Human functional corneal endothelial cell and application thereof

Abstract

To provide a method for producing a cultured human corneal endothelial cell useful for an extremely long period of time for improving corneal opacity and hydration edema of a patient with corneal endothelial dysfunction.SOLUTION: A method for producing a human functional corneal endothelial cell capable of inducing a human corneal function when injected into an anterior chamber of a human eye, comprising (b) a step of proliferating and / or differentiating / maturing a corneal endothelial progenitor cell under a culture condition capable of minimizing culture stress such as proliferation stress.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to 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, a pharmaceutical containing the cells, a method for producing the same, and the like. [Background technology]

[0002] The current treatment for corneal endothelial disorders, such as bullous keratopathy, is keratoplasty using donor corneas, but the long-term clinical outcomes of this procedure are unsatisfactory. Furthermore, visual acuity after corneal transplantation is often poor in terms of patient satisfaction due to the induction of irregular corneal astigmatism. Approximately 60% of corneal transplant patients have corneal endothelial dysfunction, including bullous keratopathy and early Fuchs corneal dystrophy. 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 corneal endothelial dystrophy. The potential prevalence of a genetic predisposition to Fuchs corneal endothelial dystrophy in Europe and the United States has been reported to be approximately 5% or higher. Corneal transplantation requires a donor cornea to treat one diseased eye, and therefore does not address the ongoing donor shortage. Considering the large number of potential patients worldwide, there is a strong global demand for an innovative medical treatment that is more versatile and simpler than corneal transplantation techniques and can be applied at a wider range of medical institutions. In addition, cell injection therapy produces a normal corneal shape without the distortion associated with corneal transplantation, resulting in the recovery of good visual function over the long term. 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 become contaminated with cell subpopulations that have different traits from those of corneal endothelial cells maintained in healthy corneal endothelial tissue due to cell phase transitions during culture (fibrosis, epithelial-mesenchymal transition, endothelial-mesenchymal transition, senescence, dedifferentiation, etc.), and by devising a technique for selectively growing subpopulations during culture, they have confirmed that a specific subpopulation, i.e., functional cells (also referred to as effector cells) that fully possess the functions of mature, differentiated human corneal endothelial cells, forms a small, hexagonal, paving stone-like shape that is ideal for cell injection therapy, and has traits similar to those of corneal endothelial cells maintained in healthy corneal endothelial tissue.

[0004] In this disclosure, we have intensively investigated a technology for testing cell traits that identifies cultured human corneal endothelial cells that have been confirmed to have early clinical effects and long-term stable clinical effects in clinical trials, and have achieved the world's first invention of this technology.Based on this newly invented technology, we have completed an invention that identifies and provides cultured human corneal endothelial cells that ensure early clinical effects and long-term stable clinical effects by defining mitochondrial function with various metabolic enzymes. This disclosure provides a world-first, groundbreaking, patient-friendly medical treatment that reconstructs homogeneous, compact, and highly cell-dense corneal endothelial tissue over the long term after transplantation using a significantly less costly manufacturing method that enables stable production, an absolute requirement for consistently providing the same medical treatment, and widespread application worldwide.These are cultured human corneal endothelial cells with enhanced mitochondrial-dependent oxidative respiration.They have been found to be effective even in patients who experience rejection responses to corneal transplants. Furthermore, the production method disclosed herein avoids the activation of multiple epigenetic genes, such as metabolic products produced by enzymes involved in the TCA metabolic pathway that act in the cytoplasm or nucleus, particularly the acetylation of histones by acetyl coenzyme A (AcCoA), and as a result, phase transition of cultured cells does not occur, and metabolic reprogramming is inclined toward maintaining mitochondrial function. This innovative invention minimizes the generation of the above-mentioned impure subpopulation cells, and provides highly functional endothelial cells that are extremely useful in the long term for improving corneal opacity and hydration edema in patients with general corneal endothelial dysfunction.

[0005] Thus, the present disclosure provides: (Item 1) A method for producing human functional corneal endothelial cells capable of inducing human corneal function when injected into the anterior chamber of a human eye, comprising: (b) A step of proliferating and / or differentiating and maturing corneal endothelial precursor cells under culture conditions that can minimize culture stress, such as proliferation stress. A method comprising: (Item 2) The method described in (Item 1), wherein the human corneal function includes corneal endothelial cell functional characteristics. (Item 3) (a) The method according to (Item 1) or (Item 2), further comprising the step of dedifferentiating cells derived from human corneal endothelial tissue to obtain the corneal endothelial precursor cells. (Item 4) A method for producing functional human corneal endothelial cells that can induce human corneal function, particularly corneal endothelial cell functional properties, when injected into the anterior chamber of a human eye, the method comprising the steps of: proliferating and / or differentiating and maturing human corneal endothelial precursor cells in the presence of a cell growth factor in an amount less than that at which transformation occurs; (Item 1) to (Item 3). (Item 5) The method according to any one of (Item 1) to (Item 4), wherein the cell growth factor comprises epidermal growth factor (EGF). (Item 6) The method according to any one of (Item 1) to (Item 5), wherein the transformation comprises endothelial-mesenchymal transition. (Item 7) The method according to any one of (Item 1) to (Item 6), wherein the step of proliferation and / or differentiation / maturation is carried out in the presence of a ROCK inhibitor. A method according to any one of (Item 1) to (Item 6), comprising a step of confirming that the cells obtained in (Item 8)(b) have increased mitochondria-dependent oxidative phosphorylation in mitochondria, no increased expression of acetyl-CoA in the cytoplasm or nucleus, and that epigenetic multigene expression mediated by histone acetylation by acetyl-CoA has not been induced. (Item 9) The method according to any one of (Item 1) to (Item 8), comprising a step of confirming that the cells obtained in (b) are cells expressing one or more metabolic enzymes selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malic enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain ketoacid dehydrogenase 2 (BCKDH2) in mitochondria. (Item 10) The method according to any one of (Item 1) to (Item 9), comprising a step of confirming that the cells obtained in (b) do not express or barely express ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malic enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), and / or lactate dehydrogenase (LDH). The method according to any one of the preceding items, wherein the expression of IDH1 and / or ACSS2 in the cells obtained in (Item 10A)(b) is attenuated compared to non-human functional corneal endothelial cells. (Item 10B) A method according to any one of the preceding items, wherein ATP citrate lyase (ACLY) is not expressed in the cells obtained in (b). (Item 10C) The method according to any one of the preceding items, wherein the expression of IDH2 in the cells obtained in (b) is enhanced compared to non-human functional corneal endothelial cells. (Item 11) The method according to any one of (Item 1) to (Item 10), comprising a step of confirming that the cells obtained in (b) show expression of ion channels and / or monocarboxylic acid transporters that lead to corneal endothelial (cell) functional properties that improve corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density over a sustained, long-term period and leading to improved vision. (Item 12) The method according to any one of (Item 1) to (Item 11), comprising a step of confirming that the expression of sodium-hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1) is enhanced in the cells obtained in (b). (Item 13) The method according to any one of (Item 9) to (Item 10), comprising a step of confirming that the cells obtained in (b) have increased expression of bicarbonate anhydrase 5B (CA5B). (Item 14) The method according to any one of (Item 1) to (Item 12), comprising a step of confirming that the cells obtained in (b) have the property that metabolic enzymes involved in the TCA cycle and metabolic products such as Acetyl CoA are not present in the cytoplasm or nucleus, but are organelle-selectively localized in mitochondria, so as not to lead to the generation of contaminating phase transition cells. (Item 15) The method according to any one of (Item 1) to (Item 14), wherein the human functional corneal endothelial cells are produced from cells selected from the group consisting of corneal endothelial tissue-derived cells, 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 produced by a direct programming method.

[0006] The following inventions are also provided: (Item 16) Human functional corneal endothelial cells in which expression of functional proteins is observed that leads to corneal endothelial (cell) functional characteristics that improve corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density over a sustained long period of time and leading to improved vision, or in which proteins that inhibit the corneal endothelial (cell) functional characteristics are not induced or are reduced. (Item 17) The cells are human functional corneal endothelial cells that can induce human corneal endothelial functional properties when injected into the anterior chamber of the human eye, and the cells described in (Item 16) express one or more metabolic enzymes selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malic enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain ketoacid dehydrogenase 2 (BCKDH2) in their mitochondria. (Item 18) The cells are human functional corneal endothelial cells that can induce human corneal endothelial functional characteristics when injected into the anterior chamber of the human eye, and include at least one selected from the group consisting of cells in which mitochondria-dependent oxidative phosphorylation is increased or acetyl-CoA expression in the cytoplasm or nucleus is not increased, and epigenetic multigene expression via histone acetylation by acetyl-CoA is not induced. (Item 19) The cell is a human corneal endothelial cell, and is a cell described in any one of (Item 16) to (Item 18), in which ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malic enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), and / or lactate dehydrogenase (LDH) are not expressed or are substantially not expressed. (Item 20) The cells described in any one of (Item 16) to (Item 19), wherein the expression of sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1) is enhanced in the human functional corneal endothelial cells. (Item 21) The cells according to any one of (Item 16) to (Item 20), wherein the expression of bicarbonate anhydrase 5B (CA5B) is enhanced in the human functional corneal endothelial cells. (Item 22) The cell according to any one of (Item 16) to (Item 21), wherein the human functional corneal endothelial cells comprise all of the following selected from the group consisting of: (i) the property that metabolic enzymes involved in the TCA cycle and the like and metabolic products such as Acetyl CoA are not present in the cytoplasm or nucleus but are organelle-selectively localized in the mitochondria so as to prevent the generation of contaminating phase transition cells; (ii) an increase in mitochondria-dependent oxidative phosphorylation in mitochondria; (iii) a decrease (including absence of induction) in epigenetic multigene expression mediated by histone acetylation by acetyl CoA; (iv) an increase in the expression of sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1); and (v) an increase in the expression of bicarbonate anhydrase 5B (CA5B). (Item 23) Cells described in any one of (Item 16) to (Item 22), in which endothelial-mesenchymal transition has not occurred or has not substantially occurred. (Item 24) Human functional corneal endothelial cells that can induce human corneal endothelial functional properties when injected into the anterior chamber of the human eye, wherein endothelial-mesenchymal transition has not occurred or has not substantially occurred. (Item 25) The cells according to any one of (Item 16) to (Item 24), wherein the human functional corneal endothelial cells are produced from cells selected from the group consisting of corneal endothelial tissue-derived cells, 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 produced by a direct programming method. (Item 26) A cell population comprising cells produced by the method according to any one of (Item 1) to (Item 15) and / or the cells according to any one of claims 16 to 25. (Item 27) A method for quality control or process control of human functional corneal endothelial cells that can induce human corneal endothelial functional properties when injected into the anterior chamber of a human eye, the method comprising a step of confirming that one or more metabolic enzymes selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malic enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain ketoacid dehydrogenase 2 (BCKDH2) are expressed in the mitochondria of the cells. (Item 28) The method described in (Item 27), further comprising a step of confirming that the expression of acetyl-CoA in the cytoplasm and nucleus of the cells and epigenetic multigene expression mediated by histone acetylation by acetyl-CoA are not induced. (Item 29) A method described in any one of (Item 27) or (Item 28), further comprising a step of confirming that the expression of sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1) is increased in the cells. (Item 30) A method according to any one of (Item 27) to (Item 29), further comprising a step of confirming that expression of bicarbonate anhydrase 5B (CA5B) is increased in the cells. (Item 31) A method for quality control or process control of human functional corneal endothelial cells that can induce human corneal endothelial functional properties when injected into the anterior chamber of a human eye, or a method for detecting non-functional corneal endothelial cells mixed in with human functional corneal endothelial cells, comprising: (1) On the day of transplantation, phase contrast images were used to visually inspect the tissue and found no fibroblasts, foreign matter, discoloration, or other abnormalities. (2) The cell count was 1.5 × 10 cells 2 weeks before and / or on the day of transplantation. 6 Cells / 450 μL (3) Cell viability is 85% or more as determined by trypan blue staining (4) Purity test of cell supernatant by ELISA PDGF-BB: 100pg / mL or more (5) Purity test by FACS of cell supernatant collected 2 weeks before and / or on the day of transplantation. CD166 + >99% CD24 + <5% CD26 + <5% CD200 + <5% CD44 high <5% CD44 low >90% CD105 -~weak >90% CD90 + <5% (6) Effector cell (E-ratio) > 90% (7) Pump function (Na+ / K+ ATPase) 2 days before transplantation: Positive (8) Barrier function (ZO-1) 2 days before transplantation: Positive (9) BSA negative test less than 125ng / μL (10) ECD on the day of transplantation is 1500 cells / mm 2 End (11) miR184 expression (12) Lactic acid production (13) Cell size less than 250 μm The method includes a step of confirming one or more of the following items. (Item 32) A cell population of human functional corneal endothelial cells that can induce human corneal endothelial functional properties when injected into the anterior chamber of a human eye, comprising: (1) On the day of transplantation, phase contrast images were used to visually inspect the tissue and found no fibroblasts, foreign matter, discoloration, or other abnormalities. (2) The cell count was 1.5 × 10 cells 2 weeks before and / or on the day of transplantation. 6 Cells / 450 μL (3) Cell viability is 85% or more as determined by trypan blue staining (4) Purity test of cell supernatant by ELISA PDGF-BB: 100pg / mL or more (5) Purity test by FACS of cell supernatant collected 2 weeks before and / or on the day of transplantation. CD166 +>99% CD24 + <5% CD26 + <5% CD200 + <5% CD44 high <5% CD44 low >90% CD105 -~weak >90% CD90 + <5% (6) Effector cell (E-ratio) > 90% (7) Pump function (Na+ / K+ ATPase) 2 days before transplantation: Positive (8) Barrier function (ZO-1) 2 days before transplantation: Positive (9) BSA negative test less than 125ng / μL (10) ECD on the day of transplantation is 1500 cells / mm 2 End (11) miR184 expression (12) Lactic acid production (13) Cell size less than 250 μm A cell population that satisfies one or more of the above criteria.

[0007] The following inventions are also provided: (Pharmaceuticals) (Item A1) A pharmaceutical containing human functional corneal endothelial cells, which improves corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density over a sustained long period of time and leading to improved vision, and in which expression of functional proteins is observed that lead to corneal endothelial (cell) functional characteristics, or in which proteins that inhibit the corneal endothelial (cell) functional characteristics are not induced or are reduced. (Item A12) The pharmaceutical described in (Item A1), wherein the cells are human functional corneal endothelial cells that can induce human corneal endothelial functional characteristics when injected into the anterior chamber of the human eye, and in which one or more metabolic enzymes selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malic enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain ketoacid dehydrogenase 2 (BCKDH2) are expressed in the mitochondria. (Item A3) The pharmaceutical according to (Item A1) or (Item A2), wherein the cells are human functional corneal endothelial cells that can induce human corneal endothelial functional characteristics when injected into the anterior chamber of the human eye, and include at least one selected from the group consisting of cells in which mitochondrial-dependent oxidative phosphorylation is increased in mitochondria, or expression of acetyl-CoA in the cytoplasm or nucleus is not increased, and epigenetic multigene expression mediated by histone acetylation by acetyl-CoA is not induced. (Item A4) The pharmaceutical according to any one of (Item A1) to (Item A3), wherein the cells are human corneal endothelial cells, and ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malic enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), and / or lactate dehydrogenase (LDH) are not expressed or are substantially not expressed. (Item A5) The cells described in any one of (Item A1) to (Item A4), wherein the expression of sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1) is enhanced in the human functional corneal endothelial cells. (Item A6) The pharmaceutical agent according to any one of (Item A1) to (Item A5), wherein the expression of bicarbonate anhydrase 5B (CA5B) is enhanced in the human functional corneal endothelial cells. (Item A7) The pharmaceutical according to any one of (Item A1) to (Item A6), wherein the human functional corneal endothelial cells comprise all of the following selected from the group consisting of: (i) the property that metabolic enzymes involved in the TCA cycle and the like and metabolic products such as Acetyl CoA are not present in the cytoplasm or nucleus but are organelle-selectively localized in the mitochondria so as to prevent the generation of contaminating phase transition cells; (ii) an increase in mitochondria-dependent oxidative phosphorylation in mitochondria; (iii) a decrease (including no induction) in epigenetic multigene expression mediated by histone acetylation by acetyl CoA; (iv) an increase in the expression of sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1); and (v) an increase in the expression of bicarbonate anhydrase 5B (CA5B). (Item A8) The pharmaceutical according to any one of (Item A1) to (Item A7), comprising the cells in which endothelial-mesenchymal transition has not occurred or has not substantially occurred. (Item A9) A pharmaceutical comprising human functional corneal endothelial cells that can induce human corneal endothelial functional properties when injected into the anterior chamber of the human eye, wherein the cells have not undergone, or have not substantially undergone, endothelial-mesenchymal transition. (Item A10) The medicine according to any one of (Item A1) to (Item A9), wherein the human functional corneal endothelial cells are produced from cells selected from the group consisting of corneal endothelial tissue-derived cells, 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 produced by a direct programming method. (Item A11) A pharmaceutical comprising a cell population containing cells produced by the method according to any one of (Item 1) to (Item 15) and / or the cells according to any one of (Item 16) to (Item 25). (Item A12) A pharmaceutical comprising a cell population of human functional corneal endothelial cells capable of inducing human corneal endothelial functional properties upon injection into the anterior chamber of a human eye, the cell population comprising: (1) On the day of transplantation, phase contrast images were used to visually inspect the tissue and found no fibroblasts, foreign matter, discoloration, or other abnormalities. (2) The cell count was 1.5 × 10 cells 2 weeks before and / or on the day of transplantation.6 Cells / 450 μL (3) Cell viability is 85% or more as determined by trypan blue staining (4) Purity test of cell supernatant by ELISA PDGF-BB: 100pg / mL or more (5) Purity test by FACS of cell supernatant collected 2 weeks before and / or on the day of transplantation. CD166 + >99% CD24 + <5% CD26 + <5% CD200 + <5% CD44 high <5% CD44 low >90% CD105 -~weak >90% CD90 + <5% (6) Effector cell (E-ratio) > 90% (7) Pump function (Na+ / K+ ATPase) 2 days before transplantation: Positive (8) Barrier function (ZO-1) 2 days before transplantation: Positive (9) BSA negative test less than 125ng / μL (10) ECD on the day of transplantation is 1500 cells / mm 2 End (11) miR184 expression (12) Lactic acid production (13) Cell size less than 250 μm A medicine that satisfies one or more of the above items.

[0008] 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 disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Brief explanation of the drawings]

[0009] [Figure 1]Figure 1 is a conceptual diagram showing the CD44-mediated mitochondrial regulation of energy metabolism. [Figure 2] FIG. 2 is a conceptual diagram showing the mechanisms of action of various clinical effects of functional human corneal endothelial cells (standard cells) according to one embodiment of the present disclosure. [Figure 3] FIG. 3 is a graph showing the results of investigating the effects of changing the method for producing functional human corneal endothelial cells according to one embodiment of the present disclosure. [Figure 4] FIG. 4 is a conceptual diagram showing induction of differentiated, mature, and functional human corneal endothelial cells according to one embodiment of the present disclosure via a dedifferentiation pathway from somatic (stem) cells. [Figure 5] Figure 5 is a conceptual diagram showing the antagonistic effects that occur when differentiation and phase transitions including EMT occur in parallel. [Figure 6] FIG. 6 shows cell photographs at P2 in one embodiment of the present disclosure, in which the EGF concentration added when producing functional human corneal endothelial cells of the present disclosure was no addition (-), 0.5 ng / mL, 1 ng / mL, or 5 ng / mL. [Figure 7] FIG. 7 shows the FACS results at P3 when the concentration of EGF added during production of functional human corneal endothelial cells of the present disclosure was either no addition (−) or 0.5 ng / mL in one embodiment of the present disclosure. [Figure 8] FIG. 8 shows the FACS results at P3 when the EGF concentration added during production of functional human corneal endothelial cells of the present disclosure was 1 ng / mL or 5 ng / mL in one embodiment of the present disclosure. [Figure 9] FIG. 9 shows the FACS results at P4 when the concentration of EGF added during production of functional human corneal endothelial cells of the present disclosure was either no addition (−) or 0.5 ng / mL in one embodiment of the present disclosure. [Figure 10] FIG. 10 shows the FACS results at P4 when the EGF concentration added during production of human functional corneal endothelial cells of the present disclosure was set to 1 ng / mL or 5 ng / mL in one embodiment of the present disclosure. [Figure 11]FIG. 11 shows the FACS results for cases in which no EGF was added (-) or 0.5 ng / mL was added at P0 to examine the effect of adding EGF from the time of primary culture when producing functional human corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 12] FIG. 12 shows FACS results for an embodiment of the present disclosure, in which no EGF was added (-) at P0 and no EGF was added (-) or 0.5 ng / mL at P1, in order to investigate the effect of adding EGF from the time of primary culture when producing functional human corneal endothelial cells of the present disclosure. [Figure 13] FIG. 13 shows FACS results for an embodiment of the present disclosure, in which EGF was added at 0.5 ng / mL in P0 and not added (-) or at 0.5 ng / mL in P1, to investigate the effect of adding EGF from the time of primary culture when producing functional human corneal endothelial cells of the present disclosure. [Figure 14] FIG. 14 shows FACS results for an embodiment of the present disclosure, in which no EGF was added (-) at P0 and no EGF was added (-) or 0.5 ng / mL at P2, in order to investigate the effect of adding EGF from the time of primary culture when producing functional human corneal endothelial cells of the present disclosure. [Figure 15] FIG. 15 is a schematic diagram showing case studies for measuring intracellular gene variations of miR378, miR146, miR34, and miR184. [Figure 16] FIG. 16 is a table showing the results of FACS measurement and photographic evaluation with and without EGF and with and without Y27632. [Figure 17] FIG. 17 is a graph showing the results of changes in miR184 gene expression with and without EGF and with and without Y. [Figure 18] FIG. 18 is a graph showing the results of changes in miR34a-5p gene expression with and without EGF and with and without Y. [Figure 19] FIG. 19 is a schematic diagram showing an example of the hierarchy of metabolites. [Figure 20]FIG. 20 is a graph showing the results of confirming the metabolite characteristics of human functional corneal endothelial cells of the present disclosure (target cells) and other cells (non-target cells) in one embodiment of the present disclosure. [Figure 21] FIG. 21 is a graph showing the results of confirming the metabolite characteristics of human functional corneal endothelial cells of the present disclosure (target cells) and other cells (non-target cells) in one embodiment of the present disclosure. [Figure 22] FIG. 22 is a graph showing the results of confirming the metabolite characteristics of human functional corneal endothelial cells of the present disclosure (target cells) and other cells (non-target cells) in one embodiment of the present disclosure. [Figure 23] FIG. 23 is a table showing culture conditions for testing the effects of various additives in one embodiment of the present disclosure. [Figure 24] FIG. 24 shows photographs of CT09 P5 cells under conditions 1 and 2 in FIG. [Figure 25] FIG. 25 shows photographs of CT09 P5 cells under conditions 3 and 4 in FIG. [Figure 26] FIG. 26 is a photograph of CT09 P5 cells under condition 5 in FIG. [Figure 27] FIG. 27 shows the FACS results under conditions 1 and 2 in FIG. [Figure 28] FIG. 28 shows the FACS results under conditions 3 and 4 in FIG. [Figure 29] FIG. 29 shows the FACS results under condition 5 in FIG. [Figure 30] Figure 30 shows the sample list of CT09 P4 and P5 culture supernatants for PDGF-bb and IL-8 ELISA. [Figure 31] FIG. 31 is a graph showing the results of classifying PDGF-bb by additives in one embodiment of the present disclosure. [Figure 32] FIG. 32 is a graph showing the results of PDGF-bb categorized by week in one embodiment of the present disclosure. [Figure 33] FIG. 33 is a graph showing the results of classifying IL-8 by additive in one embodiment of the present disclosure. [Figure 34] FIG. 34 is a graph showing the results of IL-8 categorized by week in one embodiment of the present disclosure. [Figure 35] FIG. 35 shows the results of examining the mitochondrial respiratory capacity of human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 36] FIG. 36 shows the results of examining the mitochondrial respiratory capacity of human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 37] FIG. 37 shows the results of examining the mitochondrial respiratory capacity of human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 38] FIG. 38 is a table showing donor information for investigating the effect of adding a ROCK inhibitor to human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 39] FIG. 39 is a table showing culture conditions for examining the effect of adding a ROCK inhibitor to human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 40] FIG. 40 is a table showing additives and the timing of supernatant collection for investigating the effect of the addition of a ROCK inhibitor on human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 41] FIG. 41 is a table summarizing the FACS results obtained by investigating the effect of adding a ROCK inhibitor to human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 42] FIG. 42 shows cell photographs in which the effect of adding a ROCK inhibitor to human functional corneal endothelial cells of the present disclosure was investigated in one embodiment of the present disclosure. [Figure 43] FIG. 43 shows the results of FACS investigating the effect of adding a ROCK inhibitor to human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 44] FIG. 44 shows the results of FACS investigating the effect of adding a ROCK inhibitor to human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 45] FIG. 45 shows the results of FACS investigating the effect of adding a ROCK inhibitor to human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 46] FIG. 46 is a graph showing the ELISA PDGF-bb measurement results (for each item) in the culture supernatant of #190719 in one embodiment of the present disclosure. [Figure 47] FIG. 47 is a graph showing the ELISA PDGF-bb measurement results (for each item) in the culture supernatant of #190318 in one embodiment of the present disclosure. [Figure 48] FIG. 48 is a graph showing the results of ELISA PDGF-bb measurement (weekly) in the culture supernatant of #190719 in one embodiment of the present disclosure. [Figure 49] FIG. 49 is a graph showing the results of ELISA PDGF-bb measurement (weekly) in the culture supernatant of #190318 in one embodiment of the present disclosure. [Figure 50] FIG. 50 is a graph showing the results of ELISA IL-8 measurement (weekly) in the culture supernatant of #190719 in one embodiment of the present disclosure. [Figure 51] FIG. 51 is a graph showing the results of ELISA IL-8 measurement (weekly) in the culture supernatant of #190318 in one embodiment of the present disclosure. [Figure 52] FIG. 52 is a graph showing the ELISA PDGF-bb and IL-8 measurement results (for each item) in the culture supernatant of #190802 in one embodiment of the present disclosure. [Figure 53] FIG. 53 is a graph showing the results (for each item) of cytokine measurement (BioPlex) in the culture supernatant of #190318. [Figure 54] FIG. 54 is a table showing additive conditions for investigating the effect of adding a ROCK inhibitor to human functional corneal endothelial cells of the present disclosure in one embodiment of the present disclosure. [Figure 55] FIG. 55 shows cell images at P4 with and without ROCK inhibitor #190719 in one embodiment of the present disclosure. [Figure 56]FIG. 56 shows FACS results at P4 with and without ROCK inhibitor #190719 in one embodiment of the present disclosure. [Figure 57] FIG. 57 shows FACS results at P4 with and without a ROCK inhibitor for #190719 in one embodiment of the present disclosure. [Figure 58] FIG. 58 is a graph showing the results of PDGF-bb and IL-8 by ELISA in one embodiment of the present disclosure. [Figure 59] FIG. 59 is a graph showing weekly results of PDGF-bb and IL-8 by ELISA in one embodiment of the present disclosure. [Figure 60] FIG. 60 shows cell photographs showing the results of an investigation into whether enhanced adhesion by a ROCK inhibitor is related to the cell production of the present disclosure, in one embodiment of the present disclosure. [Figure 61] FIG. 61 shows cell photographs showing the results of an investigation into whether enhanced adhesion by a ROCK inhibitor is related to the cell production of the present disclosure, in one embodiment of the present disclosure. [Figure 62] FIG. 62 shows cell photographs showing the results of an investigation into whether enhanced adhesion by a ROCK inhibitor is related to the cell production of the present disclosure, in one embodiment of the present disclosure. [Figure 63] FIG. 63 shows cell photographs showing the results of investigating whether enhanced adhesion by a ROCK inhibitor is related to the cell production of the present disclosure, in one embodiment of the present disclosure. [Figure 64] Figure 64 is a schematic diagram showing epigenetic control by metabolites and the disruption of cell senescence and cell differentiation. [Figure 65] FIG. 65 is a list of enzymes expressed in human functional corneal endothelial cells (differentiated mature cells) of the present disclosure in one embodiment of the present disclosure. [Figure 66] FIG. 66 is a schematic diagram showing the culture conditions of HCEC for DAVID analysis in one embodiment of the present disclosure. [Figure 67] FIG. 67 shows FACS results at P1 of HCEC for DAVID analysis in one embodiment of the present disclosure. [Figure 68] Figure 68 shows FACS results of HCECs at P4 for DAVID analysis in one embodiment of the present disclosure. [Figure 69] FIG. 69 is a cell photograph of HCEC at P4 for DAVID analysis in one embodiment of the present disclosure. [Figure 70] FIG. 70 shows a procedure for DAVID analysis of proteomics in one embodiment of the present disclosure. [Figure 71] FIG. 71 shows the results of a three-group analysis in one embodiment of the present disclosure. [Figure 72] FIG. 72 shows the results of a GOTERM analysis in one embodiment of the present disclosure. [Figure 73] FIG. 73 is a table showing comparison results for mitochondria after DAVID analysis in one embodiment of the present disclosure. [Figure 74] FIG. 74 is a table showing clustering results of a comparison regarding mitochondria after DAVID analysis in one embodiment of the present disclosure. [Figure 75] FIG. 75 is a table showing clustering results of a comparison regarding mitochondria after DAVID analysis in one embodiment of the present disclosure. [Figure 76] FIG. 76 is a schematic diagram comparing the protein expression intensities of enzymes and substrates involved in metabolic pathways between human functional corneal endothelial cells (standard cells) of the present disclosure and non-standard cells in one embodiment of the present disclosure. [Figure 77] FIG. 77 is a schematic diagram comparing the protein expression intensities of enzymes and substrates involved in metabolic pathways between human functional corneal endothelial cells (standard cells) of the present disclosure and non-standard cells in one embodiment of the present disclosure. [Figure 78] FIG. 78 is a schematic diagram comparing the protein expression intensities of enzymes and substrates involved in metabolic pathways between human functional corneal endothelial cells (standard cells) of the present disclosure and non-standard cells in one embodiment of the present disclosure. [Figure 79] FIG. 79 is a schematic diagram comparing the protein expression intensities of enzymes and substrates involved in metabolic pathways between human functional corneal endothelial cells (standard cells) of the present disclosure and non-standard cells in one embodiment of the present disclosure. [Figure 80] FIG. 80 is a schematic diagram comparing the protein expression intensities of enzymes and substrates involved in metabolic pathways between human functional corneal endothelial cells (standard cells) of the present disclosure and non-standard cells in one embodiment of the present disclosure. [Figure 81] FIG. 81 is a schematic diagram comparing the protein expression intensities of enzymes and substrates involved in metabolic pathways between human functional corneal endothelial cells (standard cells) of the present disclosure and non-standard cells in one embodiment of the present disclosure. [Figure 82] FIG. 82 is a schematic diagram comparing the protein expression intensities of enzymes and substrates involved in metabolic pathways between human functional corneal endothelial cells (standard cells) of the present disclosure and non-standard cells in one embodiment of the present disclosure. [Figure 83] FIG. 83 is a schematic diagram comparing the protein expression intensities of enzymes and substrates involved in metabolic pathways between human functional corneal endothelial cells (standard cells) of the present disclosure and non-standard cells in one embodiment of the present disclosure. [Figure 84] Figure 84 is a list of antigens used to compare the protein expression intensities of enzymes and substrates involved in metabolic pathways between human functional corneal endothelial cells (standard cells) of the present disclosure and non-standard cells in one embodiment of the present disclosure. [Figure 85] FIG. 85 is a photograph showing the results of cell staining for investigating the ion channel simple carboxylic acid transporter system in one embodiment of the present disclosure. [Figure 86] FIG. 86 is a photograph showing the results of cell staining for investigating the ion channel simple carboxylic acid transporter system in one embodiment of the present disclosure. [Figure 87] FIG. 87 is a photograph showing the results of cell staining for investigating the ion channel simple carboxylic acid transporter system in one embodiment of the present disclosure. [Figure 88] FIG. 88 is a photograph showing the results of cell staining for investigating the ion channel simple carboxylic acid transporter system in one embodiment of the present disclosure. [Figure 89] FIG. 89 is a graph showing the FACS results and intracellular pH at P2 for #190719 (standard cells) in one embodiment of the present disclosure. [Figure 90]FIG. 90 is a cell photograph of #190719 (standard cells) at P2 in one embodiment of the present disclosure. [Figure 91] FIG. 91 is a graph showing the FACS results and intracellular pH at P3 for #190802 (non-standard cells) in one embodiment of the present disclosure. [Figure 92] FIG. 92 is a cell photograph of #190802 (non-standard cells) at P3 in one embodiment of the present disclosure. [Figure 93] FIG. 93 is a graph showing the results of comparing the intracellular pH of #190719 (standard cells) and #190802 (non-standard cells) in one embodiment of the present disclosure. [Figure 94] FIG. 94 shows FACS and cell photographs showing the results of examining the effects of additives in one embodiment of the present disclosure. [Figure 95] FIG. 95 is a schematic diagram showing culture conditions for examining the effects of additives in one embodiment of the present disclosure. [Figure 96] FIG. 96 shows the FACS results for cells under each culture condition in one embodiment of the present disclosure. [Figure 97] FIG. 97 shows the FACS results for cells under each culture condition in one embodiment of the present disclosure. [Figure 98] FIG. 98 shows the FACS results for cells under each culture condition in one embodiment of the present disclosure. [Figure 99] FIG. 99 is a graph showing the results of measuring cytokines in cells under each culture condition in one embodiment of the present disclosure. [Figure 100] FIG. 100 is a graph showing the results of measuring cytokines in cells under each culture condition. [Figure 101] FIG. 101 is a schematic diagram illustrating the enhancement of mitochondrial oxidative phosphorylation respiration. [Figure 102] Figure 102 is a graph showing the enhancement of mitochondrial oxidative phosphorylation respiration in one embodiment of the present disclosure. [Figure 103] Figure 103 is a schematic diagram showing the relationship between enhanced mitochondrial oxidative phosphorylation respiration and clinical pharmacological effects. [Figure 104] FIG. 104 is a schematic diagram showing induction of differentiated, mature, and functional human corneal endothelial cells via a dedifferentiation pathway from somatic (stem) cells. [Figure 105] Figure 105 is a conceptual diagram illustrating that mitochondrial function is influenced by intracellular pH, and that the differentiated or dedifferentiated state of cells is determined. [Figure 106] FIG. 106 shows the FACS results and photographs of #CR04 (standard cells) at P3 in one embodiment of the present disclosure. [Figure 107] FIG. 107 shows the results of confirming selective expression of ion channels by cell immunostaining in one embodiment of the present disclosure. [Figure 108] Figure 108 shows results showing increased acetylation of histones in non-standard cells in one embodiment of the present disclosure. [Figure 109] Figure 109 shows the results of immunoblotting in standard and non-standard cells in one embodiment of the present disclosure. [Figure 110] FIG. 110 shows the FACS results and photographs of #191224S (standard cells) at P4 in one embodiment of the present disclosure. [Figure 111] FIG. 111 shows the FACS results and photographs of #200313 (non-standard cells) at P1 in one embodiment of the present disclosure. [Figure 112] FIG. 112 shows the FACS results and photographs of #191224S (standard cells) at P4 in one embodiment of the present disclosure. [Figure 113] FIG. 113 shows the FACS results and photographs of #191224S (standard cells) at P4 in one embodiment of the present disclosure. [Figure 114] FIG. 114 is a graph showing the results of measuring HAT / HDAC activity in one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present disclosure 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 disclosure belongs. In the event of a conflict, the present specification (including definitions) will prevail.

[0011] First, we explain the terms and general techniques used in this disclosure.

[0012] As used herein, "about" means ±10% of the preceding numerical value.

[0013] 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.

[0014] 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 collectively referred to as "corneal endothelial progenitor cells."

[0015] As used herein, "human functional corneal endothelial cells capable of inducing human corneal function when injected into the anterior chamber of a human eye" refer to cells capable of inducing corneal function, that is, cells capable of inducing human corneal function when injected into the anterior chamber of a human eye (when referring to humans, they are referred to as "human corneal function" and, without any particular limitation, are simply referred to as "human corneal function" in this specification). "Capable of inducing human corneal function" can encompass the ability to induce corneal endothelial function characteristics (for example, improvement of corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density sustainably over a long period of time and leading to improved vision, etc.).

[0016] As used herein, "human functional corneal endothelial cells capable of inducing corneal endothelial functional properties upon injection 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 properties (when referring to humans, these are referred to as "human corneal endothelial functional properties"; and, without any particular limitation, are referred to herein simply as "corneal endothelial functional properties") upon injection into the anterior chamber of a human eye. Typical functions include the improvement of corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density over a sustained long period of time and leading to improved vision. When specifically abbreviated, these are also referred to as "functional cells equipped with corneal endothelial properties of the present disclosure." When referring to human cells, they are referred to as "human functional corneal endothelial cells capable of inducing human corneal endothelial functional properties upon injection into the anterior chamber of a human eye." Because the present disclosure primarily relates to human corneal cells, it is understood that the term refers to human cells unless otherwise specified. As used herein, functional cells having corneal endothelial properties according to the present disclosure 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, after being used in a similar manner or after being injected, exhibit functions equivalent to those of functional mature differentiated corneal endothelial cells.

[0017] As used herein, "corneal endothelial (cell) functional properties" refer to (cellular) functional properties that are useful for maintaining visual function and that corneal endothelial cells present in corneal endothelial tissue possess under normal conditions. Note that, in this specification, the terms "corneal endothelial cell functional properties" and "corneal endothelial functional properties" have the same meaning, and in many cases, the former is referred to in in vitro discussions.

[0018] As used herein, the term "functional mature differentiated corneal endothelial cells" refers to mature differentiated corneal endothelial cells present in healthy human corneal endothelial tissue and any cells having the same function (typically, the above-mentioned corneal endothelial (cell) functional characteristics), and in the case of human cells, these are referred to as functional mature differentiated human corneal endothelial cells. In particular, the corneal endothelial (cell) functional characteristics are confirmed by forming a small, hexagonal, paving stone-like shape, utilizing an energy metabolic system based on mitochondrial function, and maintaining traits that improve corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density over a sustained, long-term period and leading to improved vision, and can be determined by whether or not the cells have a therapeutic effect when injected (for example, into the anterior chamber of the human eye), but are not limited to this. The corneal endothelial functional characteristics can also be determined using a surrogate marker as an indicator. Such surrogate markers can be determined by any one of the following 10 criteria or a combination thereof: (1) maintenance of endothelial pump and barrier function (including Claudin expression), (2) adhesion and binding to specific laminins, (3) secreted cytokine profile, (4) produced microRNA (miRNA) profile, (5) produced metabolite profile, (6) expression of ion channels and monocarboxylate transporters, which are linked to the above-mentioned corneal endothelial (cell) functional characteristics, such as improvement of corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density over the long term and leading to improved vision, (7) the organelle-selective localization of metabolic enzymes involved in the TCA cycle, which are not present in the cytoplasm or nucleus and are instead localized in mitochondria, leading to the generation of phase transition cells, (8) saturation cell density during in vitro culture, (9) spatial size and distribution of cells obtained during culture, and (10) cell maintenance when cells are injected into mouse corneas after liquid nitrogen cryoinjury.

[0019] (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-form endothelium, as described in Wigham C, Hodson S.: Current Eye Research, 1, 37-41, 1981; Hodson S, Wigham C.: J. Physiol., 342:409-419, 1983; and Hatou S., Yamada M., Akune Y., Mochizuki H., Shiraishi A., Joko T., Nishida T., Tsubota K.: Investigative Ophthalmology & Visual Science, 51, 3935-3942, 2010. Claudin expression can be confirmed using techniques known in the art, such as immunological techniques. Claudin expression can be confirmed using any immunological technique known in the art. However, since the cells of the present disclosure are expected to be injected in a suspension, in that case, it is preferable to evaluate corneal endothelial function by applying Claudin expression or any one of (2) to (10) or a combination thereof.

[0020] (2) The adhesiveness and binding ability to specific laminins can be determined by measuring the adhesiveness 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 increased expression of integrins (e.g., α3β1, α6β1, etc.) that bind to these. Such methods can be performed by cell adhesion assays.

[0021] 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 a subunit 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 in NCBI accession numbers NM_002292 and NP_002283, respectively. OMIM accession number 150325 is used. The "γ1 chain" (LAMC1) of the laminin γ chain is a subunit of the cell adhesion molecule protein (laminin) in the extracellular matrix and is referred to as LAMC1; LAMB2, etc. The gene and protein sequences of human LAMC1 are registered in NCBI accession numbers NM_002293 and NP_002284, respectively. OMIM accession number 150290 is used.

[0022] (3) The secreted cytokine profile can be determined by measuring the production levels of cytokines in the serum or aqueous humor, as described elsewhere herein. These 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, cytokine analysis can be performed using a cytokine measurement kit and analysis system, such as Bio-Plex, for integrated cytokine analysis.

[0023] (4) The resulting microRNA (miRNA) profile can be measured using the "miRNA profile" analysis method described elsewhere in this specification. For example, this can be achieved using a microRNA expression profile analysis method, such as 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., 16 hours at 32°C). After washing and drying in an ozone-free environment, the microRNA chip can be scanned using a scanner such as the 3D-Gene scanner 3000 (Toray Industries Inc., Tokyo, JAPAN) and analyzed using 3D-Gene Extraction software (Toray).

[0024] (5) To obtain a metabolite profile, for example, a metabolic extract of intracellular metabolites is prepared from the cHCEC culture vessel with methanol containing an internal standard reagent such as Internal Standard Solution (Human Metabolome Technologies; HMT, Inc., Tsuruoka, Japan). The medium is replaced, and the cell extract is processed and analyzed by CE-MS. Metabolomic analysis can be performed according to the method developed 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), and analyzed using 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, respectively). Based on m / z values ​​measured by CE MT and TOFMS, 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.

[0025] (6) Improvement of corneal opacity and hydration edema. As a result, the corneal endothelial tissue cell density is maintained sustainably over the long term, leading to improved vision. The expression of ion channels and monocarboxylate transporters, which are linked to the above-mentioned corneal endothelial (cell) functional characteristics, can be measured using any method described herein or known in the art. For example, as described in the Examples, cells obtained from corneal tissue can be fixed, immunostained with specific antibodies, and observed under a fluorescent microscope or the like. Alternatively, their expression can be measured by measuring the function of ion channels and monocarboxylate transporters.

[0026] (7) The property that metabolic enzymes involved in the TCA cycle, which leads to the generation of phase transition cells, are not present in the cytoplasm or nucleus but are organelle-selectively localized in mitochondria can also be measured using any method described herein or known in the art. For example, DAVID analysis can be used to observe the organelle-selective localization of metabolic enzymes in mitochondria, but is not limited to this.

[0027] (8) Saturation cell density during in vitro culture can be determined by measuring cell density using appropriate culture conditions described herein. It may be measured in parallel with cell size. Phase-contrast microscopic images can be acquired 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), and quantified using cell counting software (e.g., BZ-H3C Hybrid cell counting software (Keyence)). Preferred saturation cell densities in this disclosure are described elsewhere herein.

[0028] (9) 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, and measuring the spatial size and distribution of the cells, which can be achieved using crude image processing software such as BZ-H3C Hybrid cell counting software (Keyence). Preferred spatial size and distribution of cells in this disclosure are described elsewhere herein.

[0029] (10) The evaluation of cell retention after cell injection following liquid nitrogen cryoinjury to mouse corneas can be performed by creating a mouse model. Specifically, a central region (e.g., 2 mm) of the cornea of ​​an appropriate mouse (e.g., BALB / c) is cryoinjured and the endothelial cells are removed to create a model. The cells to be evaluated are then injected into the anterior chamber of the model. The characteristics of corneal transparency are clinically observed, corneal thickness is assessed by pachymetry, and HCEC adhesion is examined histopathologically using human nuclear staining to confirm whether the cells are functional.

[0030] Cells that are not derived from corneal endothelial tissue (including cells obtained by dedifferentiation of corneal endothelial tissue) (for example, cells produced by allowing stem cells (for example, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells) or the like) to mature and differentiate into corneal endothelial progenitor cells or corneal endothelial cells) fall within the scope of the functional corneal endothelial cells or functional mature differentiated corneal endothelial cells disclosed herein that can induce corneal endothelial functional properties when injected into the anterior chamber of the human eye, as long as they have the corneal endothelial functional properties described in the present disclosure. In the explanations and experiments shown in this specification, the human functional mature differentiated corneal endothelial cells disclosed herein may also be referred to as "human functional mature differentiated corneal endothelial cells," "functional mature differentiated human corneal endothelial cells," "functional cells," "target cells," "specification cells," etc., but all of these terms are used interchangeably.

[0031] As used herein, "non-functional corneal endothelial cells" refer to cells other than functional cells having corneal endothelial properties disclosed herein (i.e., "functional mature differentiated corneal endothelial cells"), and may be referred to as "non-target cells," "rejected cells," "non-target cells," "non-functional cells," "non-standard cells," etc.

[0032] As used herein, the term "cell indicator" refers to any indicator that indicates that a certain cell is a functional cell having the corneal endothelial characteristics of the present disclosure (for example, a functional mature 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 the mature differentiated human corneal endothelium and its functions. The specific characteristics are also referred to as "cell functional characteristics."

[0033] As used herein, "culture conditions that can minimize culture stress" refers to any culture conditions that can minimize (minimize) stress on cells during culture, such as proliferation stress. "Culture stress" is defined as the presence of 5-20% or more large, irregularly shaped cells that have undergone phase transition when observed under a phase contrast microscope, or a cell density of 1000 cells / mm2 or less between the 30th and 40th days of culture. 2 The culture stress can be measured using the index of whether the stress level is below a certain level. If preliminary experiments show that culture stress can be minimized using that index for a certain donor, those conditions can be applied when actually preparing and administering cells. Such culture conditions can be achieved, for example, but are not limited to, by culturing cells at an amount of cell growth factor, such as epidermal growth factor, below the amount at which transformation occurs. This amount can vary depending on the age of the donor, the cell density at the time of cell seeding, and the concentration of growth factor added, but those skilled in the art can determine such conditions (e.g., amount, etc.) taking into account various available information, donor information, etc.

[0034] As used herein, the term "transformation" refers to a change in the phenotype of a cell to an abnormal state, and includes the meaning of normal cells undergoing unlimited division, i.e., canceration, and particularly dynamic metaplasia (dedifferentiation of stem cells and changes beyond the boundaries of the tissue's basic form). Examples of transformation include cell state phase transitions (CSTs) such as EMT, fibrosis, epithelial-mesenchymal transition, senescence, dedifferentiation, and endothelial-mesenchymal transition. Corneal endothelial cells often undergo transformation such as epithelial-mesenchymal transition and often cease to be functional, mature, differentiated corneal endothelial cells. Alternatively, transformation includes endothelial-mesenchymal transition. The production methods disclosed herein include methods that can convert cells that have undergone such epithelial-mesenchymal transition or endothelial-mesenchymal transition into functional, mature, differentiated corneal endothelial cells by dedifferentiating and then maturely differentiating them.

[0035] As used herein, "below the amount sufficient to cause transformation," when referring to a cell growth factor or the like, refers to an amount less than or not sufficient to cause transformation of the target corneal endothelial cells (e.g., endothelial-mesenchymal transition). When a cell growth factor is used at an amount less than the amount required to cause transformation, it is characterized by not inducing transformation, including endothelial-mesenchymal transition, due to proliferation stress. For example, when EGF is used as the cell growth factor, an amount less than the amount sufficient to cause transformation is less than about 1 ng / mL, preferably less than about 0.5 ng / mL, and more preferably 0 ng / mL.

[0036] As used herein, the term "endothelial-mesenchymal transition" (EndMT) refers to the transformation of endothelial cells into mesenchymal cells.

[0037] 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.

[0038] As used herein, "cell growth factor" is a general term for proteins that promote the growth and proliferation of specific cells in the animal body, and is sometimes synonymous with "growth factor" or "cell proliferation factor." Examples include epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), and transforming growth factor (TGF, etc.). One of the features of the present disclosure is that no growth factors that cause cell stress known as proliferation stress are added to cells during culture. The intensity of stress varies depending on the concentration, duration, and timing of addition.

[0039] Here, the starting cells in the various samples and production methods that can be used in this specification may be any sample that is thought 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 endothelium (also called corneal endothelial tissue-derived cells) or cells that have acquired corneal endothelial-like functions through differentiation 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, they are obtained from a corneal endothelium donor. Cells obtained by inducing differentiation in vitro can be used as cell samples. In addition, cultured cells containing functional cells with corneal endothelial characteristics of the present disclosure or functional mature differentiated corneal endothelial cells that have been induced to differentiate in vitro can be used as samples. Differentiation induction into functional cells with corneal endothelial characteristics of the present disclosure or functional mature differentiated corneal endothelial cells can be carried out in vitro 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.

[0040] As used herein, the terms "high expression," "moderate expression," and "low expression" of miRNAs are used to relatively express the expression intensity of cells that are typically CD44 negative to weakly positive, CD24 negative, and CD26 negative. Note that there may not be a "moderate expression" category, in which case the respective cells can be distinguished by "high expression" and "low expression."

[0041] As used herein, "cell size" is one cellular indicator of the functional cells with corneal endothelial characteristics of the present disclosure, and is measured by techniques commonly used in the field. Cell size is expressed, for example, by cell area. As used herein, "cell area" is one cellular indicator of the functional cells with corneal endothelial characteristics of the present disclosure, and can be measured by taking a photograph of the cells and using any software or the like. An example of such a measurement method is a method using image processing software such as BZ-H3C Hybrid cell counting software (Keyence). The average value is referred to as the "average cell area." Typically, the arithmetic mean is used.

[0042] As used herein, "cell density" and "(average) cell density" refer to a cellular index 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 index of functional cells with corneal endothelial characteristics or functional, mature, differentiated corneal endothelial cells of the present disclosure. The arithmetic mean is usually used as the average. It can also be measured in parallel with cell size, and can be quantified using cell counting software (e.g., BZ-H3C Hybrid cell counting software (Keyence)) or the like, by acquiring phase-contrast microscopic images using an inverted microscope system (e.g., CKX41, Olympus, Tokyo, Japan) and an instrument including an image acquisition system such as a BZ X-700 microscope system (Keyence, Osaka, Japan). Cell density can be used as an index of saturated cell culture (also referred to as (culture) confluence; in this specification, saturated cell culture and (culture) confluence are used interchangeably), as well as the density at the time of seeding, which is also used as a guide in the production method of the present disclosure. Cell density can also be used as an indicator of therapeutic outcome after injection.

[0043] As used herein, "cellular metabolite" refers to any metabolic product produced by a cell, and "biological substance related to a cellular metabolite (said product)" refers to any biological substance related to the cellular metabolite (for example, an enzyme that synthesizes the metabolic product, an enzyme that metabolizes the metabolic product, a protein related to a signal transduction pathway, etc.), and is one of the cellular indicators of functional cells with corneal endothelial characteristics or functional mature differentiated corneal endothelial cells disclosed herein. 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, lactate, 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.

[0044] Detection, identification, quality control, etc. of cells disclosed herein can be achieved by using substances that bind to marker substances or interactive molecules. In the context of this disclosure, 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 gene for 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, "reduction," "decrease," or "suppression" of an activity or expression product (e.g., a protein, a transcript (RNA, etc.)), or synonyms thereof, refers to a decrease, reduction, or activity that reduces the quantity, quality, or effect of a particular activity, transcript, or protein. When an activity, expression product, etc. is reduced to below the detection limit, this is sometimes distinguished from the term "elimination." As used herein, "elimination" is encompassed by "reduction," "reduction," or "suppression." Furthermore, "reduction" not only refers to a decrease in the amount or effect of a gene, etc., from a state in which it is already expressed, but also includes the failure to induce any expression.

[0046] As used herein, "increase" or "activation" of an activity or expression product (e.g., protein, transcript (RNA, etc.)), or synonyms thereof, refers to an increase or increasing activity in the amount, quality, or effect of a particular activity, transcript, or protein. "Increase" includes cases where the amount, quality, or effect of a particular activity, transcript, or protein is present in the pre-comparison state (a relative increase in the amount already present), as well as cases where the amount, quality, or effect of a particular activity, transcript, or protein is not present (from none to present).

[0047] As used herein, "mitochondrial-dependent oxidative phosphorylation" refers to the oxidative phosphorylation reaction dependent on the respiratory activity of mitochondria, also known as "mitochondrial OXPHOS." Reactions related to "mitochondrial OXPHOS" can be measured using an extracellular flux analyzer, which analyzes the state of intracellular metabolism. The oxygen consumption rate (OCR), an index of OXPHOS activity, and the extracellular acidification rate (ECAR), an index of glycolytic activity, can be measured using an extracellular flux analyzer.

[0048] As used herein, "expression of acetyl-CoA in the cytoplasm or nucleus" refers to the expression of acetyl-CoA in the cytoplasm or nucleus. The expression of acetyl-CoA can be measured by any known method for analyzing protein expression or by cell immunostaining using an antibody.

[0049] As used herein, "epigenetic multigene expression mediated by histone acetylation by acetyl-CoA" refers to the regulation of the expression of multiple genes through the acetylation of histones by acetyl-CoA. The expression of these multiple genes triggers the cellular phase transition (CST). Herein, epigenetic multigene expression can be measured using techniques such as those disclosed in CellMetab. 2015 Mar3;21(3):349-50, Trends in Cell Biology, June 2017, Vol. 27, No. 6, and Sheikhet et al. Nature Rev. Genetics, 2019. Furthermore, the relationship between metabolite-mediated epigenetic regulation and the disruption of cellular senescence and differentiation can be illustrated as shown in the schematic diagram in Figure 64.

[0050] As used herein, "a functional protein that leads to corneal endothelial (cell) functional properties that improve corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density sustainably over the long term, and leading to improved vision" is also referred to as "a functional protein related to corneal endothelial (cell) functional properties" or "a functional protein (of the present disclosure)," and refers to any protein that has the function of improving corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density sustainably over the long term, and leading to improved vision, and is understood to include any protein described herein, such as AQP1, Na-KATPase, NHE1, and other proteins as well. It is understood that functional proteins include, for example, sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1), bicarbonate anhydrase 5B (CA5B) (expression is increased), as well as mitochondrial citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malic enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain ketoacid dehydrogenase 2 (BCKDH2).

[0051] Furthermore, the "expression" of a "functional protein" also includes not only the expression of the "functional protein" but also the absence of an increase in mitochondria-dependent oxidative phosphorylation in mitochondria, or the absence of expression of acetyl-CoA in the cytoplasm and nucleus, or the absence of epigenetic multigene expression mediated by histone acetylation by AcCoA.

[0052] In addition, "expression" of a "functional protein" includes not only the expression of the "functional protein," but also reduced or no expression of proteins with functions opposite to those of the "functional protein," such as no or very little expression of ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malic enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), and / or lactate dehydrogenase (LDH).

[0053] Here, the term "not substantially or hardly expressed" when used in the presently disclosed invention can be determined based on whether or not the functional properties of human corneal endothelium can be induced.

[0054] From the above, "improvement of corneal opacity and hydration edema, which results in the maintenance of corneal endothelial tissue cell density over a sustained, long-term period, and the expression of functional proteins that lead to corneal endothelial (cell) functional characteristics that lead to improved vision" can also be expressed as "improvement of corneal opacity and hydration edema, which results in the maintenance of corneal endothelial tissue cell density over a sustained, long-term period, and the expression of functional proteins that lead to corneal endothelial (cell) functional characteristics that lead to improved vision, or proteins that inhibit the corneal endothelial (cell) functional characteristics are not induced or are reduced."

[0055] In the present disclosure, "Rho kinase" or "ROCK" (Rho-associated coiled-coil forming kinase: Rho-binding kinase) refers to a serine / threonine kinase that is activated upon activation of Rho. Examples include ROKα (ROCK-II: Leung, T. et 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.

[0056] ROCK inhibitors (also called 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 / 022542, WO 2004 / 022543, WO 2004 / 022544, WO 2004 / 022545, WO 2004 / 022546, WO 2004 / 022547, WO 2004 / 022548, WO 2004 / 022549 ... 2004 / 108724, 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 may be suitably used as commercially available products (Wako Pure Chemical Industries, Ltd., Asahi Kasei Pharma, etc.). As used herein, ROCK inhibitors are particularly used in the process of proliferating and / or differentiating / maturing cultured human corneal endothelial cells. In one embodiment, a ROCK inhibitor may not be used in the step of obtaining corneal endothelial precursor cells by dedifferentiation.Furthermore, without wishing to be bound by theory, a ROCK inhibitor may or may not be used in the step of obtaining corneal endothelial progenitor cells by dedifferentiation, depending on the cell-specific properties such as the passage number of the corneal endothelial cells or corneal endothelial progenitor cells to be dedifferentiated or differentiated / matured, or the age of the donor from which they are derived. For example, in the case of cells with a low passage number (e.g., passage number of about 1, 2, or 3) and / or cells derived from a young donor, a ROCK inhibitor may be used only in the proliferation and / or differentiation / maturation step, whereas in the case of cells with a high passage number (e.g., passage number of 4, 5, 6, etc.), a ROCK inhibitor may also be used at the time of cell seeding. Thus, in certain embodiments, the discovery that a level of cells comparable to that obtained when a ROCK inhibitor is administered throughout the entire period can be obtained by using a ROCK inhibitor only in the proliferation and / or differentiation / maturation step can be considered an achievement of the present disclosure.

[0057] (Preferred embodiment) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure 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 disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0058] In various aspects of the present specification, WO 2017 / 141926 is incorporated herein by reference as appropriate. Accordingly, it is understood that the contents of WO 2017 / 141926 are incorporated herein by reference where appropriate.

[0059] (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 of the present disclosure, the present disclosure provides human functional corneal endothelial cells in which expression of functional proteins is observed that leads to corneal endothelial (cell) functional properties that improve corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density sustainably over the long term and leading to improved vision, or in which proteins that inhibit the corneal endothelial (cell) functional properties are not induced or are reduced. Such expression of functional proteins or the non-induction or reduction of proteins can be appropriately carried out by those skilled in the art in accordance with the disclosures herein and in light of the examples in this specification. In the present disclosure, human functional corneal endothelial cells in which expression of functional proteins is observed that leads to corneal endothelial (cell) functional characteristics that improve corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density sustainably over the long term and leading to improved vision, or in which proteins that inhibit the corneal endothelial (cell) functional characteristics are not induced or are reduced, are sometimes expressed as "human functional corneal endothelial cells in which expression of functional proteins is observed that leads to corneal endothelial (cell) functional characteristics that improve corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density sustainably over the long term and leading to improved vision," but these terms are synonymous unless otherwise noted.

[0060] In one embodiment, the present disclosure provides human functioning corneal endothelial cells that can induce human corneal endothelial functional characteristics upon injection into the anterior chamber of a human eye, the cells comprising at least one selected from the group consisting of (i) an increase in mitochondria-dependent oxidative phosphorylation in mitochondria, (ii) no increase in acetyl-CoA expression in the cytoplasm or nucleus, and (iii) a decrease in epigenetic multigene expression mediated by histone acetylation by acetyl-CoA. In this case, the increase in acetyl-CoA expression includes the expression of acetyl-CoA that was not expressed in non-functioning corneal endothelial cells, and also includes the absence of any decrease in epigenetic multigene expression. Furthermore, in one embodiment of the present disclosure, such cells may also include all of the following selected from the group consisting of: (i) an increase in mitochondria-dependent oxidative phosphorylation in mitochondria; (ii) organelle-selective expression of acetyl-CoA in mitochondria, without expression of acetyl-CoA in the cytoplasm or nucleus; (iii) expression of ion channels and monocarboxylic acid transporters, which lead to the above-mentioned corneal endothelial (cell) functional characteristics, such as improvement of corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density over a sustained long period of time and leading to improved vision; and (iv) characteristics in which metabolic enzymes involved in the TCA cycle, etc., which lead to the generation of phase transition cells, are not present in the cytoplasm or nucleus, but are organelle-selectively localized in mitochondria; and, in particular, a decrease or absence of epigenetic multigene expression mediated by histone acetylation by acetyl-CoA.

[0061] In one embodiment of the present disclosure, the human functional corneal endothelial cells of the present disclosure have not undergone, or have not substantially undergone, endothelial-mesenchymal transition.

[0062] Furthermore, in one embodiment of the present disclosure, the human functional corneal endothelial cells of the present disclosure have in their mitochondria an enzyme selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malic enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain ketoacid dehydrogenase 2 (BCKDH2). In another embodiment, the cell does not express or substantially does not express at least one enzyme selected from the group consisting of ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malic enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), and lactate dehydrogenase (LDH).

[0063] Furthermore, in one embodiment of the present disclosure, the human functional corneal endothelial cells of the present disclosure have increased expression of sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1), and in another embodiment, the human functional corneal endothelial cells of the present disclosure have increased expression of bicarbonate anhydrase 5B (CA5B).

[0064] Furthermore, in one embodiment of the present disclosure, the human functional corneal endothelial cells of the present disclosure can also be produced from cells selected from the group consisting of corneal endothelial tissue-derived cells, 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 produced by the direct programming method.

[0065] Furthermore, in one embodiment of the present disclosure, there is provided a method for quality control or process control of human functional corneal endothelial cells that can induce human corneal endothelial functional properties when injected into the anterior chamber of a human eye, or a method for detecting non-functional corneal endothelial cells mixed in with human functional corneal endothelial cells, comprising the steps of: (1) On the day of transplantation, phase contrast images were used to visually inspect the tissue and found no fibroblasts, foreign matter, discoloration, or other abnormalities. (2) The cell count was 1.5 × 10 cells 2 weeks before and / or on the day of transplantation. 6 cells / 450μL (3) Cell viability is 85% or more as determined by trypan blue staining. (4) Purity test of cell supernatant by ELISA PDGF-BB: 100pg / mL or more (5) Purity test by FACS of cell supernatant collected 2 weeks before and / or on the day of transplantation. CD166 + >99% CD24 + <5% CD26 + <5% CD200 + <5% CD44 high <5% CD44 low >90% CD105 -~weak >90% CD90 + <5% (6) Effector cell (E-ratio) > 90% (7) Pump function (Na+ / K+ ATPase) 2 days before transplantation: Positive (8) Barrier function (ZO-1) 2 days before transplantation: Positive (9) BSA negative test less than 125ng / μL (10) ECD on the day of transplantation is 1500 cells / mm 2 End (11) miR184 expression (12) Lactic acid production (13) Cell size less than 250 μm The method includes the step of verifying one or more of the following:

[0066] In yet another embodiment, a cell population of human functional corneal endothelial cells capable of inducing human corneal endothelial functional properties upon injection into the anterior chamber of a human eye, comprising: (1) On the day of transplantation, phase contrast images were used to visually inspect the tissue and found no fibroblasts, foreign matter, discoloration, or other abnormalities. (2) The cell count was 1.5 × 10 cells 2 weeks before and / or on the day of transplantation. 6 Cells / 450 μL (3) Cell viability is 85% or more as determined by trypan blue staining (4) Purity test of cell supernatant by ELISA PDGF-BB: 100pg / mL or more (5) Purity test by FACS of cell supernatant collected 2 weeks before and / or on the day of transplantation. CD166 + >99% CD24 + <5% CD26 + <5% CD200 + <5% CD44 high <5% CD44 low >90% CD105 -~weak >90% CD90 + <5% (6) Effector cell (E-ratio) > 90% (7) Pump function (Na+ / K+ ATPase) 2 days before transplantation: Positive (8) Barrier function (ZO-1) 2 days before transplantation: Positive (9) BSA negative test less than 125ng / μL (10) ECD on the day of transplantation is 1500 cells / mm 2 End (11) miR184 expression (12) Lactic acid production (13) Cell size less than 250 μm In this case, a cell population that satisfies all of the items (1) to (13) can also be provided.

[0067] In actual operation, it is not necessary to adopt all of the items, and it is sufficient to adopt only some of (1) to (13) as quality standards.

[0068] For example, among (3) and (5), CD44 high <5% CD44 low >90%, CD105 -~weak >90%, CD90 + <5%; (6), (11), (12), (13) can be adopted as quality standards.

[0069] Furthermore, an embodiment that employs all of (1) to (13) can be employed, for example, when conducting bioequivalence testing when some kind of accident occurs and the identity of the cells can no longer be guaranteed.

[0070] Additionally, in addition to or instead of (1) to (13), one or more other evaluation items disclosed herein can be included in the test. In this way, the present disclosure can be said to provide an extremely useful technology in that it not only produces cultured human corneal endothelial cells that show extremely excellent clinical effects, but also clarifies a technology that can identify these cells.

[0071] In another aspect, the present disclosure provides human functional corneal endothelial cells (also referred to as functional cells with corneal endothelial characteristics of the present disclosure) that are capable of inducing human corneal endothelial functional characteristics upon injection into the anterior chamber of a human eye. The functional cells with corneal endothelial characteristics of the present disclosure 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 upon injection into the anterior chamber of a human eye), and therefore can be typically referred to as human functional corneal endothelial cells that are capable of inducing corneal endothelial functional characteristics upon injection into the anterior chamber of a human eye. The functional cells with corneal endothelial characteristics of the present disclosure 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 disclosure are mature, differentiated cells that exhibit corneal endothelial functions, and effector cells, which are a subpopulation optimal for injection, form small, hexagonal, paving stone-like shapes and utilize an energy metabolic system based on mitochondrial function.

[0072] 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. Therefore, the significance of the present disclosure is great. In particular, prior to the disclosure of the present disclosure, 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 believed 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.

[0073] An application example of the present disclosure is particularly noteworthy in 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 cells of the present disclosure 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, and the safety and clinical proof of concept (POC) of human application have been demonstrated and established in clinical studies based on guidelines for clinical studies using human stem cells.

[0074] One of the factors that enabled the provision of the cells of the present disclosure was 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.

[0075] In the present disclosure, it was also found that in corneal endothelial cells, karyotypic abnormalities occur selectively in a subpopulation, and that autoantibodies that react selectively with the subpopulation are present. Functional cells with corneal endothelial characteristics of the present disclosure, particularly functional mature differentiated corneal endothelial cells, do not exhibit such abnormalities, and compared to other subpopulations, they show relatively low expression of HLA class I antigens associated with immune rejection, and are negative for expression of CD200 antigens, which have previously been thought to be cell markers. It was also found that non-target cells produce high levels of cytokines (SASP-related proteins) associated with cellular senescence.

[0076] Prior to the disclosure of the present invention, there were only a limited number of culture methods with good reproducibility, and therefore attempts to grow in vitro cultured human corneal endothelial cells that were free from cell state phase transitions (CSTs) such as fibrosis, cellular senescence, epithelial-mesenchymal transition (EMT), endothelial-mesenchymal transition, and that were free from karyotypic aneuploidy were extremely difficult, as there was no knowledge or reports whatsoever regarding the cell characteristics, or whether the cell population was composed of multiple subpopulations, or whether the cell population produced by the culture conditions exhibited stable and equitable properties, and analysis had not even been conducted from these perspectives.

[0077] Cultured HCECs undergo CST and tend to develop a senescent phenotype, EMT, and fibroblastic morphology. We identified distinct cell surface markers that distinguish these cultured contaminant cells from unsuitable transplant cells, enabling us to define a population of HCECs that can be used to reconstruct nonfunctional human corneal endothelial tissue. In the present disclosure, the cell markers described in WO2017 / 141926 can be used as needed.

[0078] In one embodiment, the functional cells with corneal endothelial characteristics of the present disclosure have corneal expression characteristics of the cell indicators defined herein.

[0079] Possible cellular indicators of functional cells having corneal endothelial characteristics of the present disclosure include cell surface markers (e.g., CD markers), cell product characteristics, cell morphology indicators, and cellular genetic characteristics. Specifically, these indicators may include cell surface markers (e.g., CD markers); characteristics of proteinaceous products and biological substances associated with those 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 associated with those products; cell size; cell density; and the presence of autoantibody-reactive cells. The cellular indicators of functional, mature, differentiated corneal endothelial cells of the present disclosure exhibit specific ranges or levels of cellular functional characteristics, or a combination thereof. Therefore, by determining specific ranges or levels of cellular functional characteristics, or a combination thereof, of specific cellular indicators, it is possible to determine whether a cell is a functional, mature, differentiated corneal endothelial cell of the present disclosure. The specific ranges or levels of cellular functional characteristics, or a combination thereof, unique to the functional, mature, differentiated corneal endothelial cells of the present disclosure have been identified for the first time in the present disclosure, making it possible to identify various cell subpopulations, perform quality control and quality testing, and ultimately achieve highly effective treatments. These cellular indicators and their specific ranges or levels of cellular functional properties or combinations thereof are specifically detailed below.

[0080] In certain embodiments, the functional cells with corneal endothelial characteristics of the present disclosure have cell functional characteristics including CD166 positivity and CD133 negativity. Another important cell functional characteristic is CD44 expression, the expression intensity of which is preferably CD44 negativity to moderate positivity, more preferably CD44 negativity to weak positivity, and even more preferably CD44 negativity, but is not limited to these. In the present disclosure, it has been discovered that in order to confirm whether corneal endothelial cells or cells differentiated into corneal endothelium-like cells are functional, their functionality can be confirmed by confirming that they are CD166 positivity and CD133 negativity. Additionally, by confirming that CD44 expression is low (CD44 negativity to moderate positivity, preferably CD44 negativity to weak positivity), it has been possible to determine with greater accuracy whether they are functional.

[0081] Therefore, in a preferred embodiment, the functional cells with corneal endothelial characteristics of the present disclosure 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

[0082] More preferably, the functional cells with corneal endothelial properties of the present disclosure have cell functional properties including CD166 positivity, CD133 negativity, and CD44 negativity. Without wishing to be bound by theory, by further limiting the cells to CD44 negativity, 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 properties.

[0083] In another embodiment, the functional cells with corneal endothelial characteristics of the present disclosure 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 disclosure, it has been found that CD200-positive cells are large cells with CST that are unsuitable 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 the careful analysis of subpopulations in the present disclosure.

[0084] In another embodiment, functional cells with corneal endothelial characteristics of the present disclosure have cell functional properties including CD166-positive, CD133-negative, CD44-negative to CD44-weakly positive, and CD90-negative to weakly positive. This further ensures cell homogeneity. 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.

[0085] The functional cells with corneal endothelial characteristics of the present disclosure 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 + The expression characteristics may include, but are not limited to, one or more 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. Alternatively, the group may be a 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.

[0086] 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.

[0087] 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.

[0088] 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 instrument settings. Therefore, under the following conditions: using PE-Cy 7-labeled anti-human CD44 antibody (BD Biosciences), setting the Blue laser area scaling factor of the FACS Canto II to 0.75 and the PE-Cy 7 voltage 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 27500 or more. In the examples herein, the mean fluorescence intensity of the negative control (isotype control) under these settings was approximately 50. (The range is 55±25. This may vary slightly depending on the cell lot, even with the same settings. Those skilled in the art will be able to understand and implement these variations.) Therefore, based on the fact that "the fluorescence intensity range for weak is less than approximately 3800, the fluorescence intensity range for medium is between approximately 3800 and 27500, and the fluorescence intensity range for strong is approximately 27500 or more," the average fluorescence intensity of the negative control (isotype control) is PE-Cy 7: approximately 50 [approximately 33 to 80], so weak: <76x, medium: 76 to 550x, strong: >550x. If the staining intensity pattern is the same as that of the negative control (isotype control), it is considered negative, and if there is even a slight shift, it is considered positive.

[0089] 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].

[0090] 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 disclosure 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.

[0091] Further cell indices used in the present disclosure include the expression intensities of MHC-1 and MHC-2, both of which are related to the absence of immune rejection. Since the present disclosure is clinically used for cell infusion therapy, it is preferable that there is no or low immune rejection.

[0092] Additional cellular indicators for use in the present disclosure include ZO-1, Na + K + These include ATPase and 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 (+).

[0093] The functional cells with corneal endothelial properties provided by the present disclosure enable the clinical application of innovative therapies, but quality control is necessary, and a highly reliable method for this purpose is needed. The present disclosure can provide a method for identifying and quality control of functional, mature, differentiated corneal endothelial cells that do not undergo cell phase transition (CST) and karyotype abnormalities (aneuploidy).

[0094] In one embodiment, the functional cells with corneal endothelial properties of the present disclosure 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.

[0095] The cells of the present disclosure preferably meet the following criteria when subjected to quality testing before use.

[0096] The appearance test includes checking that the tissue has a hexagonal, paving-stone-like shape and is free of fibrosis.

[0097] In the present disclosure, the quality standard is, for example, meeting the criteria shown in the table below.

[0098] [Table 1]

[0099] The functional cells with corneal endothelial properties of the present disclosure preferably have a small cell area, i.e., are small cells. In the present disclosure, the cell area is typically 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, since 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. The present disclosure has revealed 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, examples of the preferable cell area of ​​the functional cells having corneal endothelial properties of the present disclosure that have been realized include, for example, about 150 μm 2 or more, approximately 155μm 2 or more, approximately 160μm 2 or more, approximately 165μm 2Above, approximately 170 μm 2 or more, approximately 175μm 2 or more, about 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. The area distribution can be measured by pre-treating the cells with PBS (-) three times, and then capturing phase-contrast images using a commercially available system such as the BZ X-700 microscope system (Keyence, Osaka, Japan). The area distribution can be quantified using commercially available software such as the BZ-H3C Hybrid cell counting software (Keyence).

[0100] Therefore, the functional cells with corneal endothelial properties of the present disclosure advantageously have the above-mentioned preferred values ​​for at least one cell indicator selected from the group consisting of cell size; cell density, and the presence of autoantibody-reactive cells.

[0101] It is preferred that the functional cells with corneal endothelial characteristics of the present disclosure have cellular functional characteristics homologous to those of the functional cells with corneal endothelial characteristics of the present disclosure (i.e., including functional mature differentiated corneal endothelial cells and moderately differentiated corneal endothelial cells), preferably cellular functional characteristics corresponding to functional mature differentiated corneal endothelial cells, in at least one cellular indicator selected from the group consisting of cell surface markers; proteinaceous products and biological substances associated with these products; SASP-related proteins; intracellular or secreted miRNA; exosomes; cellular metabolic products including amino acids and biological substances associated with these products, as described herein, cell size; cell density, and the presence of autoantibody-reactive cells. Preferred cellular indicators in the present disclosure include, for example, any of the specific numerical values, ranges, and levels described in the explanation of each indicator in this specification, and combinations thereof 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 disclosure, 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 functional cells with corneal endothelial characteristics of the present disclosure 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 (e.g., into the anterior chamber of the eye). Furthermore, without being limited thereto, surrogate marker-like indicators are also effective.Such indicators include: (1) maintenance of endothelial pump and barrier function, including positive Claudin expression; (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 the reference value; (4) determination based on the profile of microRNA (miRNA) produced; (5) determination based on the profile of metabolites produced; and (6) improvement of corneal opacity and hydration edema, resulting in sustained long-term maintenance of corneal endothelial tissue cell density. The following indicators can also be used: (1) the expression of ion channels and monocarboxylate transporters, which are linked to the above-mentioned corneal endothelial (cell) functional characteristics that lead to improved vision; (2) the characteristic that metabolic enzymes involved in the TCA cycle, which lead to the generation of phase transition cells, are not present in the cytoplasm or nucleus but are preferentially localized in mitochondria; (3) the saturation cell density during in vitro culture; (4) the spatial size and distribution of cells obtained during culture; and (5) the adhesiveness to the corneal endothelial surface when cells are injected into a mouse cornea after liquid nitrogen cryoinjury. In particular, without wishing to be bound by theory, proteinaceous products or biological substances related to these products can be used to roughly identify CST cells; miRNAs can be used to partially or completely remove non-target cells; cellular metabolic products or biological substances related to these products can be used to distinguish between moderately differentiated corneal endothelial cells and functional, mature, differentiated corneal endothelial cells, allowing for the selective proliferation of higher quality functional corneal endothelial cells in culture.

[0102] In a preferred embodiment, the functional cells with corneal endothelial properties disclosed herein, particularly mature differentiated corneal endothelial cells, do not have karyotypic abnormalities. The biggest obstacle to applying cHCECs to cell-injection regenerative medicine is that, as shown by Miyai et al., cHCECs often exhibit aneuploidy during culture over several passages (Miyai T, et al., Mol Vis. 2008; 14:942-50). The aneuploidy observed in cHCECs is induced during culture due to cell division. Here, the present inventors provide a novel finding that the presence or absence of aneuploidy in cHCECs is closely related to specific cell subpopulations that predominate among cHCECs. The present inventors found that specific cell subpopulations without karyotypic abnormalities exhibit specific surface phenotypes that parallel those of functional, mature, differentiated corneal endothelial cells present in corneal tissue. We have succeeded in establishing sophisticated culture conditions for selectively growing a cell subpopulation consisting almost entirely of functional, mature, differentiated corneal endothelial cells without karyotypic abnormalities, making it possible to provide a safe and stable regenerative medicine for the treatment of corneal endothelial disorders such as bullous keratopathy by injecting functional, mature, differentiated corneal endothelial cells in the form of a cell suspension into the anterior chamber. Thus, the present disclosure has discovered that karyotypic abnormalities occur selectively in a subpopulation, something that was not previously clear. Furthermore, by using the technology of the present disclosure, it has become possible to select a subpopulation that is substantially free of karyotypic abnormalities.

[0103] In another aspect, the present disclosure provides a cell population comprising functional cells having the corneal endothelial properties of the present disclosure, in particular functional mature differentiated corneal endothelial cells.

[0104] The cell population of the present disclosure has an average cell density of at least about 1500 cells / mm when in 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 2or more. It is understood that the cell population containing functional cells with corneal endothelial characteristics of the present disclosure is provided at a considerably high density due to the small cell size. Cell density is a characteristic found in association with high-quality corneal endothelial functional characteristics, and conversely, measuring such cell density can be used as an index for selecting high-quality functional, mature, differentiated corneal endothelial cells. Because cell density is a value directly related to cell area, it can be calculated similarly by measuring the cell area using any method known in the art. As described above, a representative example is a measurement method using phase-contrast microscope images, which can be captured using a commercially available system such as an inverted microscope system (CKX41, Olympus, Tokyo, Japan). Furthermore, for area distribution measurement, the target cells can be pretreated for easy measurement, for example, by washing them three times with PBS(-), and then phase-contrast microscope images can be acquired using a commercially available system such as a BZ X-700 microscope system (Keyence, Osaka, Japan). Furthermore, the area distribution can be quantified using commercially available software such as BZ-H3C Hybrid cell counting software (Keyence).

[0105] In preferred embodiments, the cell populations of the present disclosure have 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 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 / mm2 , about 3900 pieces / mm 2 , about 4000 pieces / mm 2 etc. can also be realized as the upper limit. 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 disclosure.

[0106] 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 trial application by the phase contrast image quantification technique of cultured cells using the hybrid cell counting method. It has been found that the functional mature differentiated corneal endothelial cells of the present disclosure have a small area per cell and achieve the highest cell density in culture. As illustrated in the examples, the cultured human corneal endothelial cells produced by the manufacturing method of the present disclosure also have a cell area of ​​216 μm 2 , cell density was 2582 cells / mm 2 The cell area and cell density are at the same levels as those of endothelial cells in normal corneal endothelial tissue.

[0107] In one embodiment, the cell population of the present disclosure is characterized in that the functional cells with corneal endothelial characteristics of the present disclosure are present at a ratio that is higher than the ratio occurring in nature. This is because, by providing a cell population in which the ratio of cells capable of inducing corneal endothelial functional characteristics is higher than the ratio occurring 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 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 disclosure (e.g., functional mature differentiated corneal endothelial cells or moderately differentiated corneal endothelial cells).

[0108] 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 disclosure are advantageously functional cells with corneal endothelial characteristics of the present disclosure. Here, the cells contained in these cell populations may have the functional characteristics of corneal endothelial cells described herein. 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 disclosure is effective is that when the cell population contains a certain level of functional cells with corneal endothelial properties of the present disclosure, a good therapeutic or preventive effect is shown when the cell population is injected into a subject. In a preferred embodiment, the cell population of the present disclosure advantageously contains about 70% or more of the cells as functional cells with corneal endothelial properties of the present disclosure. The presence of this level of functional cells with corneal endothelial properties of the present disclosure allows the cell density (for example, about 2300 cells / mm) to be reached, which 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 disclosure advantageously comprises functional cells with corneal endothelial properties of the present disclosure 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 disclosure, 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 have been almost 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 or more, preferably at least about 1700 cells / 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 More than, preferably less Approximately 2300 pieces / 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.

[0109] In a further preferred embodiment, the cell population of the present disclosure 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 human corneal endothelial functional properties 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 naturally available corneal endothelial cells. The proportion of such high-quality functional cells can be increased because a technology has been provided that enables the identification and selection of the functional mature differentiated corneal endothelial cells of the present disclosure from numerous subpopulations.

[0110] In a preferred embodiment, it is advantageous that 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 disclosure are functional mature differentiated corneal endothelial cells. This ratio of 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 the functional properties of corneal endothelial cells described herein. For example, cells that are CD166-positive, CD133-negative, and CD44-negative to weakly-positive (preferably CD44-negative) may be selected as cells to be contained in these cell populations. Alternatively, CD166-positive, CD133-negative, and CD200-negative cells may be selected. Without wishing to be bound by theory, the reason why the cell population of the present disclosure, which has an even higher 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 disclosure 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 disclosure 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. Attempting to achieve this level of abundance ratio of functional cells cannot be achieved by chance, but requires the establishment of 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 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, providing an unprecedented rapid and high-quality treatment technique.

[0111] In one embodiment, functional cells with corneal endothelial characteristics (including functional mature differentiated corneal endothelial cells) or cell populations disclosed herein are characterized by lower expression of HLA class I antigens involved in immune rejection reactions and cell degeneration-associated antigens compared to other subpopulations. Furthermore, functional cells with corneal endothelial characteristics disclosed herein, 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.

[0112] In another aspect, the present disclosure provides a product containing the functional cells or cell populations with corneal endothelial properties of the present disclosure. Such products may be in any form, including, but not limited to, cell-processed products prepared for administration to humans. Such cell products preferably have no unintended transformation, are free from or have little effect from physiologically active substances produced by the cells or tissues, are free from or have little effect on normal cells or tissues, are free from or have little potential for ectopic tissue formation, are free from or have little potential for undesired immune responses, are free from or have little potential for tumor formation or canceration, and, if gene transfer has been performed, have undergone safety evaluation as stipulated in the Guidelines for Gene Therapy Products and have passed general toxicity tests, etc.

[0113] In another aspect, the present disclosure provides a method for preserving functional cells having corneal endothelial properties, functional mature differentiated corneal endothelial cells, or a cell population of the present disclosure, comprising passaging the cells or a cell population by changing the medium. Here, it has been clarified in the present disclosure that this medium change maintains and preserves the functional properties 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 method for producing cells described herein.

[0114] In another embodiment, the present disclosure provides a method for delivering functional cells with corneal endothelial characteristics, functional mature differentiated corneal endothelial cells, or cell populations of the present disclosure, 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 disclosure.

[0115] Although sorting is a typical example of a procedure for separating functional cells with corneal endothelial characteristics and functional mature differentiated corneal endothelial cells of the present disclosure, other methods, for example, a method of selectively inducing apoptosis in non-target cells (miRNA switch method) or inducing necrosis (glucose starvation, etc.) by utilizing the difference in cellular characteristics between target cells and non-target cells, can also be used. However, in the present disclosure, the purity of functional cells with corneal endothelial characteristics or functional mature differentiated corneal endothelial cells of the present disclosure is usually increased by the production method of the present disclosure.

[0116] In one embodiment, the present disclosure provides a cell bank comprising the functional cells or cell population having corneal endothelial properties of the present disclosure. 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 or businesses.

[0117] In another aspect, the present disclosure provides a product containing the functional cells or cell populations with corneal endothelial properties of the present disclosure. Such products may be in any form, including, but not limited to, cell-processed products prepared for administration to humans. Such cell products preferably have no unintended transformation, are free from or have little effect from physiologically active substances produced by the cells or tissues, are free from or have little effect on normal cells or tissues, are free from or have little potential for ectopic tissue formation, are free from or have little potential for undesired immune responses, are free from or have little potential for tumor formation or canceration, and, if gene transfer has been performed, have undergone safety evaluation as stipulated in the Guidelines for Gene Therapy Products and have passed general toxicity tests, etc.

[0118] In another aspect, the present disclosure provides a pharmaceutical comprising functional corneal endothelial cells (functional cells having corneal endothelial characteristics of the present disclosure) or functional, mature, differentiated corneal endothelial cells that can induce human corneal tissue function (particularly, human corneal endothelial functional characteristics) upon injection into the anterior chamber of a human eye. The cells used in the pharmaceutical of the present disclosure can include any cells described elsewhere in this specification. The present disclosure also significantly improves other therapeutic evaluation parameters, such as corneal thickness and visual acuity. For example, when evaluating corneal thickness, the therapeutic effect is achieved earlier than with conventional methods, the E-ratio is increased, and the corneal thickness is sufficiently reduced, resulting in significant improvement. The pharmaceutical of the present disclosure also significantly improves other parameters, such as visual acuity, stromal edema, and the total score thereof. Furthermore, no serious adverse events and almost no non-serious adverse events are observed, and it is understood that the pharmaceutical of the present disclosure provides good therapeutic results. The pharmaceutical of the present disclosure can be used to inject cultured corneal endothelial cells into patients with corneal disorders such as bullous keratopathy.

[0119] In one specific embodiment, the medicament of the present disclosure is for the treatment of corneal endothelial dysfunction, corneal endothelial disorder or corneal endothelial disease. Such corneal endothelial dysfunction, corneal endothelial disorder, or corneal endothelial disease includes, but is not limited to, at least one selected from the group consisting of Grade 3 corneal endothelial disorder and Grade 4 corneal endothelial disorder (typically, 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, congenital anterior chamber angle hypoplasia syndrome, etc.). The grading system used herein is based on the severity classification of corneal endothelial diseases based on the Japanese Journal of Ophthalmology 118: 81-83, 2014. For example, one example of bullous keratopathy is post-laser iridotomy bullous keratopathy. This procedure involves drilling a laser into the iris of a patient whose intraocular pressure is difficult to control with glaucoma medication alone, improving the flow of aqueous humor. It is believed that the aqueous humor strikes the corneal endothelium, damaging it. The pharmaceuticals disclosed herein are believed to be highly effective in treating this condition. Fuchs' corneal dystrophy is a congenital genetic disease that is said to affect 4-5% of people over the age of 40-50 in Europe and the United States. The central corneal endothelium sheds, resulting in opacity. It is the leading cause of corneal transplants in Europe and the United States. The pharmaceuticals disclosed herein are also believed to be highly effective in treating Fuchs' corneal dystrophy. They are also effective in treating multiple OP-BK, a type of bullous keratopathy caused by multiple surgeries of unknown etiology. A typical example of such multiple surgeries is the post-operative period when vitreoretinal surgery and cataract surgery + intraocular lens insertion are performed simultaneously, which is commonly referred to as "triple surgery." The pharmaceutical composition of the present disclosure can be administered to a subject in any form. However, in a preferred embodiment, the cells contained in the pharmaceutical composition of the present disclosure are administered intracamerally. Techniques for injecting cultured corneal endothelial cells into the anterior chamber have been established, and while not wishing to be bound by theory, the concept of regenerating corneal endothelium by intracameral injection is (1) minimally invasive, (2) does not require the use of artificial materials, and (3) allows for the use of highly functional corneal endothelial cells derived from young individuals with minimal aging as master cells. Furthermore, intracameral injection most efficiently regenerates corneal endothelial function. Furthermore, during the course of this disclosure, it has been demonstrated that the injection of a cell suspension into the anterior chamber of a patient (e.g., with bullous keratopathy) after in vitro culture and expansion has established safety and clinical proof-of-concept for human application through research based on guidelines for clinical research using human stem cells (reconnaissance and exploratory clinical research).

[0120] The pharmaceutical agent of the present disclosure may be administered together with an additional drug in addition to the cells. Such additional drugs may be drugs commonly used in ophthalmic treatment (e.g., steroids, antibiotics, antibacterial substances, NSAIDs). Such additional drugs may be included in the cell pharmaceutical agent of the present disclosure as a pharmaceutical agent, or may be provided in a form to be administered separately. When provided or administered separately, such drugs may be provided as a kit or a combination drug. When used as a kit or a combination drug, such drugs may be combined with an accompanying document or the like that describes how to use the kit or combination drug.

[0121] With respect to "additional drugs," it is possible to inject the cells described herein, either alone or together with the cells, instead of injecting the cells, and the present disclosure also includes cells and their products suitable for such administration.

[0122] The medicaments, pharmaceutical compositions, or agents (such as therapeutic or prophylactic agents) of the present disclosure can be provided as kits. In certain embodiments, the present disclosure provides pharmaceutical packs or kits comprising one or more containers filled with one or more components of the cells or medicaments of the present disclosure. Optionally, such containers can also be associated with information indicating approval by a government agency for the manufacture, use, or sale for human administration, in a manner prescribed by the government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products.

[0123] The medicament of the present disclosure may further contain a cell infusion vehicle. Such a cell infusion vehicle may be provided mixed with the cells of the present disclosure, or may be provided separately. When provided or administered separately, it may be provided as a kit or a combination drug. When used as a kit or a combination drug, it may be combined with an attached document or the like that describes how to use it.

[0124] As used herein, the term "kit" refers to a unit in which the components to be provided (e.g., test agents, diagnostic agents, therapeutic agents, antibodies, labels, instructions, etc.) are provided, usually separated into two or more compartments. This kit form is preferred when the purpose is to provide a composition that, for reasons of stability, should not be provided in a mixed state, but is preferably mixed immediately before use. Such a kit advantageously includes instructions or instructions describing how to use the components to be provided (e.g., test agents, diagnostic agents, therapeutic agents) or how to handle the reagents. When the kit is used herein as a reagent kit, the kit usually includes instructions describing how to use the test agents, diagnostic agents, therapeutic agents, antibodies, etc.

[0125] As used herein, "instructions" refer to written instructions for a physician or other user on how to use the present disclosure. The instructions include instructions for the detection method, use of a diagnostic agent, or administration of a medicine or the like according to the present disclosure. The instructions may also include instructions for administration to the eyeball or the anterior chamber (e.g., by injection) as the administration site. The instructions are prepared in accordance with a format specified by the regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and clearly state that they have been approved by the regulatory agency. The instructions are what is known as a package insert (label), and are usually provided in paper form, but are not limited thereto and may also be provided in the form of, for example, electronic media (e.g., a website provided on the Internet, e-mail, etc.).

[0126] As used herein, the term "cell infusion vehicle" refers to any liquid capable of supporting cells, including those used as ocular irrigation solutions. Examples of cell infusion vehicles include Opti-MEM, its additive-supplemented forms, OPeguard-MA, and OPeguard+F. The cell infusion vehicle used in the present invention may further contain at least one of albumin, ascorbic acid, and lactic acid. Based on the findings obtained in this specification, patients can be stratified using metabolites and the like as indicators, and the cells of the present disclosure can be appropriately prepared according to the pathological condition of the stratified patients, allowing for appropriate treatment.

[0127] (Method for producing functional human corneal endothelial cells that can express the functional properties of human corneal endothelial cells when injected into the anterior chamber of the human eye) In one aspect, the present disclosure provides a method for producing functional human corneal endothelial cells that can induce human corneal function when injected into the anterior chamber of a human eye, the method comprising: (b) a step of proliferating and / or differentiating and maturing the corneal endothelial progenitor cells under culture conditions that can minimize culture stress such as proliferation stress.

[0128] In another aspect, there is provided a method for producing human functional corneal endothelial cells that can induce human corneal functional characteristics when injected into the anterior chamber of the human eye, the method comprising: (a) a step of dedifferentiating cells derived from human corneal endothelial tissue to obtain corneal endothelial precursor cells; and (b) a step of proliferating and / or differentiating and maturing the corneal endothelial precursor cells under culture conditions that can minimize culture stress such as proliferation stress.

[0129] In yet another aspect, there is provided a method for producing human functional corneal endothelial cells that can induce human corneal functional properties when injected into the anterior chamber of a human eye, the method comprising: (a) a step of dedifferentiating human corneal endothelial tissue-derived cells to obtain corneal endothelial progenitor cells; and (b) a step of proliferating and / or differentiating and maturing the corneal endothelial progenitor cells in the presence of a cell growth factor in an amount less than that causing transformation.

[0130] In yet another aspect, there is provided a method for producing human functional corneal endothelial cells that can induce human corneal functional properties when injected into the anterior chamber of a human eye, the method comprising a step of proliferating and / or differentiating and maturing corneal endothelial precursor cells in the presence of a cell growth factor in an amount less than that at which transformation occurs.

[0131] In a preferred embodiment, the human corneal function includes a corneal endothelial cell function characteristic, and more preferably, the human corneal function is a corneal endothelial cell function characteristic.

[0132] In one embodiment of the present disclosure, the expression intensity of the CD44 antigen determines the function of the human functional corneal endothelial cells of the present disclosure. The CD44-mediated mitochondrial energy metabolism control effect can be considered as shown in Figure 1.

[0133] For example, cultured human corneal endothelial cells that have been confirmed to exhibit early clinical efficacy and long-term stable clinical efficacy as determined by the mitochondrial localization of the following metabolic enzymes are an example of the cells disclosed herein:

[0134] [Table 2]

[0135] In one embodiment of the present disclosure, with regard to the functional characteristics of cultured human corneal endothelial cells that lead to clinical effects, as shown in Figure 2, in standard cells, mitochondrial OXPHOS is activated as a cellular function that leads to clinical effects, and the cation-anion balance and intracellular pH are maintained, thereby enhancing the water excretion function, leading to improvement of corneal opacity, improvement of hydration edema, miniaturization of corneal endothelial cells, and increased cell density in the endothelial tissue.

[0136] In one preferred embodiment of the present disclosure, when preparing the human functional corneal endothelial cells of the present disclosure, a culture method can be used in which a ROCK inhibitor (e.g., Y-27632) is continuously added to the culture medium. Alternatively, a ROCK inhibitor is not added during dedifferentiation or the early stages of culture, and is present only during the proliferation and / or differentiation / maturation steps, thereby producing high-quality standardized cells or a highly pure cell population of high-quality standardized cells. In another embodiment, a culture method can be used in which a TGF-β inhibitor (e.g., SB-431542) is not added to the culture medium. In one embodiment, analysis at the subpopulation level has shown that cell phenotypes are better when TGF-β activity is not suppressed. In another embodiment, a culture method can be used in which a p38 MAP kinase inhibitor (e.g., SB203580) is not added to the culture medium or is added only during the final maturation step of culture. In one embodiment, analysis at the subpopulation level has shown that cell phenotypes are better when p38 MAP kinase is not suppressed under conditions without cellular stress. One of the useful aspects of the present disclosure is that it has been discovered that by using a minimum amount of reagents such as a ROCK inhibitor, a TGF-β inhibitor, a p38 MAP kinase inhibitor, and / or EGF, it is possible to provide cells of comparable quality at an economically low cost. Another useful feature is that it is now possible to obtain cells at the same level as those obtained in living tissues using in vitro production methods, making it possible to estimate the quality of cells in living tissues. In a non-limiting preferred embodiment, without wishing to be bound by theory, it is preferred that a TGF-β inhibitor (eg, SB-431542) is not added throughout the culture period. In a non-limiting preferred embodiment, without wishing to be bound by theory, a p38 MAP kinase inhibitor (e.g., SB203580) may not be added throughout the culture period, or may be added only during the final 28-35 days of culture.

[0137] Furthermore, in one embodiment of the present disclosure, when preparing the human functional corneal endothelial cells of the present disclosure, it is possible not to use human mesenchymal stem cell conditioned medium. This is because donor / lot differences in human MSCs pose a barrier to widespread medical use, as the contained SASP destabilizes quality, and while miRs paracrinely inhibit phase transition, there is significant lot-to-lot variation. In a non-limiting preferred embodiment, without wishing to be bound by theory, it can be understood that it is sufficient to not add human mesenchymal stem cell conditioned medium (MSC-CM) throughout the entire culture period.

[0138] Furthermore, in one embodiment of the present disclosure, when preparing the human functional corneal endothelial cells of the present disclosure, EGF, a type of cell growth factor, can be omitted, thereby enabling stable production of target cells. Furthermore, target cells can be obtained at a high yield even during long-term passage, such as P5. In one embodiment, in the culture method (production method) of the present disclosure, it is preferable not to use EGF throughout the culture period. In another embodiment, a culture method in which EGF is added only in the early stage of culture (e.g., within 7 days) may be used. It is understood that in an alternative embodiment, EGF may be used at a concentration that does not cause culture stress.

[0139] In one embodiment of the present disclosure, even when the production method was changed as described above, a comprehensive search for changes in gene expression and an assay of mitochondrial respiratory function using a flux analyzer revealed no changes that affected the function of cultured human corneal endothelial cells ( FIG. 3 ).

[0140] In one embodiment of the present disclosure, induction of differentiated, mature, and functional human corneal endothelial cells from somatic (stem) cells via a dedifferentiation pathway can be performed as shown in Figure 4. According to this method, the proliferation rate and number of possible passages is at the P6:83,000-fold level in young donors, and approximately 10,000 eyes' worth of cells can be obtained from one eye. This method also broadens the range of donor ages, and even middle-aged donors can obtain approximately 600 eyes' worth of cells from one eye at P4. In addition, it is possible to maintain good cell morphology and shape, maintain high density, and achieve a cell area of ​​approximately 200 mm and small distribution.

[0141] In one embodiment of the present disclosure, without wishing to be bound by theory, the production method as shown in FIG. 5 involves causing differentiation and phase transitions including EMT in parallel, which can result in inconveniences due to antagonistic effects. However, as disclosed herein, the step of proliferating and / or differentiating and maturing corneal endothelial precursor cells under culture conditions that can minimize culture stress such as proliferation stress, and / or the step of proliferating and / or differentiating and maturing corneal endothelial precursor cells is carried out in the presence of regulated trace amounts of cell growth factors, thereby making it possible to resolve side effects due to antagonistic effects against the differentiation effect induced by transformation.

[0142] In one embodiment of the present disclosure, the transformation comprises endothelial-mesenchymal transition, and the proliferation and / or differentiation / maturation step may be performed in the presence of a ROCK inhibitor.

[0143] Furthermore, in one embodiment of the present disclosure, the method may further comprise the step of confirming at least one characteristic selected from the group consisting of: (i) the property that metabolic enzymes involved in the TCA cycle and the like and metabolic products such as Acetyl CoA are not present in the cytoplasm or nucleus but are organelle-selectively localized in the mitochondria so as to prevent the generation of contaminating phase transition cells; (ii) increased mitochondria-dependent oxidative phosphorylation in mitochondria; (iii) decreased (including absence of) epigenetic multigene expression mediated by histone acetylation by acetyl CoA; (iv) increased expression of sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1); and (v) increased expression of bicarbonate anhydrase 5B (CA5B) in the candidate cells obtained after the proliferation and / or differentiation / maturation step; and identifying the candidate cells as human functional corneal endothelial cells when the candidate cells contain at least one of the characteristics.

[0144] One feature of the present disclosure is that it provides cells defined by traits such as the expression of carbonic anhydrase (CA), an ion channel involved in the control of intracellular pH, the expression of intracellular transporters for NHE-1 and monocarboxylic acids (metabolites such as pyruvate and lactate), and enhanced expression of AQP1, which is involved in water excretion and is directly linked to clinical effects such as the reduction of corneal opacity and hydration edema. As a result of possessing these characteristics, the present disclosure provides cells with precisely defined intracellular pH, and as a result, defined cell size and functions involved in the control of mitochondrial function.

[0145] Therefore, in one embodiment of the present disclosure, the method may further comprise the step of confirming whether or not one or more metabolic enzymes selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malic enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain ketoacid dehydrogenase 2 (BCKDH2) are expressed in the mitochondria of the candidate cells obtained after the proliferation and / or differentiation / maturation step, and identifying the candidate cells as human functional corneal endothelial cells if the expression is confirmed.

[0146] Furthermore, in another embodiment of the present disclosure, the method may further comprise the step of confirming whether or not at least one enzyme selected from the group consisting of ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malic enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), and lactate dehydrogenase (LDH) is expressed in the candidate cells obtained after the proliferation and / or differentiation / maturation step, and identifying the candidate cells as human functional corneal endothelial cells if the enzyme is not expressed or is not substantially expressed.

[0147] Furthermore, in another embodiment, the method may further comprise the step of confirming whether or not expression of sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1), which is a water channel, is enhanced in the candidate cells obtained after the proliferation and / or differentiation / maturation step, and, if such enhancement is confirmed, identifying the candidate cells as human functional corneal endothelial cells; in another embodiment, the method may further comprise the step of confirming whether or not expression of bicarbonate anhydrase 5B (CA5B) is enhanced in the candidate cells obtained after the proliferation and / or differentiation / maturation step, and, if such enhancement is confirmed, identifying the candidate cells as human functional corneal endothelial cells.

[0148] Furthermore, in one embodiment of the present disclosure, the method may further comprise the step of confirming whether the human functional corneal endothelial cells have been produced using as their origin cells selected from the group consisting of corneal endothelial tissue-derived cells, 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 produced by the direct programming method, and in another embodiment, the method may comprise the step of producing corneal endothelial progenitor cells using as their origin cells selected from the group consisting of corneal endothelial tissue-derived cells, 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 produced by the direct programming method.

[0149] As used herein, "corneal endothelial tissue-derived cells or corneal endothelial precursor cells", as defined elsewhere in this specification, respectively refer to cells derived from corneal endothelial tissue and cells which become functional cells having the characteristics of corneal endothelium or functional mature differentiated corneal endothelial cells of the present disclosure by differentiating through a dedifferentiation process, but these include, in addition to cells obtained from the corneal endothelium of a donor, any cells such as cells differentiated from iPS cells, ES cells, etc. into corneal endothelial cells, and precursor cells before differentiating into corneal endothelial cells, and also include moderately differentiated corneal endothelial cells as defined herein.

[0150] In the manufacturing method of the present disclosure, corneal endothelial tissue-derived cells that can be used as a starting material can be collected from a living organism, or the starting material can be corneal endothelial progenitor cells, for example, cells differentiated from stem cells or progenitor cells. Such differentiated cells can include, but are not limited to, cells differentiated from various stem cells (e.g., induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), fertilized eggs, and somatic stem cells). Therefore, in a specific embodiment, the corneal endothelial tissue-derived cells or corneal endothelial progenitor cells used (as a starting material) in the present disclosure can include, but are not limited to, pluripotent stem cells, mesenchymal stem cells, corneal endothelial progenitor cells collected from corneal endothelium, corneal endothelial cells collected from corneal endothelium, and corneal endothelial progenitor cells and corneal endothelial-like cells prepared by the direct programming method. Here, examples of pluripotent stem cells include, but are not limited to, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and the like. Therefore, it is understood that the corneal endothelial cells or their precursor cells (as starting materials) used in the present disclosure include cells prepared by differentiating induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), etc. into corneal endothelium-like cells. Techniques for differentiating induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), etc. into corneal endothelium-like cells are known in the art, and examples thereof include, but are not limited to, the AMED method (Ueno et al., supra), WO2013 / 051722 (Keio University), etc.

[0151] When cells that have not been differentiated into corneal endothelium-like cells are used, it is preferable to include a step of differentiating or maturing the cells into corneal endothelium-like cells.

[0152] (Quality control) In one embodiment of the present disclosure, a method for quality control or process control of human functional corneal endothelial cells capable of inducing human corneal endothelial functional characteristics upon cell injection into the anterior chamber of a human eye can include a step of confirming the expression in the mitochondria of the cells of one or more metabolic enzymes selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malic enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain ketoacid dehydrogenase 2 (BCKDH2). In this case, the method can further include a step of confirming the expression of acetyl-CoA in the cytoplasm and nucleus of the human functional corneal endothelial cells and the absence of epigenetic multigene expression mediated by histone acetylation by acetyl-CoA.

[0153] In another embodiment, the quality control may further include the step of confirming that ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malic enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), and / or lactate dehydrogenase (LDH) are not expressed or are substantially not expressed in the human functioning corneal endothelial cells, and in another embodiment, the quality control may further include the step of confirming that expression of sodium / hydrogen exchanger 1 (NHE1) and / or aquaporin 1 (AQP-1), or bicarbonate anhydrase 5B (CA5B) is increased in the human functioning corneal endothelial cells. In another embodiment, the quality control may further include the step of measuring intracellular pH in the human functioning corneal endothelial cells.

[0154] (Other embodiments) Although the determination method and analysis method according to one or more aspects of the present disclosure have been described based on the embodiments, the present disclosure is not limited to these embodiments. As long as they do not deviate from the spirit of the present disclosure, various modifications conceivable by those skilled in the art to the present embodiments and forms constructed by combining components of different embodiments may also be included within the scope of one or more aspects of the present disclosure.

[0155] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is stated that "within a range of two values," the range includes the two values ​​themselves.

[0156] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims. [Example]

[0157] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to these examples.

[0158] The experimental methods and materials used in the present disclosure are described below. Note that although the following experimental methods are used in the present embodiment, similar results can be obtained by using other experimental methods.

[0159] The following examples were conducted in compliance with medical ethical regulations such as the Declaration of Helsinki, regulations such as the GCH, and regulations established by Kyoto Prefectural University of Medicine and other institutions, and were approved by the ethics committee of the institution to which the inventor belongs. The following experiments were conducted after obtaining the necessary informed consent.

[0160] (Example 1: I. Experiment to Examine the Concentration of Added EGF) In this example, the concentration of epidermal growth factor (EGF) used in producing functional human corneal endothelial cells was investigated. Details are shown below.

[0161] Methods and Materials Donor information was as follows: #202, ABG-404 OSCN / ODCN (right eye / left eye), age 64, endothelial cell density (ECD) = 3104 / 3070, cause of death (COD): Metastatic Neuroendocrine Carcinoma of the Uterus, DP (time from death of corneal donor to placing cornea in storage solution) = 12:15, DC (time from death of corneal donor to placing in cell culture) = 8D. The concentration of added EGF was examined under the following culture conditions, the details of which are as follows: 0+Y, cultured with SB2, EGF, and Asc (ascorbic acid) ↓ P1 +Y, cultured with SB2, EGF, and Asc ↓ P2 EGF(-), 0.5ng / mL, 1ng / mL, 5ng / mL culture (+Y, SB2, Asc) ↓ P2 Passage from each condition P3: Cultured in 4 groups: EGF(-), 0.5ng / mL, 1ng / mL, 5ng / mL (+Y, SB2, Asc) ↓ P3 Passage from each condition P4 EGF(-), cultured in 4 groups: 0.5ng / mL, 1ng / mL, 5ng / mL (+Y, SB2, Asc) *Trypsin (TrypLE, ThermoFisher) concentration at detachment x10, seeded cell number ECD800 (In the above, P indicates the passage number.)

[0162] The culture conditions were as follows: SB2, EGF, and Asc refer to SB203580, epidermal growth factor, and ascorbic acid, respectively. Epidermal growth factor was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and SB203580 (SB2) was obtained from Cayman Chemical (Ann Arbor, MI). Dulbecco's Modified Eagle Medium High Glucose (DMEMHG) and fetal bovine serum were obtained from Gibco Industries, Inc. (Langley, OK), and plastic culture plates were obtained from Corning. Unless otherwise indicated, all other chemicals were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). EGF(-), 0.5 ng / mL, 1 ng / mL, and 5 ng / mL are the above SB2, EGF, and Asc to which EGF at the respective concentrations has been added, or to which EGF has not been added.

[0163] (result) Four photographs of P2 taken at D41 are shown in Figure 6. The results of FACS at P3 and P4 are shown in Figures 7 to 10. From these results, without wishing to be bound by theory, it was found that judging from both cell morphology under microscopic observation and cell surface CD antigen phenotype, the non-addition of EGF is desirable at any passage from P2 to P4. Furthermore, the significance of releasing proliferation stress by not adding the growth factor EGF is thought to be that if EGF is not added when producing the human functional corneal endothelial cells disclosed herein, the cellular phase transition CST, including EMT, is released, the cellular senescence circuit is avoided, and mature differentiated cells are efficiently produced.

[0164] (Example 2: II. Effect of adding EGF from the time of primary culture) Next, we investigated the effect of adding EGF during primary culture. Details are shown below.

[0165] Methods and Materials Next, we investigated the effect of adding EGF from the P0 primary culture stage. Donor information was as follows: #214, ABS-355OSCN / ODCN, NancyY(+), age 18, endothelial cell density (ECD) = 3571 / 3401, cause of death: multitrauma 2' MVA, DP = 12:32, DC = 7D. The culture conditions are as follows: P0: Two eyes were cultured in two ways: EGF(-) and 0.5ng / mL (+Y, SB2, Asc). ↓ P0 passages from each condition P1: Cultured in two ways: EGF(-), 0.5ng / mL (+Y, SB2, Asc) ↓ P1 Passage from each condition P2: Cultured in two ways: EGF(-), 0.5ng / mL (+Y, SB2, Asc) *TrypLE concentration at time of detachment x 10, ECD800 at time of seeding

[0166] Y, SB2, and Asc refer to Y27632, SB203580, and ascorbic acid, respectively. The Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Y) and epidermal growth factor were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and SB203580 (SB2) was obtained from Cayman Chemical (Ann Arbor, MI). Dulbecco's Modified Eagle Medium High Glucose (DMEMHG) and fetal bovine serum were obtained from Gibco Industries, Inc. (Langley, OK), and plastic culture plates were obtained from Corning. Unless otherwise indicated, all other chemicals were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). EGF(-), 0.5ng / mL is the above Y, SB2, Asc plus EGF at each concentration. The same applies to Example 1 except for the specific matters described above.

[0167] (result) Furthermore, the results of FACS at P0 to P2 are shown in Figures 11 to 14. Without wishing to be bound by theory, since the proportion of corneal endothelial cells of the present disclosure increases when EGF is not added, it is understood that when EGF is not added (or when the concentration is low), culture stress is reduced and more preferable cells are produced, and this is preferable for producing human functional corneal endothelial cells that can induce human corneal function when injected into the anterior chamber of the human eye.

[0168] Therefore, from these facts as well, it can be understood that in the present disclosure, it is important to proliferate and / or differentiate / maturate corneal endothelial precursor cells under culture conditions that can minimize culture stress such as proliferation stress, in order to culture cells with a high proportion of standardized cells.

[0169] (Example 3: III. Assay of mIR Expression Without Addition of EGF, Effect of Addition of ROCK Inhibitor) In this example, we tested whether EGF-free cell cultures could be assayed by mIR expression, and also examined the effect of adding a ROCK inhibitor.

[0170] (material and method) The cases were divided as shown in Figure 15, and intracellular gene fluctuations of miR378, miR146, miR34, and miR184 were measured. Y27632 was used as the ROCK inhibitor. The culture conditions were the same as those in Examples 1 and 2.

[0171] (result) The results of FACS measurement and photographic evaluation are shown in Figure 16. As a result, it was found that the influence of age differences was superimposed on Y+ / -.

[0172] The results of qRT-PCR showing changes in the expression of miR184, miR34, miR378, and miR146 genes in the presence or absence of EGF and in the presence or absence of Y are shown in Figures 17 and 18.

[0173] As a result, without wishing to be bound by theory, we found that miR184 (1) has an inversely proportional relationship with the amount of intracellular CD44 gene expression, (2) expression levels are higher in younger people than in middle-aged people, (3) expression levels are higher with the addition of ROCK inhibitor than without ROCK inhibitor, and (4) expression levels are higher with the addition of EGF than with the addition of EGF.

[0174] Furthermore, without wishing to be bound by theory, miR34a-5p is expressed in the following ways: (1) within the same donor, Although there is an inverse correlation with intracellular CD44 gene expression, comparisons across donors suggest that the correlation with CD44 may be reversed; (2) there is no change in expression level in younger than in middle-aged individuals; (3) expression level is higher with the addition of ROCK inhibitor than without it; and (4) expression level is higher without the addition of EGF than with the addition of EGF.

[0175] From these results, without wishing to be bound by theory, it was confirmed that the absence of EGF was preferable, based on the expression intensities of miR184 and 34a, which are cell characteristic indicators. It was also confirmed that the expression levels of miR184 and 34a increased with the addition of a ROCK inhibitor.

[0176] From these facts, it can be understood that in the present disclosure, it is important to proliferate and / or differentiate and mature corneal endothelial precursor cells under culture conditions that can minimize culture stress such as proliferation stress.

[0177] Example 4: IV. Cellular Function Characterization Using Metabolites in the Absence of EGF, and the Effect of Adding a ROCK Inhibitor Next, we assayed the metabolites characteristic of cell function in the cells of the present disclosure without the addition of EGF, and also evaluated the effect of adding a ROCK inhibitor.

[0178] (material and method) In this example, the differences between the metabolic product characteristics of human functional corneal endothelial cells and those of non-target cells were confirmed. (Metabolite Assay) Measurement of metabolites in the culture medium For the measurement of metabolites in culture supernatant (CS), 20 μL of CS and internal standard solution 1 (H3304-1002; HumanMetabolome Technologies, Inc., Yamagata, Japan) were thoroughly mixed in an 80 μL Milli-Q (Merckl KGaA, Darmstadt, Germany) flask. Cationic compounds were measured by CE time-of-flight mass spectrometry (CE-TOFMS) in positive mode, and anionic compounds were measured by CE tandem mass spectrometry (CE-MS / MS) in positive and negative modes. Hierarchical cluster analysis (HCA) was performed using our proprietary software, "PeakStat." Differences between values ​​were statistically analyzed using Student's t-test or one-way analysis of variance (ANOVA) with Bonferroni's post-hoc test (GraphPad Prism 6.0, GraphPad Software). A p value of <0.05 was considered statistically significant. The effect of adding a ROCK inhibitor was examined in accordance with Example 3.

[0179] (result) An example of the hierarchy of metabolites is shown in Figure 19. Furthermore, the metabolic characteristics of human functional corneal endothelial cells (target cells) that can induce human corneal function when injected into the anterior chamber of the human eye, which is the objective of the present disclosure, and cells that do not (non-target cells) were confirmed and are shown in Figures 20 to 22.

[0180] The results showed that the target cells showed little increase in lactic acid or the L / P (lactate / pyruvate) ratio, indicating low anaerobic glycolytic activity. Furthermore, while Gln increased in the target cells, Glu did not increase significantly, indicating that the Gln synthesis pathway was enhanced rather than glutaminonysis.

[0181] Furthermore, it was found that the target cells showed significantly low levels of BCAA, indicating that their metabolism is highly dependent on BCAA.

[0182] Furthermore, it was found that when the amount of EGF added was reduced, the production of branched-chain amino acids (Ile, Leu), which are one of the characteristics of the target cells, decreased, and this resulted in an increase in the content of the target cells.

[0183] Without wishing to be bound by theory, the above results indicate that the metabolic products of cultured endothelial cells exhibit a non-target cell profile upon addition of EGF.

[0184] Furthermore, when a smaller amount of EGF was added, the increase in lactic acid and L / P ratio was small, and the content of target cells with low anaerobic glycolytic activity increased. In other words, it was found that the addition of EGF caused cultured endothelial cells to exhibit a non-target cell profile with high anaerobic glycolytic activity.

[0185] From these facts, it can be understood that in the present disclosure, it is important to proliferate and / or differentiate and mature corneal endothelial precursor cells under culture conditions that can minimize culture stress such as proliferation stress.

[0186] (Example 5: V. Effects of Various Additives) In this example, the effects of various additives were tested, as detailed below. V-1. Evaluation of the product in a new culture method without the presence of p38 MAPK inhibitor (SB203580) In this example, a method in which the p38 MAPK inhibitor SB203580 was not added (also referred to as SB2-) was tested.

[0187] (material and method) To evaluate the product using the new SB2- culture method, we tested the effects of additives using lot CT09 P5. First, we used CT09 P4 (excess cells for FBS lot testing). Culture supernatants were collected the day before subculture (Day 42). CT09 P5 was then subcultured at ECD400 (181219) and cultured in Nancy medium (FBS #1652794, containing ascorbic acid) under conditions 1–5 shown in Figure 23 (two wells per 6-well plate). Culture supernatants were collected from weeks 1 through 5, and one well was analyzed by FACS on Day 34 (190122). IL-8 and PDGF-bb levels were measured by ELISA in the culture supernatants.

[0188] Regarding the experimental conditions, a list of culture supernatant samples from CT09 P4 and P5 for ELISA PDGF-bb and IL-8 is shown in FIG.

[0189] (result) Photographs of CT09 P5 under each condition are shown in Figures 24 to 26, and the results of FACS on Day 34 are shown in Figures 27 to 29, respectively. The ELISA PDGF-bb and IL-8 culture supernatant sample lists for CT09 P4 and P5 are shown in Figure 30. The results for PDGF-bb categorized by additives are shown in Figure 31, and the results for week are shown in Figure 32. The results for IL-8 categorized by additives are shown in Figure 33, and the results for week are shown in Figure 34. From the above results, without wishing to be bound by theory, it was found that, overall, the effects of additives were such that SB4, no EGF, Y from day 10 onwards, and no SB2 addition or its addition only during the final stage of culture were desirable.

[0190] V-2. Mitochondrial respiratory capacity In this example, mitochondrial respiratory capacity was examined.

[0191] (material and method) Corneal endothelial cells (HCECs) were obtained from donor corneas provided by SightLife (Seattle, WA, USA) and cultured according to a published protocol (Toda M, Ueno M, et al. Invest Ophthalmol Vis Sci. 2017;58:2011) with some modifications. HCECs from passages 2–5 were used for all experiments. SPs containing CSTs were subcultured at low cell density. SPs of cHCECs were identified by cell surface markers (CD24, 44, 105, 166) using phase-contrast microscopy and flow cytometry.

[0192] (result) The results are shown in Figures 35 to 37. These results indicate that mitochondrial respiratory capacity is high in standard cells. Furthermore, EGF addition reduces the proportion of standard cells, which in turn reduces mitochondrial respiratory capacity and oxidative phosphorylation (OXPHOS).

[0193] Example 6: VI. Addition of ROCK inhibitors In this example, the effect of adding a ROCK inhibitor (e.g., Y27632) on the production of human functional corneal endothelial cells (target cells) that can induce human corneal function when injected into the anterior chamber of the human eye was investigated.

[0194] (material and method) Donor information is shown in Figure 38. Regarding culture conditions, the timing of Y addition was investigated at P1 and P2 for #190719, and at P1 for #190802, so culture supernatants were collected from 1 week to 5 weeks under the conditions shown in Figure 39. For #190318, culture supernatants were collected at P1 and P2 under the conditions shown in Figure 40.

[0195] (result) The FACS results are shown in Figures 41 and 43 to 45, and photographs of the cultured cells are shown in Figure 42. These results show that in all three experiments, the proportion of standardized cells was higher when a ROCK inhibitor was added 10 days after the start of culture. Without wishing to be bound by theory, these results demonstrate that culturing in the presence of a ROCK inhibitor is preferable in the proliferation and / or differentiation / maturation steps. These findings also demonstrate that the present disclosure demonstrates the induction of efficient differentiation, which is essential for obtaining corneal endothelial progenitor cells.

[0196] Figure 46 shows the results of ELISA PDGF-bb measurements (by parameter) in the culture supernatant of #190719. In both experiments, P1 and P2, even when a ROCK inhibitor was added on day 10 after the start of culture, PDGF, one of the parameters for mature cells, increased as the cells progressed from differentiation to maturation. This indicates that adding expensive Y for adhesion purposes from the first day of culture is pointless under these culture conditions. Adding it during the differentiation phase likely prevents dedifferentiation. Furthermore, in P1, PDGF peaked at 4 weeks and then slightly decreased at 5 weeks.

[0197] The ELISA PDGF-bb measurement results (for each item) in the culture supernatant of #190318 are shown in Figure 47. As a result, it was found that when the p38 MAPK inhibitor SB2 was not added, PDGF levels were high at 3 weeks, indicating early induction of differentiation.

[0198] ELISA PDGF-bb measurement results (weekly) in the culture supernatant of #190719 are shown in Figure 48, and ELISA PDGF-bb measurement results (weekly) in the culture supernatant of #190318 are shown in Figure 49. Without wishing to be bound by theory, these results indicate that the addition of Y27632 increased PDGF levels in #190719's P1 and P2 cultures, but slightly decreased levels in the P1 proliferation phase. Without wishing to be bound by theory, we also found that PDGF was an independent factor, largely unrelated to the presence or absence of SB203580.

[0199] The results of ELISA IL-8 measurement (per item) in the culture supernatant of #190719 are shown in Figure 50. The results showed that in both P1 and P2 experiments, even when a ROCK inhibitor was added on day 10 after the start of culture, IL-8, one of the parameters for mature standard cells, reached its lowest level as the cells progressed from differentiation to maturation. Furthermore, in P1, the level was high during the proliferation phase and decreased as differentiation progressed, and in P2, the level also decreased as differentiation progressed.

[0200] The results of ELISA IL-8 measurement (weekly) in the culture supernatant of #190318 are shown in Figure 51. The results showed that IL-8 fluctuated almost dependently on the presence or absence of SB2.

[0201] The ELISA PDGF-bb and IL-8 measurement results (for each item) in the culture supernatant of #190802 are shown in Figure 52. The results showed that PDGF increased in a manner dependent on differentiation induction by a ROCK inhibitor (Y27632), and that IL-8 decreased in a manner dependent on a p38 MAPK inhibitor (SB203580).

[0202] The results of cytokine measurement (BioPlex) in the culture supernatant of #190318 (by item) are shown in Figure 53. The results showed that all three, IL-6, IL-8, and MIP-1b, were dependent on the p38 MAPK inhibitor (SB203580).

[0203] These findings also demonstrate that the present disclosure induces efficient differentiation, which is essential for obtaining corneal endothelial precursor cells.

[0204] Example 7: VII. Addition of a ROCK inhibitor In this example, conditions related to ROCK inhibitors were further investigated following Example 6. Details are given below.

[0205] (material and method) Culture supernatants were collected from #190719 P4 at 1-5 weeks and the effects of the presence or absence of a ROCK inhibitor were examined. The additive conditions are shown in Figure 54. Donor information was #190719S / D, AEB-301, OSCN / ODCN, Age = 40 / Gender = M, ECD = 3448 / 3289, COD:Bleomycin Lung Toxicity 2 / 2 ChemoTreatments, DP = 11:28, DC = 8D.

[0206] (result) Photographs taken on DAY 35 are shown in Figure 55, and the FACS results are shown in Figures 56 and 57. The ELISA results for PDGF-bb and IL-8 are shown in Figure 58, and the weekly results are shown in Figure 59.

[0207] As a result of the above, even in a three-group comparison experiment, no mature standard cells were detected by FACS even when a ROCK inhibitor was added for three days at the start of culture. Furthermore, PDGF, one of the standard cells, was found to have low levels and did not meet the standard reference value. IL-8 levels were also higher than the standard, indicating the need to add it during differentiation.

[0208] From the above, it can be understood that in one embodiment, in the step of proliferation and / or differentiation / maturation, it is preferable to culture in the presence of a ROCK inhibitor.

[0209] Example 8: VIII. Enhancement of Adhesion by ROCK Inhibitors Next, we tested the adhesion enhancement effect of ROCK inhibitors. To investigate whether the adhesion enhancement effect of ROCK inhibitors is related to the production of standardized cells, cells cultured under the following conditions were compared at P0 with or without Y addition.

[0210] (material and method) AEM-510 (62Y, male), ECD ODCN:3058, OSCN:3058, were used and stored at 4°C on January 30th. Corneal treatment was performed at 10:00 AM on January 31st, collagenase treatment at 10:45 AM, and collagenase treatment at OS at 11:05 AM. Seeding began at 2:45 PM, and the OD cells were cultured in Y- and ascorbic acid (A)+ medium, while the OS cells were cultured in Y+ and A+ medium. The medium was changed on February 4th at 10:00 AM, and both eyes were photographed (phase contrast x4, x10).

[0211] (result) The results are shown in Figures 60 to 63. In the embodiment shown in this example, the ROCK inhibitor Y27632 certainly enhances adhesion to the culture vessel after cell seeding, but no difference was observed on day 14 of culture even when Y27632 was added for the first time after day 4. This demonstrates that standardized cells can also be provided by culturing in the presence of a ROCK inhibitor in the proliferation and / or differentiation / maturation step.

[0212] Example 9: IX. Epigenetic regulation by signal transduction from mitochondria to the nucleus In this example, epigenetic regulation by signal transduction from mitochondria to the nucleus was investigated. Epigenetic control through signal transmission from mitochondria to the nucleus is discussed in detail in Cell Metab. 2015 Mar3;21(3):349-50., Trends in Cell Biology, June 2017, Vol. 27, No. 6, and Sheikh et al. Nature Rev. Genetics, 2019. Epigenetic control by metabolites and the breakdown of cell senescence and differentiation can be thought of as illustrated in the schematic diagram in Figure 64. Organelle-localized enzymes are also considered, as shown in Figure 65. The isozymes expressed in the dedifferentiated progenitor cells mentioned above are present in the cytoplasm and nucleus, and acetylate histones via nuclear-translocated AcCoA.

[0213] In this example, we verified that the expression of proteins such as metabolic enzymes differs significantly between standard cells and non-standard cells that have undergone phase transition, and demonstrated clear differences in the organelle-selective localization of isozymes.

[0214] (material and method) The so-called DAVID analysis was performed as follows: (HCEC) HCECs were cultured according to a published protocol with some modifications. Descemet's membrane containing CECs was removed from donor corneas and digested with 1 mg / mL collagenase A (Roche Applied Science, Penzberg, Germany) for 2 hours at 37°C. HCECs obtained from a single donor cornea were seeded in a single well of a type I collagen-coated 6-well plate (Corning, Inc., Corning, NY). Culture medium was prepared according to the published protocol. When HCECs reached confluence, they were harvested and passaged using 10x TrypL Select (ThermoFisher Scientific, Inc., Waltham, MA) for 12 minutes at 37°C. The donor information for the HCECs used in all experiments was as follows: #184, ABF-956 OSCN / ODCN, Age = 11, Gender = Female, ECD = 3207 / 3588; and #225, ABZ-612 OSCN / ODCN, Age = 27, Gender = Male, ECD = 3195 / 3425. The culture conditions were as shown in Figure 66. The FACS results for P1 and P4 are shown in Figures 67 and 68, and photographs of the cells are shown in Figure 69.

[0215] (Cultured human corneal endothelial cells (cHCECs) passaging and preparation of cell suspension) All work other than microscopic photography was carried out in a safety cabinet, and upon entering the culture room, participants washed their hands thoroughly and wore masks and gloves. PBS(-) and culture medium (Nancy medium) were preheated to 37°C, and the added reagents (gray boxes) were allowed to return to room temperature. The cells were removed from the culture, the lot number confirmed, and the cells were observed under a phase-contrast microscope. Images were taken at 40x magnification (24-well: 1 location, 12-well: 1 location, 6-well: 2 locations, T-25: 3 locations) and 100x magnification (24-well: 2 locations, 12-well: 2 locations, 6-well: 3 locations, T-25: 3 locations) using a phase-contrast microscope camera. The cells were placed in a safety cabinet, and the culture supernatant was collected into tubes (1.5 mL or 15 mL) using a Pipetman or disposable pipette. PBS(-) (24-well: 500 mL, 12-well: 1 mL, 6-well: 2.5 mL, T-25: 7 mL) was injected (first time) and the wells were gently shaken to wash. The PBS(-) in the wells was removed with a disposable pipette, and PBS(-) was added (24-well: 500 mL, 12-well: 1 mL, 6-well: 2.5 mL, T-25: 7 mL) (second time). The wells were gently shaken to wash. The PBS(-) in the wells was removed with a disposable pipette, and PBS(-) was added (24-well: 500 mL, 12-well: 1 mL, 6-well: 2.5 mL, T-25: 7 mL) (third time). A 10-minute timer was started, and photographs were taken at 40x magnification (24-well: 1 location, 12-well: 1 location, 6-well: 2 locations, T-25: 3 locations) and 100x magnification (24-well: 2 locations, 12-well: 2 locations, 6-well: 3 locations, T-25: 3 locations) using a phase-contrast microscope camera. The plates were placed in a CO2 incubator for 10 minutes (until the timer sounded). TrypLE Select (10x) was warmed to 37°C in an incubator and placed in a safety cabinet before the 10-minute timer expired. TrypLE Select (10x) was diluted to 5x with PBS (-) (1:1 dilution), and the PBS (-) was removed using a disposable pipette and completely removed using a P-1000. TrypLE Select (5x) was then injected (24-well: 200 mL, 12-well: 400 mL, 6-well: 1 mL, T-25: 2.5 mL) and placed in a CO2 incubator for 15 minutes.A phase-contrast microscope was used to confirm that more than half of the cells had become rounded, and the cells were then detached from the bottom by tapping. If less than half of the cells were rounded, the plate was placed in a CO2 incubator for 2-5 minutes again (the maximum enzyme treatment time with TrypLE was 20 minutes in total, and the plate was used for the next step even if less than half of the cells were rounded). Next, the wells were placed in a safety cabinet and repeatedly aspirated and dispensed with a P-1000 tip to detach the cells, which were then collected into 1.5-mL ProteoSave tubes or 15-mL tubes. The safety cabinet light can be turned on during this process. The collected culture supernatant was added per well using a P-1000 tip (24-well: 200 mL, 12-well: 400 mL, 6-well: 1 mL, T-25: 2.5 mL) to rinse the wells, and the cells were collected into tube 21. The wells were centrifuged (300 × g; 2,100 rpm for 3 minutes in an Eppendorf #5452000034 tabletop centrifuge, or 1,200 rpm for 5 minutes in a TOMY #LC-220 tube centrifuge), and the supernatant was removed using a P-1000 tip (long). The tip was replaced with a new one, and the collected medium was added per well (24-well: 100 mL, 12-well: 200 mL, 6-well: 500 mL, T-25: 1 mL) to suspend the cells. The cells were centrifuged (300 × g; 2,100 rpm for 3 minutes in an Eppendorf #5452000034 tabletop centrifuge, or 1,200 rpm for 3 minutes in a TOMY #LC-220 tube centrifuge), and the supernatant was removed by replacing the tip with a P-1000 tip (long). The tip was replaced with a new one, and the collected medium was added per well (24-well: 200 mL, 12-well: 400 mL, 6-well: 1 mL, T-25: 2.5 mL) to suspend the cells. Using a tip attached to a P-20, 10 mL of the cell suspension was quickly extracted and placed in a 96-well plate (FALCON: 35591, 96-well assay plate, U-bottom, no lid, non-sterile polystyrene) for measurement. 10 mL of trypan blue was added to the 10 mL of cell suspension, mixed by pipetting, and 10 mL was taken and the number of viable cells was counted using a hemocytometer. Cell count calculation Number of viable cells (count value) ÷ ____ (number of compartments) × 2 × 10 4 =Number of living cells (cells / mL) Number of viable cells (cells / mL) × volume of suspension (mL) = total number of viable cells (cells) For FACS analysis, 16 x 10 4 The cells (one antibody + negative control) were dispensed into ProteoSave tubes, and the remaining cells were passaged. FACS was performed using 8 × 10 cells per antibody. 4 The passage was performed based on the endothelial cell density (ECD: cells / mm 2 ) 400 (as of 191001) seeded (24-well: 7.6 × 10 4 cells, 12-well: 15.2 × 10 4 cells, 6-well: 38 × 10 4 cells, T-25: 100 × 10 4 cells). (Adjustment of culture medium) The amount of medium + α to be used (24-well: 500 mL, 12-well: 1 mL, 6-well: 2.5 mL, T-25: 7 mL) was placed in an appropriate tube, and additive reagents (1 / 1000 volume of each based on the medium volume) were added. Because the additive reagents are sensitive to light, the lights in the safety cabinet were turned off when adjusting the reagents or changing the medium. The plate was shaken vertically and horizontally to seed the cells evenly, and the cells were cultured in a CO2 incubator.

[0216] (FACS measurement) <Necessary reagents> cHCEC suspension (16 x 10 with one FACS antibody and negative control (NC)) 4 cells required) FACS Buffer (+NaN3) BSA (globulin-free) 0.5% / NaN3 0.05% / 1xPBS 100mL FACS Buffer (-NaN3) BSA (globulin-free) 0.5% / 1x PBS 100mL Antibody for FACS (ex.CD90-FITC / CD166-PE / CD24-PerCP-Cy5.5 / CD44-PE-Cy7 / CD105-APC) Cell strainer ice chest

[0217] <FACS measurement> The FACS was started and the antibody solution (protected from light, on ice) was prepared as follows: Antigen Fluorescent dye Volume (μL) × Sample type + α (μL) CD90 FITC 4 CD166 PE 4 CD24 PerCP-Cy5.5 1 CD44 PE-Cy7 0.25 CD105 APC 1 FACS Buffer(+NaN3) 9.75 (total vol.20μL) When there were multiple samples, an additional solution was prepared. The cell suspension was centrifuged at 250 × g (1,800 rpm) for 2 minutes at 4°C, and the supernatant was removed using a P-200. 6 The concentration was adjusted to cells / mL and suspended in FACS Buffer (+NaN3). If the suspension volume was less than 30 μL, 20 μL was used for the reaction, and 10 μL was used as NC. 20 μL of antibody solution and 20 μL of suspension were mixed thoroughly by pipetting, and the tube was wrapped in aluminum foil and rotated at 4°C in the dark for 2 hours (up to a maximum of 4 hours). The tube was centrifuged at 250 × g (1,800 rpm) for 2 minutes at 4°C, the supernatant was discarded, and the tube was washed with 100 μL of FACS Buffer (+NaN3). The tube was centrifuged at 250 × g (1,800 rpm) for 2 minutes at 4°C, the supernatant was discarded, and the tube was suspended in 350 μL of FACS Buffer (-NaN3) and transferred through a cell strainer to a 5-mL tube. For NC, the remaining portion of the initial suspension was suspended in FACS Buffer (-NaN3) to a total volume of 350 μL and transferred through a cell strainer to a 5-mL tube. During FACS measurement, the sample was kept protected from light and on ice. The FACS laser settings and target cell range were determined and a file created (Open Book → Open Syringe → Green → Cytometer tab: Laser → Follow Area scaling for HCEC label: FSC 0.5 / Blue 0.75 / Red 0.80). The NC was placed in the SIT and allowed to flow for a short time (Flow rate: set to medium, Acquire data for 5 seconds → Remove the sample from the SIT and place on ice). The fluorescence parameter graph and target cell range were checked. Parameters: Checked for peaks between 102 and 103. If they were out of range, the parameter values ​​were adjusted up or down during flow, and if they were out of range, the Restart button was pressed to check and adjust. If peaks outside the graph were calculated as a maximum of 105 during sample flow, adjustments were required to ensure they did not exceed this value. Target cells: Adjustments were made to avoid dead cells or cell clumps from entering the measurement range. The NC and sample were run in this order (Acquire data → Record data → 10,000 events or 5 min → (after 5 min) Stop Recording → Stop acquire). After the sample had run, the FACS was shut down. Combined proteomics by liquid chromatography / mass spectrometry (LC / MS) Cell lysates from passage 4 cHCECs were used for proteomic analysis. High-quality (HQ) cHCECs, containing 93.9% CD44- / + mature differentiated cHCEC SPs (effector ratio = E ratio, n = 3), and low-quality (LQ) cHCECs, containing 73.8% CD44++ / +++ immature cHCEC SPs (n = 3), were analyzed. Cell lysates from three aliquots of HQ or LQ cHCECs were dried and reconstituted in 20 mmol / L HEPES-NaOH (pH 58.0), 12 mmol / L sodium deoxycholate, and 12 mmol / L sodium N-lauroylsarcosinate. After reduction with 20 mmol / L dithiothreitol at 100°C for 10 min and alkylation with 50 mmol / L iodoacetamide at ambient temperature for 45 min, the proteins were digested with immobilized trypsin (Thermo Fisher Scientific) for 6 h at 37°C with shaking at 1000 rpm. After removal of sodium deoxycholate, the resulting peptides were desalted using Oasis HLB m-elution plates (Waters) and subjected to mass spectrometry. Peptides were analyzed using an LTQ-Orbitrap-Velos mass spectrometer (Thermo Fisher Scientific) coupled with an Ulti-Mate 3000 RSLC nanoflow HPLC system (Thermo Fisher Scientific). Protein identification and quantification were performed using MaxQuant software. MS / MS spectra were searched against the Homo sapiens protein database in Swiss-Prot with a false discovery rate of 1% for both peptide and protein identification filters. Only "Razor unique peptides" were used to calculate relative protein concentrations. For protein composite analyses, all detected peaks were normalized by adjusting the median value from 1.0 to 104. (LC / MS data set analysis) The LC / MS dataset consisted of a total of 4641 proteins, which was obtained using Proteome Discoverer 2.2 software. After removing data for which abundance ratios could not be calculated, we analyzed the remaining data using the web-based program DAVID v6.8 (The Database for Annotation, Visualization, and Integrated Discovery; https: / / david.ncifcrf.gov). Finally, we obtained 4315 genes, each of which was assigned a unique DAVID gene ID for subsequent analysis. For gene expression analysis, we calculated statistical P values ​​and fold changes between the two groups, plotted volcano plots, and extracted differentially expressed genes between HQ and LQ cHCECs. Further investigation of the genes of interest and related genes / pathways suggested to be related to cHCEC metabolism was performed using DAVID and its options "BIOCARTA" and "KEGG_PATHWAY." The reference genes / pathways in the figures were slightly modified, referring to the original databases, BioCarta (https: / / cgap.nci.nih.gov / Pathways / BioCarta_Pathways) or KEGG (KyotoEncyclopediaofGenes and Genomes; https: / / www.genome.jp / kegg / ). For gene ontology (GO) analysis, the data were divided into three groups based on the range of abundance ratio (LQ / HQ), and each group was analyzed in DAVID using the "GOTERM_DIRECT" option. GO results were sorted by P value, and the top 10 GO terms in each group were displayed. For LC / MS data analysis, the significance of the difference between HQ and LQ cHCECs was assessed by Student's or Welch's t-test after confirmation by F-test.

[0218] (DAVID analysis of proteomics) Subsequently, proteomic DAVID analysis was performed in the following order (Figure 70). (1) Three group analysis (2) Analysis of ratios over 27 (3) Analysis of GOTERM only (4) Comparison of mitochondria after DAVID analysis

[0219] (1) Three group analysis Of the 4641 proteins, 323 proteins without an abundance ratio of (225) phase transition non-standard cell LQ / (184) standard cell HQ were excluded (323 proteins). The remaining 4318 proteins were divided into groups based on the abundance ratio: LQ / HQ (if data was available for even one protein, it was included in the analysis). The three groups were as shown in the table below.

[0220] [Table 3]

[0221] (2) Analysis of ratios over 27 Next, we analyzed those with a ratio of over 27. In Figure 71, we performed DAVID analysis on the two genes with an Abundance Ratio:LQ / HQ exceeding 27, resulting in cluster 0. A search in KEGG PATHWAY revealed that Nitrogen metabolism came up. We searched for genes (17 genes) included in KEGG PATHWAY's Nitrogen metabolism and the proteins (4641 genes) analyzed this time, and picked seven (CA12, CA2, CA3, CA5B, GLUD1, GLUD2, GLUL). We then ran these through DAVID to search for and confirm the KEGG PATHWAY (Nitrogen metabolism).

[0222] (3) Analysis of GOTERM only Next, we performed an analysis using only GOTERMs. DAVID analysis can use around 50 databases, and the DAVID recommended (Default) analysis uses around 10 of these (e.g., UP_KEYWORD, KEGG_PATHWAY, INTERPRO, etc.) (GOTERMs are included). This time, we performed an analysis using only three of these GOTERMs (GOTERM_BP_DIRECT, GOTERM_CC_DIRECT, GOTERM_MF_DIRECT). Biological Process (BP): Function within a cell. Cellular Component (CC): A component of a cell. Molecular Function (MF): Function of a molecule. The results are shown in Figure 72.

[0223] (4) Comparison of mitochondria after DAVID analysis Next, we performed a comparison of mitochondria after DAVID analysis. In the Annotation Cluster 1 (Enrichment Score: 72.73354929082193) of the group with an Abundance Ratio of LQ / HQ of 0.5-0.2 (398 annotations), many annotations were related to mitochondria (Figure 73). To further confirm the pathways, the 398 annotations were analyzed again using DAVID and clustered by the GOTERM, PASTHWAY, and PROTEINDOMAINS fields selected as the DAVID defaults (Figure 74). From the clustering results, pathways were selected for investigation (Figure 75). Pathways were examined and a graph of genes was inserted. Furthermore, metabolic pathways of interest were searched and visualized using KEGG Pathway (bold text), and the protein expression intensities of enzymes and substrates related to the metabolic pathways of phase transition nonstandard cells and standard cells were compared (Figures 76-83).

[0224] Example 10: X. Ion Channels: Single Carboxylate Transporter System Cell Immunostaining In this example, the ion channel simple carboxylic acid transport system was investigated.

[0225] (material and method) Cells obtained from the corneal tissues of the following donors were immunostained, and the antibody data used are shown in Figure 84. #190802S / D, ACW-134OSCN / ODCN, Age 28 / Gender: Female, ECD = 3003 / 3021, Cause of Death: ESRDD-P = 08:57, DC=5D The immunostaining results are shown in the table below.

[0226] [Table 4]

[0227] 1 is standard cells, and 2 is non-standard cells undergoing phase transition and cultured with EGF. Both were cultured from Day 0 to Day 41, 42, or 43 before use in experiments. Each cell was imaged at 200x magnification and stained in duplicate. The following items were prepared for immunofluorescent staining of cHCECs (24-well plate). Fixative (-30°C ice-cold special grade methanol (Nacalai #21915-35) or 4% PFA / phosphate buffer solution (Wako #163-20145) returned to RT) □PBS (-) □PBS(-) / 0.2%TritonX-100(0.5 mL × wells × 1.1 = mL) □1%BSA / PBS(-)(BSA: EIA / RIA grade, nacalai #01281084)(BSA g / PBS(-) mL) □ Primary antibody (including isotype control) □Fluorescently labeled secondary antibodies DAPI (Dojin Chemical, wako#340-07971)

[0228] The procedure was as follows: All volumes were measured per well of a 24-well plate. First, the cell culture supernatant was removed, and the cells were washed with 0.5 mL of PBS(-) once. 0.5 mL of fixative was added, and the cells were left to stand at room temperature for 15 minutes (-30°C for MeOH). 0.5 mL of PBS(-) / 0.2% Triton X-100 was added, and the cells were incubated at room temperature for 15 minutes. The solution was removed, and 0.5 mL of 1% BSA / PBS(-) was added, and the cells were blocked for 60 minutes at room temperature. The solution was removed, and 0.3 mL of the primary antibody diluted in 1% BSA / PBS(-) was added, and the cells were incubated overnight at 4°C. The cells were washed with 0.5 mL of PBS(-) for 5 minutes three times. The solution was removed, and 0.3 mL of the fluorescently labeled secondary antibody diluted in 1% BSA / PBS(-) was added, and the cells were incubated at room temperature for at least 60 minutes. The cells were washed with 0.5 mL of PBS(-) for 5 minutes once. The solution was removed, and 0.3 mL of DAPI diluted 200-fold with PBS(-) was added and incubated at room temperature for 5 to 15 minutes. The cells were washed twice with 0.5 mL of PBS(-) for 5 minutes. 0.5 mL of PBS(-) was added, and the cells were observed under a fluorescence microscope.

[0229] (result) The results are shown in Figures 85 to 88. As a result, it was found that ATP1A1 (Na+KLATPase) and AQP1 were highly expressed on the cell membrane in standard cells, but not in non-standard cells. The same was true for SLC4A11, NHE1, and SLC25A42. From the above, without wishing to be bound by theory, the organelle-selective localization of monocarboxylate transporters was revealed. From the above, it can be understood that standard cells express functional proteins that lead to corneal endothelial (cell) functional characteristics that improve corneal opacity and hydration edema, thereby maintaining corneal endothelial tissue cell density over a sustained, long-term period and leading to improved vision.

[0230] Example 11: XI. Transporter SLC Family Proteins Next, in this example, we observed the expression of SLC family proteins in normal cells, as described in detail below.

[0231] (material and method) In this example, an experiment was carried out on the expression of transporter SLC family proteins. We performed integrated proteomic analysis of cell lysates by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using three samples of either CD44- / + (mature) or CD44++ / +++ cHCECs predominantly composed of cell state transition (CST). The cell culture conditions were similar to those described in the previous examples. The expression patterns of cation / anion transporters (ion transporters), single carboxylate transporters and solute carrier (SLC) family proteins, as well as carbonic anhydrase (CA) were examined. MS / MS spectra were analyzed against the human protein sequence database (SwissProt) using the Mascot or SEQUEST search engines with Proteome Discoverer 2.2 software (Thermo Fisher Scientific).

[0232] (result) The abundance ratio of SLC family transporter proteins between standard and non-standard cells

[0233] [Table 5]

[0234] The results above indicated that enzymes related to nicotinic acid and nicotinamide metabolites were most upregulated in CST cHCECs, whereas only ectonucleotide pyrophosphatase was upregulated in mature cHCECs. Interestingly, the expression of glutamate-ammonia ligase (GLUL) was elevated in CST cHCECs, whereas the expression of glutamate dehydrogenase 1 and 2 (GLUD1 and 2) and glutaminase 2 (GLS2) was decreased. By integrative proteomics, we observed that the expression patterns of cation / anion transporters (ion transporters), monocarboxylate transporters (MCTs), and solute carrier (SLC) family proteins, as well as carbonic anhydrase (CA), showed organelle-selective localization between mature cHCECs (target cells) and CST cHCECs (non-target cells).

[0235] Example 12: XII. Intracellular pH Measurement In this example, the relationship between the intracellular pH and the target cells was investigated, as described in detail below.

[0236] (material and method) Subsequently, intracellular pH was measured. Donor information was as follows: Lot #190802P3: #190802S / D, ACW-134OSCN / ODCN, age 28 / gender female, endothelial cell density ECD = 3003 / 3021, cause of death: ESRD, DP = 08:57, DC = 5D

[0237] (Intracellular pH measurement) Phase contrast micrographs showing cell morphology, subpopulation analysis based on cell surface CD antigen expression profiles, and pH measurements were performed.

[0238] The intracellular pH of cultured human corneal endothelial cells is thought to determine cell size and mitochondrial function. We created standard cells and phase-transition non-standard cells under the following culture conditions and examined their pH. We verified that the intracellular pH of CD44- standard cells was lower than that of CD44++ / +++ phase-transition non-standard cells.

[0239] [Table 6]

[0240] (cHCECs intracellular pH measurement method) (Adjustment reagent) HEPES buffer (153mM NaCl, 5mM KCl, 5mMglucose, 20mMHEPES, pH7.4) Calibration buffer(130mM KCl, 10mMNaCl, 1mM MgSO4,10mM Na-MOPS)…pH6.6 / 7.0 / 7.2 / 7.4 / 7.8 / 8.2 2 mg / mL Nigercin / EtOH stock solution - store in aliquots at -30°C 1 mM BCECF-AM / DMSO solution (Dojin Chemical B221: Add 72.612 mL of DMSO to 50 mg / tube, mix, dispense, and store at -30°C)

[0241] (Preparations) Black flat-bottom 96-well plate (Thermo Fisher: 237105, individually wrapped with lid, untreated) Consider the number of cells that can be collected (1×105 cells / sample; 6 samples are used for calibration). At least 7 to 8 x 10 5 cells prepared) pH measurement reagent (HEPES, 1 mM BCECF-AM / DMSO, 2 mg / mL Nigercin / EtOH) cHCEC detachment-related reagents (PBS, TrypLE / PBS)

[0242] (method) (Preparation of cell suspension (cells to be measured)) The cells were photographed with a phase-contrast microscope camera, and cultured human corneal endothelial cells were detached with PBS(-) (cells were photographed with a phase-contrast microscope camera) and 5x TrypLE / PBS(-).

[0243] Count the number of cells (actual value: cells / mL, total cells / mL) and add 2-10 × 10 cells to one 1.5-mL ProteoSave tube. 5 The cells were suspended in HEPES buffer to a concentration of 2 x 10 cells / 1 mL of HEPES. 5 mL of 1 mM BCECF-AM / DMSO was added per mL (final concentration: 5 mM) (actual value: cells / mL x cells), and the cells were incubated for 30 minutes in a 37°C, 5% CO2 incubator in the culture room. The cells were then transferred to the laboratory bench and centrifuged at 300 x g (1,867 rpm: Eppendorf Centrifuge 5418) for 3 minutes, the supernatant was removed, and the cell pellet was suspended in 200 mL of HEPES buffer (first wash). The cells were centrifuged at 300 x g (1,867 rpm) for 3 minutes, the supernatant was removed, and the cell pellet was resuspended in 200 mL of HEPES buffer (first wash). 5 The cells were suspended in HEPES buffer at a concentration of 8 × 10 cells / mL HEPES and combined in a 15-mL tube (e.g., 8 × 10 5 (The total volume was adjusted to 8 × 10 cells / 4 mL HEPES.) Cells were then collected from this cell suspension into 500 mL / 1.5 mL ProteoSave tubes (e.g., a total volume of 8 × 10 cells / 4 mL HEPES). 5 (Two tubes for cells / 4 mL HEPES). Before fractionation, the cell suspension was mixed to make it uniform (after fractionation, proceeded to the preparation of cell suspension for calibration). Centrifuge at 300 × g (1,867 rpm) for 3 minutes (second wash), discard the supernatant, and suspend the cell pellet in 500 mL of HEPES buffer.

[0244] (Preparation of cell suspension for calibration) Calibration buffer was prepared, and cells were separated from the cell suspension into six 500 mL / 1.5 mL ProteoSave tubes. The tubes were centrifuged at 300 × g (1,867 rpm) for 3 minutes (second wash). The supernatant was discarded, and the cell pellet was suspended in 500 mL of each pH buffer. 2 mg / mL Nigercin / EtOH (2.5 mL per 500 mL) was added (final concentration: 10 mg / mL) and incubated at room temperature for 10 minutes. This calibration buffer and the cell suspension were added to 3 wells of a 96-well plate, 150 mL per well.

[0245] (measurement) Measurements were made using a fluorescent plate reader (GloMax Explorer, Promega) at an excitation wavelength of 500 nm and an emission wavelength of 530 nm (GloMax was set to excitation Blue (475 nm) and an emission filter of 500-550 nm).

[0246] (result) The results are shown in Figures 89 to 93.

[0247] (Example 13: XIII. Effect of Additives) In this example, the effect of additives was further investigated, as detailed below. (material and method) One P3 T-25 plate was passaged at 24wp and used (=P4). C37, ABH-096OSCN / ODCN, Age = 26Y / ECD = 3255 / 3137, COD:Trauma, DP = 17:57 / DC = 8D

[0248] [Table 7]

[0249] (result) The results are shown in Figures 94 to 100. Of the measurement items, IL-1b, IL-2, IL-4, IL-5, IL-7, IL-9, IL-10, IL-13, IL-15, IL-17, basicFGF, IFN-g, MIP-1a, MIP-1b, TNF-a, and VEGF were below the detection limit.

[0250] From the above Examples 1 to 13, it is understood that it is important to proliferate and / or differentiate and mature corneal endothelial precursor cells under culture conditions that can minimize culture stress, such as proliferation stress. It is also understood that expression of functional proteins that lead to corneal endothelial (cell) functional properties that lead to improvement in corneal opacity and hydration edema, and as a result, to maintaining corneal endothelial tissue cell density sustainably over the long term and improving vision, is observed in standard cells. Additionally, it is understood that in a preferred embodiment, it is preferable to culture in the presence of a ROCK inhibitor in the proliferation and / or differentiation and maturation step.

[0251] Example 14: Increased oxidative phosphorylation respiration As explained in Figure 1, Mir34a expression is reduced in non-standard cells, resulting in enhanced glycolysis in mitochondrial cells. Therefore, we forcibly introduced miR43a mimics into cells to increase miR43a expression, and confirmed that oxidative phosphorylation respiration increased (Figures 101 and 102).

[0252] When mitochondrial oxidative phosphorylation respiration (OXPHOS) is increased, water is expelled from the corneal stroma, leading to clinical pharmacological effects (Figure 103). In addition, by increasing the number of proliferative undifferentiated cells and then differentiating them, it is possible to produce standardized cells and high-quality cells with excellent pharmacological effects (Figure 104).

[0253] Whether the cells of the present disclosure become mature differentiated cells or dedifferentiate is thought to be due to the influence of intracellular pH on mitochondrial function (Figure 105). The corneal endothelial cells disclosed herein function to excrete excess water from the corneal stroma to the anterior chamber, maintaining corneal stroma transparency. Therefore, clinical effects include reduced corneal opacity and transparency, and reduced corneal thickening. Furthermore, cell-injected medically qualified cells enhance mitochondrial OXPHOS, creating a lactate concentration gradient within the endothelial cells from the stroma to the anterior chamber, resulting in osmotic pressure promoting water excretion via AQP1 channels. Therefore, cell function characteristics can be evaluated, including mitochondrial oxidative phosphorylation function and AQP1 channel expression. Furthermore, as described above, intracellular pH suppresses the decrease in dedifferentiation-inhibitory miR34a, suppresses CD44 expression, and maintains mitochondrial OXPHOS. Therefore, cell function characteristics can be evaluated, including decreased expression of intracellular miR34a and CD44. The intracellular cation-anion balance maintains intracellular pH in the neutral range of 7.0 to 7.2, thereby maintaining high levels of mitochondrial OXPHOS. Examples of cell function characteristic evaluation include Na+K+ATPase (AT1P1), NBCe1 (bicarbonate ion channel), NHE1 (Na+,H+ exchange ion channel), MCT4 (lactate transporter: releases into aqueous humor), SLC4A11, etc. Furthermore, as a functional evaluation method for the mixing of non-qualified cells with final product-qualified cells, a method can be considered to confirm that the intracellular cation-anion balance shifts the intracellular pH to the alkaline range, and cell function characteristic evaluation can confirm the presence or absence of histone acetylation and mitochondrial dysfunction.

[0254] Example 15: Immunostaining of cells In Example 10, ion channels and / or monocarboxylic acid transport systems were confirmed based on the results of proteomics, but in this Example, selective expression of ion channels was confirmed by immunostaining of cells.

[0255] FACS+ ion channel molecular immunostaining results (Lot#CR04_P3:Donor information) #CR04 ORL2002-314 LCN / RCN Age: 12 / Gender: Male ECD=3546 / 3460 Cause of death: Blunt head trauma DP=6:42 DC=--D

[0256] FACS measurement results (Lot#CR04_Day41) After sowing at ECD800-900 by CPC, transfer to the car on Day 2 The cells were then cultured until Day 41, detached with 5xTrypLE select, and used for FACS. The culture conditions are as shown in the table below.

[0257] [Table 8]

[0258] A photograph taken on the 41st day is shown in Figure 106.

[0259] Furthermore, for immunostaining, the cells were cultured until Day 41 as described above before use in the experiment, and images were taken at 200x magnification. The antibodies used are listed in the table below.

[0260] [Table 9]

[0261] As a result, ATP1A1 was localized to the plasma membrane, NHE1 was localized intracellularly and partially to the plasma membrane, AQP1 was localized to the plasma membrane, NBCe1 was localized to the plasma membrane, AE2 was localized both intracellularly and to the plasma membrane, and Acetyl-Histone H3 was underexpressed. The results are summarized in Figure 107.

[0262] Example 16: Increased histone acetylation in non-standard cells In this example, we investigated why non-standard cells are generated. As shown in Figure 108, it was suggested that intracellular pH promotes mitochondrial glycolysis, and citrate transported from mitochondria to the nucleus acetylates histones via ACLY and ACSS2, which are localized in the nucleus, and changes the composition of mitochondrial matrix proteins.

[0263] Example 17: Comparison of standard and non-standard cells In this example, we compared the expression of specific proteins in standard cells and non-standard cells (CST cells). Standard cells #191224S P4 Day 46 (+Y) were used, and non-standard cells #200313 P1 Day 74 (+SB4, EGF) were used. The results are shown in Figures 109 to 111.

[0264] To confirm that histone deacetylase activity is reduced in standard cells, which may explain the increased histone acetylation in non-standard cells, we measured HAT / HDAC activity in standard and non-standard cells as follows.

[0265] The following Lot#191224S_P4 was used as the donor. #191224S AEN-024 OSCN Age = 29 / Gender = Male ECD=2941 Cause of death: Acute Respiratory Distress Syndrome DP=07:32 DC=7D

[0266] The culture conditions were as shown in the table below.

[0267] [Table 10]

[0268] After seeding 1 with ECD400 and 2 with ECD800 (passage from non-standard cells), they were cultured until Day 42. One well from each well was detached using FACS:5xTrypLE select and used in the experiment (the same sample as for miRNA expression measurement). In addition, nuclear protein was extracted from one well, and the cells were directly recovered using the Nuclear Extraction Kit. The results are shown in Figures 112 to 114.

[0269] The substrate requirement of the mitochondrial respiratory system was found to be enhanced in CST cells compared to standard cells, demonstrating enhanced anaplerosis. Furthermore, the expression of BCAT2 and BCKDH, which are involved in mitochondrial branched-chain amino acid metabolism, was confirmed in standard cells. Interestingly, the ubiquitous mitochondrial metabolic enzymes involved in the TCA pathway, CS, ACO2, IDH2, MDH2, and ME3, and the enzymes involved in acetyl-CoA production, ACSS1 and ACAT1, were all upregulated in standard cells. Meanwhile, the expression of ubiquitous cytoplasmic isozymes, ACLY, ACO1, IDH1, MDH1, and ME1, and the enzymes involved in cytoplasmic and nuclear acetyl-CoA production, ACSS2 and ACAT2, were upregulated in non-competent CST cells. This suggests that under the influence of environmental factors such as culture stress, cytoplasmic and nuclear acetyl-CoA may epigenetically mediate histone acetylation and regulate the phenotypic expression of CST cells. The ubiquitous presence of nuclear acetyl-CoA may play an important role in the failure of differentiation of dedifferentiated cells.

[0270] (Note) While the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present disclosure should be interpreted solely by the claims. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. This application claims priority to Japanese Patent Application No. 2020-32139, filed with the Japan Patent Office on February 27, 2020, the contents of which are incorporated by reference in their entirety as if they constitute the content of this application. [Industrial Applicability]

[0271] The present disclosure finds applicability in the medical industry related to corneal endothelial regenerative medicine and related industries.

Claims

1. Human functional corneal endothelial cells cultured from human corneal endothelial progenitor cells in the presence of a Rho-related protein kinase (ROCK) inhibitor, and (i) without inducing endothelial-mesenchymal transition (CST) in the presence of epidermal growth factor (EGF) at a concentration of less than 5 ng / mL, or (ii) in the absence of EGF.

2. Human functional corneal endothelial cells according to Claim 1, satisfying one or more of the following: (I) (Ia) Increased mitochondrial-dependent oxidative phosphorylation in the mitochondria of the human functional corneal endothelial cells; (Ib) No increase in the expression of acetyl-CoA in the cytoplasm and nucleus of the human functional corneal endothelial cells; and (Ic) No epigenetic polygene expression mediated by acetyl-CoA-mediated histone acetylation is induced in the human functional corneal endothelial cells; (II) The mitochondria of the human functional corneal endothelial cells express one or more metabolic enzymes selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malate enzyme 3 (ME3), ACSS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain keto acid dehydrogenase 2 (BCKDH2); (III) In the human functional corneal endothelial cells, ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malate enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), or lactate dehydrogenase (LDH) are not expressed or are hardly expressed; (IV) The human functional corneal endothelial cells express ion channels or monocarboxylic acid transporters that are linked to the functional characteristics of corneal endothelial cells; (V) The expression of sodium / hydrogen exchanger 1 (NHE1) or aquaporin 1 (AQP-1) is enhanced in the human functional corneal endothelial cells; (VI) The expression of bicarbonate anhydrase 5B (CA5B) is upregulated in the human functional corneal endothelial cells; or (VII) (VIIa) The metabolic enzymes involved in the TCA cycle are not present in the cytoplasm or nucleus of the human functional corneal endothelial cells; or (VIIb) Metabolic enzymes involved in the TCA cycle are organelle-selectively localized in the mitochondria of the human functional corneal endothelial cells.

3. In the mitochondria of the human functional corneal endothelial cells, mitochondrial-dependent oxidative phosphorylation is increased; The expression of acetyl-CoA in the cytoplasm and nucleus of the aforementioned human functional corneal endothelial cells does not increase; and In the aforementioned human functional corneal endothelial cells, epigenetic polygene expression mediated by acetyl-CoA-mediated histone acetylation is not induced. Human functional corneal endothelial cells according to claim 1 or 2.

4. The human functional corneal endothelial cell according to claim 1 or 2, wherein the mitochondria of the human functional corneal endothelial cell express one or more metabolism-related enzymes selected from the group consisting of citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malate enzyme 3 (ME3), ACS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, and branched-chain keto acid dehydrogenase 2 (BCKDH2).

5. The human functional corneal endothelial cells according to claim 1 or 2, wherein ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malate enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), or lactate dehydrogenase (LDH) are not expressed or are hardly expressed in the human functional corneal endothelial cells.

6. The human functional corneal endothelial cells according to claim 1 or 2, wherein the human functional corneal endothelial cells express an ion channel protein or a monocarboxylic acid transporter protein that is related to the functional characteristics of corneal endothelial cells.

7. The human functional corneal endothelial cell according to claim 6, wherein the ion channel protein or monocarboxylic acid transporter protein comprises ATP1A1, AE2, ZO-1, NBCe1, NHE1, MCT4, SLC9A1, SLC4A11, SLC44A1, SLC2A13, SLC12A1, SLC25A42, SLC25A18, SLC4A1AP, or SLC16A2.

8. The human functional corneal endothelial cell according to claim 6, wherein the ion channel protein or monocarboxylic acid transporter protein comprises ATP1A1, AE2, ZO-1, NBCe1, NHE1, MCT4, SLC4A11, or SLC25A42.

9. The human functional corneal endothelial cells according to any one of claims 6 to 8, wherein, after injection of the human functional corneal endothelial cells, the expression of the ion channel protein or the monocarboxylic acid transporter protein improves corneal opacity and hydration edema, and as a result maintains corneal endothelial tissue cell density over a long period of time, leading to improved visual acuity.

10. The human functional corneal endothelial cells according to claim 1 or 2, wherein the expression of sodium / hydrogen exchanger 1 (NHE1) or aquaporin 1 (AQP-1) is enhanced in the human functional corneal endothelial cells.

11. The human functional corneal endothelial cells according to claim 1 or 2, wherein the expression of bicarbonate anhydrase 5B (CA5B) is enhanced in the human functional corneal endothelial cells.

12. The metabolic enzymes involved in the TCA cycle are not present in the cytoplasm or nucleus of the human functional corneal endothelial cells; or The metabolic enzymes involved in the TCA cycle are selectively localized to the mitochondria of the aforementioned human functional corneal endothelial cells. Human functional corneal endothelial cells according to claim 1 or 2.

13. The human functional corneal endothelial cell according to claim 12, wherein the metabolite of the metabolic enzyme is acetyl-CoA.

14. The human functional corneal endothelial cells according to any one of claims 1 to 13, wherein the human corneal endothelial progenitor cells are prepared from corneal endothelial tissue-derived cells, pluripotent stem cells, mesenchymal stem cells, cells collected from corneal endothelium, or corneal endothelial progenitor cells or corneal endothelial-like cells prepared by direct programming.

15. The human functional corneal endothelial cells according to any one of claims 1 to 14, wherein the human corneal endothelial progenitor cells are dedifferentiated from human corneal endothelial tissue-derived cells.

16. The human functional corneal endothelial cell according to any one of claims 1 to 15, wherein the ROCK inhibitor is Y-27632.

17. Human functional corneal endothelial cells according to any one of claims 1 to 16, cultured from human corneal endothelial progenitor cells in the presence of less than 1 ng / mL of EGF.

18. Human functional corneal endothelial cells according to any one of claims 1 to 17, cultured from human corneal endothelial progenitor cells in the presence of EGF, but only within the first seven days of each passage.

19. Human functional corneal endothelial cells according to any one of claims 1 to 16, cultured from human corneal endothelial progenitor cells in the absence of EGF.

20. A cell population comprising human functional corneal endothelial cells according to any one of claims 1 to 19.

21. Functional cultured human corneal endothelial cells that express one or more of ATP1A1, AE2, ZO-1, NBCe1, NHE1, MCT4, SLC9A1, SLC4A11, SLC44A1, SLC2A13, SLC12A1, SLC25A42, SLC25A18, SLC4A1AP, or SLC16A2, and that can induce human corneal endothelial functional characteristics when injected into the anterior chamber of a human eye.

22. Functional cultured human corneal endothelial cells according to claim 21, expressing one or more of ATP1A1, AE2, ZO-1, NBCe1, NHE1, MCT4, SLC4A11, or SLC25A42.

23. Functional cultured human corneal endothelial cells according to claim 21, expressing one or more of SLC9A1, SLC4A11, SLC44A1, SLC2A13, SLC12A1, SLC25A42, SLC25A18, SLC4A1AP, or SLC16A2.

24. Functional cultured human corneal endothelial cells according to any one of claims 21 to 23, wherein one or more of the following are expressed in the mitochondria of the functional cultured human corneal endothelial cells: citrate synthase (CS), aconitase 2 (ACO2), isocitrate dehydrogenase 2 (IDH2), malate dehydrogenase 2 (MDH2), malate enzyme 3 (ME3), ACS1, acetyl-CoA acetyltransferase 1 (ACAT1), pyruvate dehydrogenase (PDH), BCAT2, or branched-chain keto acid dehydrogenase 2 (BCKDH2).

25. Functional cultured human corneal endothelial cells according to any one of claims 21 to 24, wherein ATP citrate lyase (ACLY), aconitase 1 (ACO1), isocitrate dehydrogenase 1 (IDH1), malate dehydrogenase 1 (MDH1), malate enzyme 1 (ME1), ACSS2, acetyl-CoA acetyltransferase 2 (ACAT2), or lactate dehydrogenase (LDH) are not expressed or are hardly expressed in the functional cultured human corneal endothelial cells.

26. Functional cultured human corneal endothelial cells according to any one of claims 21 to 25, expressing one or more of aquaporin 1 (AQP-1) or bicarbonate anhydrase 5B (CA5B).

27. ​​A cell population comprising functional cultured human corneal endothelial cells according to any one of claims 21 to 26.

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