathological condition is significantly reduced after administration, it may be determined that the therapeutic effect has been achieved. The effective dosage can be determined by in vitro or animal model analysis. The dose-response curves obtained from the fluororespiratory test system can be used to estimate the dose-response curves.

[0089] In one embodiment of the present invention, the "patient" is primarily a human, but may also be a mammal other than a human, as long as applicable.

[0090] (Preferred embodiment) Preferred embodiments of the present invention will be described below. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present invention in consideration of the description in this specification. It is also understood that the following embodiments of the present invention can be used alone or in combination.

[0091] (Human functional corneal endothelial cells that can induce human corneal endothelial functional properties when transferred into the anterior chamber of the human eye) In one aspect, the present invention provides human functional corneal endothelial cells (also referred to as the functional cells with corneal endothelial characteristics of the present invention) that can induce human corneal endothelial functional characteristics when injected into the anterior chamber of a human eye. The functional cells with corneal endothelial characteristics of the present invention have the corneal endothelial functional characteristics of mature differentiated corneal endothelium, and are effective in cell injection therapy (for example, they can induce corneal endothelial functional characteristics when injected into the anterior chamber of a human eye). These cells can be referred to as human functional corneal endothelial cells that can induce corneal endothelial functional properties when injected into the anterior chamber of the eye. The functional cells with corneal endothelial properties of the present invention may include functional mature differentiated corneal endothelial cells as well as moderately differentiated corneal endothelial cells. The functional mature differentiated corneal endothelial cells of the present invention are mature differentiated cells that exhibit corneal endothelial function, and effector cells, which are a subpopulation optimal for injection, form small, hexagonal, pavement-like shapes and utilize an energy metabolic system based on mitochondrial function.

[0092] Although cells named "cultured corneal endothelial cells" or "cultured human corneal endothelial cells" have been reported in the past, it was not known that these were composed of multiple subpopulations, and it was not known that there was a subpopulation that was particularly optimal for cell injection therapy among them, and therefore the significance of the present invention is great. In particular, prior to the disclosure of the present invention, the issue of cellular heterogeneity associated with regenerative medicine had not been clearly recognized in human corneal endothelial cells, and the discovery and resolution of this issue is of great significance. This is because human corneal endothelial cells (HCECs) are unable to divide in vivo and are arrested in the G1 phase of the cell cycle, but are said to still retain their proliferative capacity. However, recent research has led to the understanding that it is extremely difficult to culture HCECs for long periods of time.

[0093] As an application example of the present invention, it is particularly noteworthy that it enables the regeneration of corneal endothelial function by intracameral injection using a suspension of "allo" functional, mature, differentiated human corneal endothelial cells that are high-quality, do not show karyotypic abnormalities, and do not induce immune rejection responses. The medical technology using the cells of the present invention makes it possible to treat patients with bullous keratopathy by injecting a cell suspension into the anterior chamber of the patient after ex vivo expansion and amplification of corneal endothelial cells derived from young donors. In clinical studies based on guidelines for clinical studies using human stem cells, the present invention has demonstrated and established safety and clinical proof of concept for human application.

[0094] One of the factors that enabled us to provide the cells of the present invention is the discovery that the cells used for injection therapy are a mixture of heterogeneous cell subpopulations, and that the "human functional corneal endothelial cells that can induce the functional properties of human corneal endothelium when injected into the anterior chamber of the human eye" that can be used for therapy are limited to a subset of these.

[0095] In the present invention, it was also found that in corneal endothelial cells, karyotype abnormalities occur selectively in a subpopulation, and that autoantibodies that react selectively to the subpopulation exist. The functional cells with corneal endothelial characteristics of the present invention, particularly functional mature differentiated corneal endothelial cells, do not have such abnormalities, and compared to other subpopulations, they have relatively lower expression of HLA class I antigens involved in immune rejection reactions than other subpopulations, and it was also found that expression of CD200 antigen, which had previously been thought to be a cell marker, was negative. It was also found that the cells produced a high amount of cytokines (SASP-related proteins) related to cellular senescence.

[0096] Before the disclosure of this invention, there were limited reproducible culture methods, and it was difficult to culture cells without karyotypic aneuploidy and without cell state transitions (CSTs) such as fibrosis, cellular senescence, and epithelial-mesenchymal transition (EMT). Attempts to grow human corneal endothelial cells in vitro have been extremely difficult, as there has been no knowledge or reports on the cell characteristics, whether the cell population is composed of multiple subpopulations, or whether the cell population produced by the culture conditions is stable and equitable, and analysis from these perspectives has not even been conducted.

[0097] Cultured HCECs undergo CST and tend to undergo a senescent phenotype, EMT, and fibroblastic morphology. We identified distinct cell surface markers that distinguish these phenotypes, allowing us to define HCEC populations that are applicable to the reconstruction of non-functional human corneal endothelial tissue.

[0098] Considering the reports of karyotypic aneuploidy in cHCECs and the plasticity of the metabolic profile of cHCECs, Several CD markers were selected to define SP: CD166, CD44, CD49e, CD73, CD105, CD90, CD133, CD26, and CD24, all of which are associated with mesenchymal stem cells. There is some association with phenotypic changes in MSCs, cancer stem cells (CSCs), or CSTs (Davies S, Beckenkamp A, Buffon A. Biomed Pharmacother. 2015; 71:135-8; Roberta Pang, et al., Stem Cell, 6, 2010, 603-615; Krawczyk N, et al., Biomed Res Int. 2014; 2014:415721. Epub 2014 May 8; Irollo E, Pirozzi G. Am J Transl Res. 2013 Sep 25; 5:563-81; Williams K, et al., Exp Biol Med (Maywood). 2013; 38:324-38; Zhe Shi, et al., Mol Cell Biochem (2015) 401:155-164). This selection was plausible, but it is important to note that normal stem cells are not organized. This is because CSCs are the longest-lived cells in tissues, are likely to accumulate mutations over time, and may arise from transit-amplifying cells (BJ Huntly Cancer Cell, 6 (2004), 587-596; CH Jamieson et al., N. Engl. J. Med., 351 (2004), pp. 657-667).

[0099] Prior to the disclosure of the present invention, specific cell surface markers for truly human functional, mature, differentiated corneal endothelial cells had not been identified. Glypican-4 and CD200 were identified as markers for HCECs in the corneal endothelium. It has been proposed as a marker for HCECs to distinguish them from stromal fibroblasts (Cheong YK et al., Invest Ophthalmol Vis Sci. 2013; 54: 4538-4547). However, a practical problem in culturing HCECs was found to be the contamination with fragile transformed cultured human corneal endothelial cells. This problem can be resolved by the production method provided by the present invention.

[0100] Flow cytometry analysis demonstrated the existence of several subpopulations of cultured human corneal endothelial cells, and one specific subpopulation of cultured human corneal endothelial cells, which typically exhibits surface expression of CD166-positive, CD105-negative, CD44-negative, CD24-negative, and CD26-negative, was shown to be a subpopulation lacking CST. This is the first finding that makes it possible to apply this subpopulation to clinical use, and the combination of CD markers defined herein is appropriate for quality control to ensure the functional characteristics of cultured human corneal endothelial cells for clinical application. The inventors conducted further research and discovered a cell indicator that more appropriately reflects the corneal endothelial functional properties of functional, mature, differentiated corneal endothelial cells.

[0101] In one embodiment, the functional cells with corneal endothelial characteristics of the present invention have corneal expression characteristics of the cell indicators defined herein.

[0102] Possible cell indicators of functional cells having corneal endothelial characteristics of the present invention include cell surface markers (e.g., CD markers), cell product characteristics, cell morphology indicators, and cellular genetic characteristics. Specifically, these may include cell surface markers (e.g., CD markers); characteristics of proteinaceous products and biological substances related to the products; expression characteristics of SASP-related proteins; expression of miRNAs (e.g., intracellular miRNAs, secreted miRNAs, etc.); characteristics of exosomes; expression characteristics of cellular metabolic products and biological substances related to the products; cell size; cell density; and the presence of autoantibody-reactive cells. The cell indicators of functional, mature, differentiated corneal endothelial cells of the present invention exhibit specific ranges or levels of cell functional characteristics, or combinations thereof. Therefore, by determining specific ranges or levels of cell functional characteristics, or combinations thereof, of specific cell indicators, it is possible to determine whether a cell is a functional, mature, differentiated corneal endothelial cell of the present invention. The specific ranges or levels of cell functional characteristics, or combinations thereof, unique to the functional, mature, differentiated corneal endothelial cells of the present invention have been identified for the first time in the present invention, which allows the identification of various cell subpopulations, enabling quality control and quality testing, and ultimately enabling the achievement of highly effective treatments. These cellular indicators and their specific ranges or levels of cellular functional properties or combinations thereof are specifically detailed below.

[0103] In a specific embodiment, the functional cells having corneal endothelial characteristics of the present invention are CD166-positive and The cells have cell functional characteristics including CD133 negativity. Another important cell functional characteristic is CD44 expression, the expression intensity of which is preferably CD44 negative to moderately positive, more preferably CD44 negative to weakly positive, and even more preferably CD44 negative, but is not limited to these. In the present invention, it has been discovered that in order to confirm whether corneal endothelial cells or cells differentiated into corneal endothelial-like cells are functional, it is possible to confirm whether they are CD166 positive and CD133 negative. Additionally, by confirming that CD44 expression is also low (CD44 negative to moderately positive, preferably CD44 negative to weakly positive), it has been possible to determine with higher accuracy whether they are functional.

[0104] Therefore, in a preferred embodiment, the functional cells with corneal endothelial characteristics of the present invention have cell functional characteristics including CD166 positivity, CD133 negativity, and CD44 negativity to weak positivity. Without wishing to be bound by theory, it has been confirmed that the presence of these three cell markers in corneal endothelial cells or cells differentiated into corneal endothelium-like cells is a functional mature differentiated corneal endothelial cell with high quality functionality. In the results of clinical studies, such functionality can be confirmed by a corneal endothelial cell test (specular) value of approximately 1000 (cells / mm) in a short period of time (for example, about one month). 2 ), exceeding approximately 2000 (pieces / mm 2 ), preferably at a level exceeding about 2300 (pieces / mm 2 ), more preferably at a level exceeding about 2500 (pieces / mm 2 ), and in some cases, approximately 3000 (pieces / mm 2 It has been shown that high levels of therapeutic efficacy are achieved, exceeding levels

[0105] More preferably, the functional cells of the present invention having corneal endothelial characteristics have cell functional characteristics including CD166-positive, CD133-negative, and CD44-negative. Without wishing to be bound by theory, by further limiting the cells to CD44-negative cells, it is possible to more appropriately provide high-quality cells with a high degree of guaranteed proliferation ability, etc. (sometimes referred to herein as "high-quality" functional mature differentiated corneal endothelial cells). "High-quality" functional mature differentiated corneal endothelial cells have more stable and improved corneal endothelial functional characteristics.

[0106] In another embodiment, the functional cells with corneal endothelial characteristics of the present invention have cell functional characteristics including CD166 positivity, CD133 negativity, and CD200 negativity. Regarding CD200, it has been said that CD200 positivity is a characteristic of corneal endothelial cells. However, by conducting detailed studies of each subpopulation in the present invention, it has been found that CD200-positive cells are large cells with CST that are not suitable for transplantation, and that CD200 negativity is a characteristic of functional corneal endothelial cells that can induce human corneal endothelial functional characteristics when transplanted into the anterior chamber of the human eye. These characteristics could not have been predicted based on conventional knowledge, and can be said to be the result of careful analysis of the subpopulations in the present invention.

[0107] In another embodiment, the functional cells of the present invention having corneal endothelial characteristics have cell functional properties including CD166-positive, CD133-negative, CD44-negative to CD44-weakly positive, and CD90-negative to weakly positive. This further ensures the homogeneity of the cells. Alternatively, the cell surface antigens include a CD166-positive, CD133-negative, CD44-negative to moderately positive, and CD90-negative phenotype. In another embodiment, the cell surface antigens include a CD166-positive, CD133-negative, and CD44-negative to CD44-weakly positive phenotype, or the cells express cell surface antigens including a CD44-negative to CD44-weakly positive phenotype.

[0108] The functional cells with corneal endothelial characteristics of the present invention may further have additional cell functional properties. Such cell functional properties include CD90 negative (CD90 negative to weakly positive), CD105 negative to weakly positive, CD24 negative, CD26 negative, LGR5 negative, SSEA3 negative, MHC1 weakly positive (particularly weakly positive compared to phase transition cells), MHC2 negative, PDL1 positive, ZO-1 positive, Na + K + / ATPase positive, Claudin 10 positive, and Table 1A below

[0109] [Table 1A]

[0110] (Here, the median fluorescence intensity of each marker divided by the value of the negative control (stained with isotype control antibody) 30 or more: Strongly positive 10 or more but less than 30: Moderately positive 5 or more but less than 10: Weak positive (Weak positive, medium positive, and strong positive are collectively referred to as "positive") Less than 5: Negative The group may include, but is not limited to, one or more of the expression characteristics described in 1. Alternatively, the group may be the group consisting of CD105 negative to weakly positive, CD24 negative, CD26 negative, LGR5 negative, SSEA3 negative, MHC1 weakly positive, MHC2 negative, ZO-1 positive, and Na+ / K+ ATPase positive.

[0111] The various genes used in the present invention are identified by the following accession numbers.

[0112] [Table 1B-1]

[0113] [Table 1B-2]

[0114] [Table 1B-3]

[0115] [Table 1B-4]

[0116] [Table 1B-5]

[0117] The same applies to all other proteins mentioned herein, and it is therefore understood that the name of a given protein or nucleic acid refers not only to the protein or nucleic acid as set forth in the sequence listing, but also to functionally active derivatives.

[0118] As used herein, the intensity of expression of cell indicator markers such as CD markers is expressed as negative (sometimes indicated as "-"; when "-" and "+" are used interchangeably, both are included). Here, "dull positive" is included in the negative definition. Non-negative results, i.e., results in which significant expression is observed, are expressed as positive (i.e., when the two categories of "+" and "-" are used, "+" may be used). When distinguishing expression levels, the intensity is classified into three levels: weak positive, moderate positive, and strong positive. For purposes of graphical display of FACS measurement results, these may be expressed as the number of "+" signs. Weak positive, moderate positive, and strong positive may be expressed as "+," "++," and "+++," respectively, but these are synonymous. In this case, they can be distinguished as "weak positive," "moderate positive," and "strong positive." When no distinction is made, they may simply be referred to as positive. Results less than weak positive are usually referred to as negative. These levels of intensity are used as commonly used in the art. These levels are relative and are defined as follows: For example, "-" indicates virtually no expression. Expression is classified into three levels: weakly positive, moderately positive, and strongly positive. During FACS separation, signals can be graded as negative, weakly positive, moderately positive, or strongly positive.

[0119] Specific levels can be identified using the mean fluorescent signal intensity (MFI) for the display of signal intensity in FACS, which can be negative, negative positive, weak positive, medium positive, and strong positive. The distribution of cells can be displayed in a histogram, and a relative judgment can be made to display negative, negative positive, weak positive, medium positive, and strong positive. Further, the criteria for determining specific measurement values ​​are explained below.

[0120] In this specification, the intensity of expression of cell indicator markers such as CD markers typically varies depending on the type of fluorescent label and the instrument settings. Therefore, the following conditions are used: when using PE-Cy 7-labeled anti-human CD44 antibody (BD Biosciences), the Blue laser area scaling factor of the FACS Canto II is set to 0.75, and the PE-Cy 7 voltage is set to 495, the weak fluorescence intensity range is approximately less than 3800, the medium fluorescence intensity range is approximately 3800 to less than 27500, and the strong fluorescence intensity range is approximately 1000 to 27500. The light intensity range is approximately 27500 or more. In the examples of this specification, the shadow The mean fluorescence intensity of the negative control (isotype control) was approximately 50 (within the range of 55±25; there may be some deviations depending on the cell lot even with the same settings, but those skilled in the art will be able to understand these deviations and carry out the measurements accordingly). Therefore, based on the idea that "the range of weak fluorescence intensity is less than approximately 3800, the range of medium fluorescence intensity is approximately 3800 to less than 27500, and the range of strong fluorescence intensity is approximately 27500 or more," the mean fluorescence intensity of the negative control (isotype control) was PE-Cy 7: It is about 50 [33-80], so weak: <76 times, medium: 76-550 times, strong: >550 times. Negative If the staining intensity pattern is the same as that of the control (isotype control), it is judged to be negative, and if there is even a slight shift, it is judged to be positive.

[0121] As used herein, other settings may include: Area Scaling Factor: FSC=0.5, Blue laser=0.75, Red laser=0.8 Voltage: FSC=270, SSC=400, FITC=290, PE=290, PerCP-Cy 5.5=410, PE-Cy 7=495, APC=430 The mean fluorescence intensity of the negative control (isotype control) can be: FITC: Approx. 130 [about 65~225] PE: Approx. 120 [about 73~204)] PerCP-Cy5.5: Approx. 120 [about 74~191] PE-Cy 7: Approx. 50 [about 33~80] APC: Approximately 110 [about 67~196].

[0122] In another embodiment, when using the Lyoplate experiment (Examples, Table 2), detection is performed using Alexa Measurements are performed using a Fluorescence 647-labeled secondary antibody (included in the kit). In this case, weak, moderate, and strong positives can be defined as follows: the median fluorescence intensity of each marker divided by the negative control (stained with an isotype control antibody) is the classification shown on the left. 30 or more: Strongly positive 10 or more but less than 30: Moderately positive 5 or more but less than 10: Weak positive (Weak positive, medium positive, and strong positive are collectively referred to as "positive" Less than 5: Negative The intensity of such cellular markers can be readily assessed by techniques such as, but not limited to, fluorescence-activated cell sorting and immunohistochemistry. With respect to the above markers and their expression levels, "negative" means that the marker expression is absent or at a significantly low level, and "positive" means that the expression is significant. The transition of a cell marker from "negative" to "positive" indicates a change from absent or low expression to high or significant expression. The term "weakly positive" refers to weak or low expression, and may be expressed as "low expression." "Moderately positive" refers to a medium level of expression that is easily detectable, and may be expressed as "moderate expression." "Strongly positive" refers to strong or high expression that is prominent and very easily detectable, and may be expressed as "high expression." In this case, the transition of expression from "weakly positive" to "moderately positive" ("moderately positive"), "moderately positive" to "strongly positive," or "strongly positive" to "moderately positive," or "moderately positive" to "weakly positive" can be readily confirmed. For example, non-target cells are strongly CD44 positive, progenitor cells are intermediately CD44 positive, and the functional mature differentiated corneal endothelial cells of the present invention are CD44 negative or weakly CD44 positive. For example, as shown in the Examples, cells can be classified into subpopulations using two or more cell surface markers.

[0123] Further cell indicators used in the present invention include the expression intensities of MHC-1 and MHC-2, both of which are related to the absence of immune rejection. Since the present invention is clinically used in cell infusion therapy, it is preferable that immune rejection is absent or low.

[0124] Further cellular indicators that may be used in the present invention include ZO-1, Na + K + These include ATPase. Since these are closely related properties that indicate the functionality of human corneal endothelial cells, it is preferable that all of these are clearly expressed normally (+).

[0125] In the present invention, proteinaceous products or biological substances related to said products can also be used as cell indicators. In the present invention, for example, (A) those whose expression is increased in functional cells having corneal endothelial characteristics of the present invention (including functional mature differentiated corneal endothelial cells) and (B) ) An example of a gene whose expression decreases in functional cells having corneal endothelial characteristics of the present invention (including functional, mature, differentiated corneal endothelial cells) is CD44. The proteinaceous product of the present invention or a biological substance related to the product can determine whether target cells are functional cells having corneal endothelial characteristics of the present invention (functional, mature, differentiated corneal endothelial cells or moderately differentiated corneal endothelial cells) by one or a combination of these cell indicators. In one embodiment, a combination of multiple genes selected from (A) may be used, or a combination of multiple genes selected from (B), or a combination of (A) and (B) may be used. Without wishing to be bound by theory, these genes are expressed in relatively high amounts, making it possible to clearly classify cells suitable for treatment from those not suitable for treatment. With regard to (A), it is understood that moderately differentiated corneal endothelial cells also show the same tendency as functional, mature, differentiated corneal endothelial cells.

[0126] In addition, in a preferred embodiment, one or more of the following may be used (some of which may overlap with the above):

[0127] [Table 1C]

[0128] Morphologically, these have been found to have the following characteristics, and markers can be used appropriately based on this information. Specifically, MMP9, SPP1, STEAP1, IL33, TSLP, and CDH1 have low expression intensities, requiring ingenuity when conducting quantitative experiments. COL4A1, COL4A2, COL8A1, COL8A2, CDH2, and TGF-β2 are expressed at elevated levels in functional cells with corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention. MMP1, MMP2, TIMP1, BMP2, IL13RA2, TGF-β1, CD44, COL3A1, IL6, IL8, HGF, THBS2, and IGFBP3 are expressed at elevated levels in non-functional cells. MMP4, CD105, and CD24 can be used to distinguish functional cells with corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention from non-functional cells based on their expression intensities. CD166 and IGFBP7 can also be used.

[0129] The functional cells provided by the present invention that have corneal endothelial properties enable the clinical application of innovative treatments, but quality control is necessary, and a highly reliable method for this purpose is required. The present invention has revealed that genetic diversity can be used to identify and quality control functional, mature, differentiated corneal endothelial cells that do not undergo cell phase transition (CST) and karyotype abnormalities (aneuploidy). The morphological characteristics of corneal endothelial cells are different depending on the culture protocol. One of the biggest obstacles in applying corneal endothelial cells to cell injection therapy is how to verify whether the corneal endothelial cells satisfy the cell quality required for functional cells with corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention; however, this problem can be solved in the present invention by also utilizing genetic diversity.

[0130] In one embodiment, the functional cells with corneal endothelial properties of the present invention may have properties specific to the functionality of a particular cytokine or a substance related thereto. Examples of such properties include, but are not limited to, high production of PDGF-BB, low production of IL-8, low production of MCP-1, high production of TNF-α, high production of IFNγ, high production of IL-1R antagonist, low production of VEGF, etc. Preferred indicators are cytokine levels that reflect a normal state, rather than cytokine levels that reflect a state in which inflammatory cells or the like attack others.

[0131] As used herein, the terms "high production" and "low production" are relative terms and are determined by whether the level is higher or lower than the level normally observed for each cytokine, etc. For example, when used in the present invention, the culture method exemplified in Example 4 (Opti-MEM-I (Life Technologies Corp., Carlsbad, CA, USA), 8% fetal bovine serum (FBS), 5 ng / mL epidermal growth factor, 20 μg / mL ascorbic acid, 200 mg / L calcium chloride, 0.08% Culture in basal medium containing chondroitin sulfate and 50 μg / mL gentamicin, or appropriate conditioned medium (see Nakahara, M. et al. PLOS One (2013) 8, e69009). When cultured, typically, PDGF-BB is preferably about 30 pg / ml or more, IL-8 is preferably about 500 pg / ml, MCP-1 is preferably about 3000 pg / ml or less, TNF-α is preferably about 10 pg / ml or more, IFNγ is preferably about 30 pg / ml or more, IL-1R antagonist is preferably about 40 pg / ml or more, and VEGF is preferably about 200-500 pg / ml or less.

[0132] Another advantage of the present invention is that it provides a cell indicator for evaluating the quality of the final product, functional cells with corneal endothelial characteristics or functional, mature, differentiated corneal endothelial cells of the present invention, beyond visual examination. This is because, during the research process of the present invention, it was found that defining product quality by product specifications or proteinaceous secretory products does not correspond to clinical pharmacological effects. While it was confirmed that all specifications applied in human stem cell clinical studies were fully satisfied, it was found that the proportion of non-target cells and the production amount of the protein product MCP-1 were in undesirable ranges. Therefore, in a preferred embodiment, it is preferable that the indicator using MCP-1 is also low production.

[0133] The cells of the present invention preferably satisfy the following criteria when subjected to quality testing before use.

[0134] The appearance test includes checking that the tissue has a hexagonal, paving-stone-like shape and is free of fibrosis.

[0135] [Table 1D]

[0136] The calculation method for E-ratio and the calculation method for non-target cells A, B, and C are as follows.

[0137] Calculation method of E-ratio: Because the fluorescence intensity varies depending on the type of fluorescent label and the instrument settings, the following conditions were used: PE-Cy7-labeled anti-human CD44 antibody (BD Biosciences) was used, and the blue laser area of ​​the FACS Canto II was used. For measurements with a scaling factor of 0.75 and a PE-Cy7 voltage of 495, set the gates as follows: First, in a dot plot with CD24 on the X axis and CD166 on the Y axis (Figure 68, upper left), set fractions A, B, C, and D. Here, fraction A is CD24-negative and CD166-positive, fraction B is CD24-positive and CD166-positive, fraction C is CD24-negative and CD166-negative, and fraction D is CD24-positive and CD166-negative. The percentage of fraction B relative to the control cells for analysis, taken as 100%, is taken as the content of non-target cells C [CD24-positive cells]. For fraction B, set fractions 1, 2, and 3 in a dot plot with CD44 on the X axis and CD105 on the Y axis, as shown in the lower left of Figure 68. The percentage of fraction 1 at this time is defined as the E-ratio, the sum of the percentages of fraction 1 and fraction 2 is defined as the content percentage of "functional mature differentiated corneal endothelial cells + moderately differentiated corneal endothelial cells," and the percentage of fraction 3 is defined as the content percentage of non-target cell A [strongly CD44 positive cells]. Separately from these, create a dot plot with CD44 on the X axis and CD26 on the Y axis, and set fractions a', b', c', and d' as shown in the upper right diagram of Figure 68. The percentage of fraction B, when the analysis control cells are defined as 100%, is defined as the content percentage of non-target cell B [CD26 positive cells] (see Figure 68).

[0138] In one embodiment, the present invention provides cells, particularly corneal endothelial cells, containing specific miRNAs, based on the first successful identification of the functionality of miRNAs. This study provides the first evidence that differences in miRNA types can be used to identify the functionality of cells in which they are expressed. MicroRNAs (miRNAs or miRs) are small non-coding RNAs that function as endogenous regulators of gene expression. Their disregulation has been implicated in the pathogenesis of various diseases. Increasing evidence suggests that miRNAs play important roles in various biological processes, including cell proliferation, development, and differentiation (Bartel DP. Cell. 2004; 116:281-297; Croce CM, Cell. 2005; 122:6-7). MiRNA expression is a key regulator of cell proliferation and development. It is essential for regulating many cellular processes, including the formation, maintenance, and remodeling of the extracellular matrix (ECM) (Rutnam ZJ, Wight TN, Yang BB. Matrix Biol. 2013; 32:74-85), and it is closely linked to CST in cHCECs. Regarding corneal endothelial-related miRNA expression, there is currently no information available that can identify functional, mature, differentiated corneal endothelial cells. The present inventors have identified miRNAs that can be used to identify functional, mature, differentiated corneal endothelial cells. These miRNAs can be detected using a 3D-Gene miRNA microarray platform (e.g., commercially available from Toray, Kamakura, Japan). Comparative studies of phenotypically distinct, functional, mature, differentiated corneal endothelial cells using hierarchical clustering revealed expression patterns of various miRNAs. Distinct miRNA expression patterns, including up- and down-regulated miRNA clusters, were revealed in cultured cells and corresponding culture supernatants. MiRNAs in culture supernatants, i.e., secreted miRNAs, may serve as a tool for noninvasively identifying functional, mature, differentiated corneal endothelial cells suitable for cell therapy via injection into the anterior chamber.

[0139] In a representative embodiment, for example, when the expression characteristics of a5 are defined as CD44 negative to weakly positive, CD24 negative, and CD26 negative for functional mature differentiated corneal endothelial cells (a5), moderately differentiated corneal endothelial cells (a1), and non-functioning corneal endothelial cells (a2), the expression characteristics of a1 are CD44 moderately positive, CD24 negative, and CD26 negative, and the expression characteristics of a2 are CD44 strongly positive, CD24 negative, and CD26 positive, then in the functional cells having corneal endothelial characteristics of the present invention, at least one miRNA has the a5 characteristic.

[0140] In one embodiment, the miRNA marker used in the present invention is provided as follows, that is, the characteristics of the miRNA are as follows: (A) Functional mature differentiated corneal endothelial cells (a5): Moderately differentiated corneal endothelial cells (a1): Non-functional corneal endothelial cells (a2) = High expression: High expression: Low expression: (Intracellular) miR23a-3p, miR23b-3p, miR23c, miR27a-3p, miR27b-3p, miR181a-5p, miR181b-5p, miR181c-5p, miR181d-5p (cell-secreted) miR24-3p, miR1273e; (B) a5:a1:a2 = High expression: Medium expression: Low expression: (Intracellular) miR30a-3p, miR30a-5p, miR30b-5p, miR30c-5p, miR30e-3p, miR30e-5p, miR130a-3p, miR13 0b-3p, miR378a-3p, miR378c, miR378d, miR378e, miR378f, miR378h, miR378i, miR184, miR148a-3p (cell-secreted) miR184; (C) a5:a1:a2 = High expression: Low expression: Low expression: (Intracellular) miR34a-5p, miR34b-5p (Cell-secreted) miR4419b, miR371b-5p, miR135a-3p, miR3131, miR296-3p, miR920, miR6501-3p; (D) a5:a1:a2 = Low expression: Low expression: Medium to high expression: (Intracellular) miR29a-3p, miR29b-3p, miR199a-3p, miR199a-5p, miR199b-5p, miR143-3p (cell-secreted) miR1915-3p, miR3130-3p, miR92a-2-5p, miR1260a; (E) a5:a1:a2 = Low expression: Medium expression: High expression: (Intracellular) miR31-3p, miR31-5p, miR193a-3p, miR193b-3p, miR138-5p (F) a5:a1:a2 = High expression: Low expression: High expression: (cell-secreted) miR92b-5p (G) a5:a1:a2 = Low expression: High expression: Low expression: (Cell secreted type) miR1246, miR4732-5p, miR23b-3p, miR23a-3p, miR1285-3p, miR5096 The expression level of the miRNA is determined among three types of cells. The cell surface antigen characteristics of a5 are CD44- to weakly positive, CD24- and CD26-negative, the cell surface antigen expression of a1 is CD44-moderately positive, CD24-negative, and CD26-negative, and the cell surface antigen expression of a2 is CD44-strongly positive, CD24-negative, and CD26-positive.

[0141] The expression intensity is strong > medium > low, and there is a statistically significant difference between strong and low expression.

[0142] [ka]

[0143] These strong, medium, and weak expression levels differ in absolute terms depending on the miRNA, but can be determined appropriately during actual measurements. Typically, the miR expression level (expression intensity) is determined by determining the gene whose fluorescence intensity is being measured, and then comparing the values ​​corrected so that the median expression intensity of all detected genes is the same, assuming that the total gene copy number between samples does not differ significantly. A significant difference (relative ratio of 2 or more, P-value 0.05 or less) is observed between strong and weak expression. Medium expression can also be included if necessary. Three or more groups If a third expression intensity different from the strongest and weakest is observed in the cells above, intermediate expression is also included in the evaluation.

[0144] The various miRNAs used in the present invention are identified by the following numbers. When used in the present invention, the relative intensities can be measured based on information from the mature form (MiRBase), but this is not limiting. It will be understood that those skilled in the art can similarly measure the relative intensities by appropriately processing information using Stem-Loop information.

[0145] [Table 1E-1]

[0146] [Table 1E-2]

[0147] [Table 1E-3]

[0148] In a preferred embodiment, the miRNA marker used in the present invention includes at least one selected from (B) or (C) of the above classifications. When using miRNA having the pattern of (B) or when using miRNA having the pattern of (C), it is possible to distinguish the functional cells (a5+a1) having corneal endothelial characteristics of the present invention by measuring only one marker. When distinguishing between functional mature differentiated corneal endothelial cells (a5) and moderately differentiated corneal endothelial cells (a1) and non-target cells (a2), for example, (A), (B) ), (D), or (E) can be used to distinguish one type. Of course, if you want to distinguish three types, you can use (B) or (E), or multiple miRNA markers such as a combination of (A) and (C). miRNA can be identified and its level determined by extracting RNA using a known method and then using microarray analysis as described in the Examples. For example, Toray's 3D-Gene TM Human microRNA Commercially available analytical chips, such as chips, can be used. The obtained data can be image-processed using a scanner (e.g., 3D-Gene Scanner 3000 (Toray Industries Inc., Tokyo, Japan)) and processed using processing software (e.g., 3D-Gene Extraction Software (Toray)). The obtained digitized fluorescent signals can be treated as raw data and further normalized. For example, the median value of the fluorescent intensity can be corrected to 25. Alternatively, the normalized level can be corrected so that the 100th highest ranked value matches.

[0149] In a preferred embodiment, the miRNA used is preferably a secretory type, because secretory miRNA allows for the identification of so-called non-destructive types without destroying cells.

[0150] In the present invention, the use of miRNA to identify functional cells having corneal endothelial characteristics of the present invention is advantageous in that it provides a non-invasive method, unlike methods for identifying cells such as CD166-positive, CD133-negative, CD105-negative, CD44-negative, CD24-negative, CD26-negative, and CD200-negative using cell surface markers (CD markers) and the like.

[0151] In one embodiment, it is preferable that the functional cells having corneal endothelial characteristics of the present invention do not exhibit abnormal values ​​of exosomes. Exosomes are small particles with a diameter of about 40 nm to 150 nm secreted by cells. These exosomes are membrane vesicles that contain a large amount of proteins with ribonuclease activity, and such abnormal values ​​can be investigated using related markers. Examples of such markers include CD63, CD9, CD81, and HSP70. The functional cells with corneal endothelial characteristics of the present invention preferably have low expression of these exosome-related markers. Specific levels can be exemplified by the following experiment. That is, typically, the presence of markers in exosome proteins in the culture supernatant using the Exoscreen method is examined. This method can measure whether exosomes are present in the body or not, and is an alternative to the Exoscreen method for detecting exosomal proteins. This can be performed by Western blotting, and the size of the Western detection band can be determined visually.

[0152] The functional cells with corneal endothelial properties of the present invention preferably have a small cell area, i.e., are small cells. In the present invention, the cell area is usually evaluated as the cell area under conditions in which PBS-treated cells are imaged. That is, in this specification, the measured value of the hybrid cell count is the area when gaps are formed between the cells, because images are obtained using PBS-treated cells. In other words, the measured cell area is lower than the mature differentiated state in which tight junctions are formed during saturated cell culture (confluent) in a culture medium. It has been found in the present invention that functional cells have a small area per cell and are of high quality due to the highest cell density in culture. This can be said to be the same level of cell area and cell density as endothelial cells of normal corneal endothelial tissue, or a level exceeding this. The preferred cell area of ​​PBS-treated cells during saturated cell culture (confluent) is approximately 250 μm for the average of the cell population or individual cells. 2 The following may be mentioned, and more preferably, about 245 μm 2 Below, approximately 240μm 2 Below, approximately 235μm 2 Below, approximately 230μm 2 Below, approximately 225μm 2 Below, approximately 220μm 2 Below, approximately 215μm 2 Below, approximately 210μm 2 Below, approximately 205μm 2 Below, approximately 200μm 2 On the other hand, the preferred cell area of ​​the functional cells having corneal endothelial properties of the present invention is, for example, about 150 μm 2 or more, approximately 155μm 2 or more, approximately 160μm 2 More than 165 μm 2 Above, approximately 170 μm2 or more, approximately 175μm 2 or more, approximately 180μm 2 The cell area can be measured by any method known in the art, but a representative example is a measurement method using phase contrast microscope images. Here, a commercially available system such as an inverted microscope system (CKX41, Olympus, Tokyo, Japan) is used. For area distribution measurement, for example, the target cells can be soaked in PBS. After pre-treatment to make it easier to measure, such as washing with (-) three times, a phase contrast microscope image was taken, e.g. The area distribution can be obtained using a commercially available system such as the BZ X-700 microscope system (Keyence, Osaka, Japan). The area distribution can also be obtained using the BZ-H3C Hybrid cell counting software (Keyence). This can be quantified using commercially available software.

[0153] Therefore, it is advantageous that the functional cells of the present invention having corneal endothelial properties have the above-mentioned preferred values ​​in at least one cell indicator selected from the group consisting of cell size, cell density, and the presence of autoantibody-reactive cells.

[0154] The functional cells with corneal endothelial characteristics of the present invention preferably have a cellular functional characteristic that is homologous to that of the functional cells with corneal endothelial characteristics of the present invention (i.e., including functional mature differentiated corneal endothelial cells and moderately differentiated corneal endothelial cells), preferably a cellular functional characteristic that is homologous to a5, which is a functional mature differentiated corneal endothelial cell, in at least one cellular indicator selected from the group consisting of cell surface markers, proteinaceous products and biological substances related to these products, SASP-related proteins, intracellular or secreted miRNAs, exosomes, cellular metabolic products including amino acids and biological substances related to these products, as described herein. Cell indicators preferred in the present invention include, for example, any of the specific numerical values, ranges, and levels described in the explanation of each indicator in this specification, and combinations of these may also be used. When a candidate cell exhibits values ​​for these cell indicators that should be exhibited by functional cells with corneal endothelial characteristics of the present invention, preferably functional mature differentiated corneal endothelial cells, as defined herein, the candidate cell is determined to be a moderately differentiated corneal endothelial cell that expresses human corneal endothelial functional characteristics when injected into the anterior chamber of a human eye, or a functional mature differentiated corneal endothelial cell. Furthermore, in addition to or in parallel with determination using the cell indicators listed above, the following indicators can also be referenced. In particular, the function of the functional cells with corneal endothelial characteristics of the present invention can be confirmed by forming a small, hexagonal, paving stone-like shape and utilizing an energy metabolic system based on mitochondrial function, and determination can be made based on whether or not they have a therapeutic effect when injected (for example, into the anterior chamber of the eye). Furthermore, without being limited thereto, surrogate marker-like indicators are also effective.Such indicators include any of the following eight indicators, or a combination thereof: (1) maintenance of endothelial pump and barrier function, including positive expression of Claudin; (2) high adhesion and binding to laminin 511 or its fragment E8; (3) secreted cytokine profile, including production of PDGFbb, TNFα, IFNγ, and IL-1 receptor antagonist above standard values; (4) determination based on the profile of microRNA (miRNA) produced; (5) determination based on the profile of metabolites produced; (6) saturation cell density during in vitro culture; (7) spatial size and distribution of cells obtained during culture; and (8) adhesion to the corneal endothelial surface when cells are injected into mouse corneas after liquid nitrogen cryoinjury. In particular, without wishing to be bound by theory, this is because protein products or biological substances related to the products make it possible to roughly determine whether the cells are CST cells, miRNAs make it possible to remove non-target cells partially or completely, cellular metabolic products or biological substances related to the products make it possible to distinguish between moderately differentiated corneal endothelial cells and functional, mature, differentiated corneal endothelial cells, and higher quality functional corneal endothelial cells can be selectively grown in culture.

[0155] In a preferred embodiment, the functional cells having corneal endothelial properties of the present invention, particularly the mature differentiated corneal endothelial cells, do not have karyotypic abnormalities. As shown by Miyai et al., cHCECs often undergo a number of passages during culture. (Miyai T, et al., Mol Vis. 2008; 14:942-50). Aneuploidy observed in cHCECs is induced during culture due to cell division. Here, the present inventors provide a new finding that the presence or absence of aneuploidy in cHCECs is closely related to a specific cell subpopulation that predominates among cHCECs. The present inventors discovered that specific cell subpopulations without karyotypic abnormalities appear in parallel with specific surface phenotype patterns of functional, mature, differentiated corneal endothelial cells present in corneal tissue. They successfully established refined culture conditions for selectively growing a cell subpopulation consisting almost entirely of functional, mature, differentiated corneal endothelial cells without karyotypic abnormalities. This has enabled safe and stable regenerative medicine to be provided by injecting functional, mature, differentiated corneal endothelial cells in the form of a cell suspension into the anterior chamber for the treatment of corneal endothelial disorders such as bullous keratopathy. Thus, the present invention has discovered that karyotypic abnormalities occur selectively in a subpopulation, which was previously unknown. Furthermore, the technology of the present invention has made it possible to select subpopulations that are substantially free of karyotypic abnormalities.

[0156] Furthermore, it has been revealed in the present invention that the cells equipped with functional corneal endothelial characteristics of the present invention exhibit a subpopulation-specific phenomenon in which autoantibodies are substantially absent. In the present invention, by selecting a specific subpopulation, it is possible to select a subpopulation that is substantially free of autoantibodies. Autoantibodies can be measured by techniques known in the art. For example, the following procedure can be exemplified. First, HCECs are fixed with methanol, washed twice with PBS, and then soaked in PBS-0.2% Permeabilize with Tx-100 (room temperature, 15 minutes) and block with 1% BSA / PBS (room temperature >1 hour). Then, 250 μL of normal human serum diluted 5-fold or 25-fold with 1% BSA / PBS was added to the wells and allowed to stand at 4°C overnight. After washing four times with PBS-0.2% Tx-100, the wells were resuspended in 1% Alexa Fluor 488-labeled anti-human IgG (5 μg / mL) and Alexa Fluor 647-labeled anti-human IgM (5 μg / mL). BSA / PBS was added (250 μL / well). The plate was then left to stand at room temperature for 1 hour, washed twice with PBS-0.2% Tx-100, and once with PBS. Nuclei were stained with DAPI (5 μg / mL) for 15 minutes at room temperature, washed with PBS, and then examined under an inverted fluorescence microscope (BZ-9000) (see Figure 10C).

[0157] In another aspect, the present invention provides a cell population comprising functional cells having corneal endothelial characteristics of the present invention, in particular functional mature differentiated corneal endothelial cells.

[0158] The cell population of the present invention has an average cell density of at least about 1500 cells / mm when the cell is in a saturated cell culture (confluent). 2 or more, at least about 1600 pieces / mm 2 or more, at least about 1700 pieces / mm 2 or more, at least about 1800 pieces / mm 2 or more, at least about 1900 pieces / mm 2 or more, or at least about 2000 / mm 2 or more is preferable. It is understood that the cell population containing functional cells with corneal endothelial properties of the present invention is provided at a considerably high density due to the small size of the cells. Cell density is a characteristic found in association with high-quality corneal endothelial functional properties, and conversely, measuring such cell density can be used as an index for selecting high-quality functional, mature, differentiated corneal endothelial cells. Since cell density is a value directly related to cell area, it can be calculated in the same way by measuring the cell area using any method known in the field. As mentioned above, a representative example is a measurement method using phase-contrast microscope images, and in this case, images can be taken using a commercially available system such as an inverted microscope system (CKX41, Olympus, Tokyo, Japan). In addition, area distribution measurement For the determination, for example, the target cells are pretreated to facilitate measurement by washing them three times with PBS(-), and then the phase contrast microscopic images are taken using, for example, a BZ X-700 microscope system (Keyence, Osaka, Japan). The area distribution can be obtained using a commercially available system such as the BZ-H3C Hybrid cell counting software (Keyence). can.

[0159] In a preferred embodiment, the cell population of the invention has an average cell density of at least about 2100 cells / mm 2 or more, at least about 2200 pieces / mm 2 or more, at least about 2300 pieces / mm 2 or more, at least about 2400 pieces / mm 2 or more, at least about 2500 pieces / mm 2 That's all. The upper limit can be any feasible value, but is not limited to these. For example, about 3000 pieces / mm 2 Approximately 3100 pieces / mm 2 , about 3200 pieces / mm 2 , about 3300 pieces / mm 2 , about 3400 pieces / mm 2 , about 3500 pieces / mm 2 , about 3600 pieces / mm 2 , about 3700 pieces / mm 2 , about 3800 pieces / mm 2 , about 3900 pieces / mm 2 , about 4000 pieces / mm 2 It is understood that any combination of these upper and lower limits can be used as a preferred cell density range for the cell population of the present invention.

[0160] Such characteristics of cell density or cell area can be applied to the evaluation of the suitability of the final cultured cell product for clinical trials by the phase contrast image quantification technique of cultured cells using the hybrid cell counting method. The functional mature differentiated corneal endothelial cells of the present invention have a small area per cell and are suitable for culture. As shown in the examples, the cell density of cultured human corneal endothelial cells prepared by the production method of the present invention is highest at a cell area of ​​216 μm. 2 , cell density was 2582 cells / mm 2The cell area and cell density are at the same levels as those of endothelial cells in normal corneal endothelial tissue.

[0161] In one embodiment, the cell population of the present invention is characterized in that the functional cells with corneal endothelial characteristics of the present invention are present at a ratio that is higher than the ratio that occurs in nature. By providing a cell population in which the ratio of cells capable of inducing corneal endothelial functional characteristics is higher than the ratio that occurs in nature, it is possible to provide a more effective treatment than using a population of corneal endothelial cells that are available in nature. The reason why such a ratio of cells capable of inducing corneal endothelial functional characteristics can be increased is because a technology has been provided that can identify and select numerous subpopulations of functional cells with corneal endothelial characteristics of the present invention (for example, functional mature differentiated corneal endothelial cells or moderately differentiated corneal endothelial cells).

[0162] In a preferred embodiment, at least 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more of the cells in the cell population of the present invention are advantageously functional cells with corneal endothelial characteristics of the present invention. Here, the cells contained in these cell populations may have cell functional characteristics belonging to either the so-called a5 or a1 category. For example, cells contained in these cell populations may have cell functional characteristics including CD166 positivity and CD133 negativity, and, if necessary, cells with CD44 negativity to intermediate positivity may be selected. Without wishing to be bound by theory, the reason why the cell population of the present invention is effective is that, when the cell population contains a certain level of functional cells with corneal endothelial properties of the present invention, good therapeutic or preventive effects are exhibited when the cell population is injected into a subject. In a preferred embodiment, the cell population of the present invention advantageously contains about 70% or more of the cells as functional cells with corneal endothelial properties of the present invention. The presence of this level of functional cells with corneal endothelial properties of the present invention allows the cell density (for example, about 2300 cells / mm) to be increased to a level that is considered to be an indicator of the success of corneal cell injection therapy. 2 ) can be achieved. In a more preferred embodiment, the cell population of the present invention advantageously comprises functional cells with corneal endothelial properties of the present invention in an amount of at least about 90%. When attempting to achieve this level of abundance ratio of functional cells with corneal endothelial properties of the present invention, it is not possible to do so by chance, but rather it is necessary to establish techniques and information that can accurately and reliably identify and separate cell subpopulations, which can be said to be largely impossible with conventional techniques. The cell density, which is used as a measure of the success of corneal cell injection therapy, can be calculated by measuring the average cell density of cells that have taken root on the surface of human corneal endothelium after injection of the cell population. Such a cell density is at least about 1,000 cells / mm 2 or more, preferably at least about 1100 cells / mm 2or more, preferably at least about 1200 cells / mm 2 or more, preferably at least about 1300 cells / mm 2 or more, preferably at least about 1400 cells / mm 2 or more, preferably at least about 1500 / mm 2 or more, preferably at least about 1600 cells / mm 2 That's all, I prefer At least about 1700 pieces / mm 2 or more, preferably at least about 1800 cells / mm 2 or more, preferably at least about 1900 cells / mm 2 or more, preferably at least about 2000 / mm 2 or more, preferably at least about 2200 cells / mm 2 or more, preferably at least about 2300 cells / mm 2 or more, preferably at least about 2400 cells / mm 2 or more, preferably at least about 2500 / mm 2 or more, preferably at least about 2600 cells / mm 2 or more, preferably at least about 2700 cells / mm 2 or more, preferably at least about 2800 cells / mm 2 or more, preferably at least about 2900 cells / mm 2 or more, preferably at least about 3000 / mm 2 It could be more than that.

[0163] In a further preferred embodiment, the cell population of the present invention is characterized in that the proportion of functional mature differentiated corneal endothelial cells is higher than that found in nature. Functional mature differentiated corneal endothelial cells express the functional properties of human corneal endothelium when injected directly into the anterior chamber of the eye, and by providing a cell population in which the proportion of high-quality cells is higher than that found in nature, it is possible to provide a more effective treatment than using a population of corneal endothelial cells that are naturally available. The reason why the proportion of such high-quality functional cells can be increased is because a technology has been provided that enables the identification and selection of the functional mature differentiated corneal endothelial cells of the present invention from numerous subpopulations.

[0164] In a preferred embodiment, at least 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 98% or more, or about 99% or more of the cells in the cell population of the present invention are advantageously functional mature differentiated corneal endothelial cells. The ratio of such functional mature differentiated corneal endothelial cells may be referred to herein as the "E-ratio" (also referred to as the "E ratio"). The method for calculating the E-ratio is described elsewhere in this specification. Herein, the cells contained in these cell populations may have functional properties belonging to the so-called a5 category. For example, cells that are CD166-positive, CD133-negative, and CD44-negative to weakly-positive (preferably CD44-negative) can be selected as cells to be contained in these cell populations. Alternatively, CD166-positive, CD133-negative, and CD200-negative cells can be selected. Without wishing to be bound by theory, the reason why the cell population of the present invention with improved quality is effective is that the inclusion of a certain level of functional, mature, differentiated corneal endothelial cells results in an even better therapeutic or preventive effect when the cell population is injected into a subject. In a preferred embodiment, the cell population of the present invention advantageously contains about 40% or more of the cells as functional, mature, differentiated corneal endothelial cells. The presence of this level of functional cells allows for a high-quality cell density (for example, about 1,000 cells / mm of cells engrafted on the corneal endothelial surface) that is considered to be an indicator of the success of corneal cell injection therapy. 2 or more, preferably about 2000 cells / mm 2 , usually about 2300 cells / mm 2) can be more reliably achieved. In a more preferred embodiment, the cell population of the present invention advantageously comprises at least about 70% or more, more preferably at least 80% or more, and even more preferably at least about 90% or more of the cells being functional mature differentiated corneal endothelial cells. When attempting to achieve an abundance ratio of functional cells at this level, it is not possible to do so by chance, but rather it is necessary to establish techniques and information that can precisely and reliably identify and separate cell subpopulations, which can be said to have been almost impossible with conventional techniques. Note that by using the technology of the present invention, the ratio of functional mature differentiated corneal endothelial cells can be further increased, and it is also possible to provide a cell population in which, for example, at least about 95% or more, at least about 96% or more, at least about 97% or more, at least about 98% or more, or at least about 99% or more of the cells are functional mature differentiated corneal endothelial cells. Furthermore, by providing a cell population containing such functional mature differentiated corneal endothelial cells, it is possible to achieve a ratio of about 2,300 cells / mm 2 More than (e.g., approximately 3000 cells / mm 2 It has also been demonstrated that such therapeutic results can be achieved within one month of injection. It has been proven that the system provides unprecedentedly fast and high-quality treatment techniques.

[0165] In one embodiment, the functional cells with corneal endothelial characteristics of the present invention (including functional mature differentiated corneal endothelial cells) or cell population are characterized by lower expression of HLA class I antigens and cell degeneration-associated antigens involved in immune rejection reactions compared to other subpopulations. Furthermore, the functional cells with corneal endothelial characteristics of the present invention, particularly functional mature differentiated corneal endothelial cells, do not contain autoantibodies found in other subpopulations, and therefore can be said to be immunologically stable cells.

[0166] In one embodiment, the cellular metabolic products and related biological substances used in the present invention include the following substances: succinate, Pro, Gly, glycerol 3-phosphate, Glu, lactate, arginine. Succinic acid, xanthine, N-carbamoyl aspartic acid, isocitric acid, cis-aconitic acid Acid, Ala-citric acid, 3-phosphoglyceric acid, hydroxyproline, malic acid, uric acid, Examples of amino acids that may be used include ATP, ATP, ATP-dependent amino acids, ... Alternatively, the cellular metabolic products of the present invention include, but are not limited to, the following: Substances decreased by culture (substances taken up by cells): Arg, creatine, total amino acids, Ty r, carnosine, Asp, total essential amino acids, total ketogenic amino acids, Trp, Val, total oxalate Acid-related amino acids, total glutamic acid-related amino acids, total acetyl-CoA-related amino acids, total succinyl-CoA-related amino acids, citrulline, total BCAAs, Fischer ratio, hypoxanthine, Leu, Asn, Ile, 2-hydroxyglutaric acid, pyruvate, Ser, citrulline / ornithine ratio, uric acid, β-alanine. Substances increased by culture (substances excreted from cells). : Sarcosine, sedoheptulose 7-phosphate, spermidine, spermine, total adenylate, total glutathione, total guanylate, UDP-glucose, XMP, xylulose 5 -phosphate, cis-aconitic acid, citric acid, betaine, glucose 6-phosphate, lactic acid / pyruvic acid, glycerol 3-phosphate, Ala, lactic acid, 2-oxoisovaleric acid, arginine Succinic acid, Glu, hydroxyproline, xanthine, ornithine, total pyruvate-related acids Amino acid, Pro, Gly, N,N-dimethylglycine, choline, urea, folic acid, His, creatinine N, Met, Lys, Thr, succinic acid, γ-aminobutyric acid, Phe, total non-essential amino acids, total fumaric acid-related amino acids, total aromatic amino acids, total glucogenic amino acids In particular, in the present invention, an increase in serine, alanine, proline, glutamine or the citrate / lactic acid ratio, particularly the citrate / lactic acid ratio, in the culture supernatant can be used to perform quality control of the functional cells having corneal endothelial characteristics or functional, mature, differentiated corneal endothelial cells of the present invention.

[0167] The ability to use metabolites allows for non-invasive identification of cellular characteristics. Metabolomic analysis conducted as part of the present invention revealed that the energy metabolic characteristics of each subpopulation of cultured human corneal endothelial cells, a so-called "heterogeneous population" conventionally used, differ significantly. It was found that the glycolytic energy metabolic pathway was enhanced in the cell culture supernatant of a subpopulation of cells (transformed cells) that had undergone phase transition, while the mitochondrial energy metabolic pathway was enhanced in a subpopulation of effector cells (functional, mature, differentiated corneal endothelial cells) without karyotypic abnormalities. Mitochondria are responsible for regulating cellular energy production in the majority of somatic cells, and all cell types in a particular state can have different metabolic characteristics. While proliferative cells, such as stem cells, tend to favor glycolysis, mature, differentiated cells, such as the functional, mature, differentiated corneal endothelial cells of the present invention, are said to be more under the regulation of oxidative phosphorylation (OXPHOS). From these findings, it can be said that by examining information on the metabolic profile of either an increased dependency on OXPHOS activity during differentiation or a shift to glycolytic metabolism during cell proliferation, the quality of the cells of the present invention can be improved, and this contributes to the optimization of the conditions for culturing and producing the cells of the present invention. "Cultured human corneal endothelial cells" as a "heterogeneous population" can exhibit senescence phenotypes, endothelial-mesenchymal transition (EMT), and other conditions. T) and a tendency to undergo cellular phase transition (CST) to a transformed fibroblast-like cell morphology. We discovered a way to distinguish subpopulations among cHCECs based on their secreted metabolites using culture supernatants. The CST subpopulation utilizes anaerobic lysis instead of mitochondria-dependent OXPHOS. The cells exhibit a tendency toward sugars. In the form of a cell suspension, metabolically defined, functional, mature, differentiated corneal endothelial cells can be used to provide a safe and stable product for regenerative medicine.

[0168] In the present invention, for example, an extracellular flux analyzer is purchased and used to continuously track metabolic products and oxygen consumption in the culture medium in addition to proteinaceous products and secreted miRNAs as a process control method. This demonstrates that the mitochondrial energy metabolism system is most enhanced in the functional mature differentiated corneal endothelial cells of the present invention. In the present invention, lactate, pyruvate, lactate / pyruvate, citrate / lactate, Ser, Pro / Ser, Leu, Ile (branched-chain amino acids), and Gln are utilized in the culture medium, and the present invention provides a method for process control during product production that should be applied to clinical trials and manufacturing. Its usefulness as an evaluation method can be verified in practice.

[0169] In another embodiment, the functional mature differentiated corneal endothelial cells or cell population of the present invention have a specific gene morphology that is superior to other subpopulations. Examples of the "gene characteristic" include genes corresponding to any of the cell functional characteristics exhibited by functional cells having corneal endothelial characteristics of the present invention (e.g., functional mature differentiated corneal endothelial cells or moderately differentiated corneal endothelial cells) in any of the gene products described above in the section on proteinaceous products.

[0170] In another embodiment, the functional mature differentiated corneal endothelial cells or cell population of the present invention are characterized by not substantially eliciting any unintended biological responses, including serum cytokine profiles, after administration to a living body. Conventional low-quality cells have been shown to elicit no inflammatory cytokines even two days after injection, resulting in the results shown in Figures 61-A to 61-B.

[0171] On the other hand, it is understood that the functional cells having corneal endothelial characteristics, functional mature differentiated corneal endothelial cells or cell population of the present invention do not induce abnormal or inflammatory cytokines after 2 days, after 1 week, or thereafter, as shown in Figures 62 and 63 .

[0172] The "untargeted" cytokine profile used herein includes, but is not limited to, RANTES, PDGF-BB, IP-10, MIP-1b, VEGF, EOTAXIN, IL-1ra, IL-6, IL-7, IL-8, IL-0, IL-10, IL-12(p70), IL-13, IL-17, FGFbasic, G-CSF, GM-CSI, IFN-γ, MCP-1, MIP-1a, TNF-α, etc. The "untargeted cytokine profile" is a profile in which, when its production is detected in a higher amount than normal, it indicates that the cells are not functional cells having corneal endothelial characteristics of the present invention and are therefore "untargeted."

[0173] As used herein, the term "unintended biological response" refers to the induction of a level exceeding that normally induced in at least one of the above-mentioned "unintended" cytokine profiles.

[0174] In one aspect, the present invention provides a cell bank containing functional cells or cell populations having corneal endothelial properties of the present invention. A cell bank refers to an institution or system that stores "cells" (usually cultured cells) that have been produced or collected through research or the like, and provides them to other researchers and businesses.

[0175] In another aspect, the present invention provides a product comprising the functional cells or cell population of the present invention having corneal endothelial properties. Such a product may be in any form, and includes, but is not limited to, a cell-processed product prepared for administration to humans. Such a cell product preferably has not undergone unintended transformation and has no physiological activity produced by the cells or tissue. There should be no or little effect from harmful substances, no or little effect on normal cells or tissues, no or little possibility of forming ectopic tissue, no or little possibility of causing undesirable immune reactions, no or little possibility of tumor formation and canceration, and if gene transfer is used, it is desirable that the safety evaluation specified in the Gene Therapy Product Guidelines has been conducted and that general toxicity tests, etc. have been passed.

[0176] In another aspect, the present invention provides a method for preserving functional cells having corneal endothelial characteristics, functional mature differentiated corneal endothelial cells, or cell populations of the present invention, comprising passaging said cells or cell populations by changing the medium. Here, it has been clarified in the present invention that this medium change maintains and preserves the functional characteristics of the cells. Any medium can be used as the medium used here, but it is preferably advantageous to use the components and medium used in the cell production methods described herein.

[0177] In another embodiment, the present invention provides a method for delivering functional cells with corneal endothelial characteristics, functional mature differentiated corneal endothelial cells, or cell populations of the present invention, comprising the step of carrying out the method for preserving functional cells with corneal endothelial characteristics, functional mature differentiated corneal endothelial cells, or cell populations of the present invention.

[0178] Although sorting is a typical example of a procedure for separating functional cells having corneal endothelial properties and functional mature differentiated corneal endothelial cells of the present invention, other methods can also be used, such as selective apoptosis induction (miRNA switch method) or necrosis induction (glucose starvation, etc.) in non-target cells by utilizing the difference in cellular properties between target cells and non-target cells. However, in the present invention, the purity of functional cells having corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present invention is usually increased by the production method of the present invention.

[0179] (Method for producing functional human corneal endothelial cells that can express human corneal endothelial functional properties when injected into the anterior chamber of the human eye) In one aspect, there is provided a method for producing human functional corneal endothelial cells (functional cells with corneal endothelial characteristics of the present invention) or functional mature differentiated corneal endothelial cells that are capable of inducing human corneal endothelial functional characteristics when injected into the anterior chamber of a human eye, the method comprising a step of maturing and differentiating corneal endothelial tissue-derived cells or corneal endothelial precursor cells. This method may be carried out after a dedifferentiation step in addition to the maturation and differentiation step. As described elsewhere in this specification, the functional cells with corneal endothelial characteristics of the present invention include not only "functional mature differentiated corneal endothelial cells" that have corneal endothelial functional characteristics in their original state, but also "functional mature differentiated corneal endothelial cells" that lack some of the functions but are used in the same way or that exhibit functions equivalent to those of functional mature differentiated corneal endothelial cells after cell injection.

[0180] In another aspect, the present invention provides a method for producing human functional corneal endothelial cells (functional cells having corneal endothelial characteristics of the present invention) or functional mature differentiated corneal endothelial cells that can induce human corneal endothelial functional characteristics when injected into the anterior chamber of the human eye, which method comprises the step of culturing corneal endothelial tissue-derived cells or corneal endothelial precursor cells under conditions that cause actin depolymerization. Culturing by a step that includes actin depolymerization achieves mature differentiation, thereby enabling the cells to express corneal endothelial functional characteristics when injected into the anterior chamber of the human eye.

[0181] As used herein, the term "differentiated to maturity" may strictly differ from the ordinary meaning used in this field. That is, as used herein, "differentiated to maturity," when referring to corneal endothelium, refers to differentiated cells that exhibit corneal endothelial function, and that have been confirmed to form small, hexagonal, paving stone-like shapes that are optimal for cell injection and utilize an energy metabolic system based on mitochondrial function.

[0182] The present invention was accomplished by discovering that when corneal endothelial cells, cells differentiated in this way, or their precursor cells are exposed to conditions that cause actin depolymerization, they undergo maturation and differentiation in the strict sense described above.

[0183] In one embodiment, actin depolymerization or conditions for achieving actin depolymerization used in the present invention are achieved by at least one agent selected from the group consisting of a ROCK inhibitor, an HDAC inhibitor, an actin polymerization inhibitor, a PPARγ inhibitor, an MMP2 inhibitor, a p53 activator, and miRNA.

[0184] In a specific embodiment, actin polymerization inhibitors used in the present invention include, but are not limited to, latrunculin A and swinholide A. Here, latrunculin A can be used at, for example, about 0.4 μM, and swinholide A can be used at, for example, about 0.1 μM.

[0185] Examples of HDAC inhibitors include trichostatin A (TSA) and vorinostat. Trichostatin A can be used at approximately 0.5 μM. Examples of PPARγ inhibitors include rosiglitazone and pioglitazone. Examples of MMP2 inhibitors include resveratrol. Examples of p53 activators include those used in cancer research. Examples of miRNAs include, but are not limited to, those associated with activation in human functional corneal endothelial cells that can induce corneal endothelial functional characteristics upon injection into the anterior chamber of the human eye, such as miRNA 34, 1246, 1273, and 4732.

[0186] In another specific embodiment, the agent used for actin depolymerization or the conditions for achieving actin depolymerization is present in the medium in the step at a concentration effective to achieve actin depolymerization, such as about 1 to about 30 μM, e.g., about 10 μM, for the ROCK inhibitor Y-27632, or about 1 to about 30 μM, e.g., about 10 μM, for the HDAC inhibitor trichostatin. The concentration is about 100 to 2000 nM, for example, 500 nM, but is not limited thereto. By measuring cellular function (eg, using surrogate markers) or by identifying cellular indicators, concentrations can be adjusted accordingly.

[0187] In a further aspect, the method of the present invention for producing functional cells having corneal endothelial characteristics or functional mature...

Claims

[Claim 1] The invention described in the examples.

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