Method for preparing high-purity corneal endothelial cell population

Abstract

One aspect of the present invention relates to a method for preparing a high-purity corneal endothelial cell population. The method for preparing a high-purity corneal endothelial cell population according to one aspect can not only increase the efficiency of corneal endothelial cell differentiation from stem cells, but can also improve the function, survival rate, and purity of corneal endothelial cells directly differentiated from stem cells.

Description

Method for producing high-purity corneal endothelial cell population

[0001] This relates to a method for producing a high-purity corneal endothelial cell population.

[0002] The cornea is the outermost structure of the eye and is one of the primary organs responsible for refracting light. It is composed of six layers: the corneal epithelium, Bowman's layer, corneal stroma, Dua's layer, Descemet's membrane, and the corneal endothelium.

[0003] The corneal endothelium is a single layer of hexagonal cells on the posterior surface of the cornea and contains physiological ion pumps. The average density of human corneal endothelial cells (CECs) at birth is approximately 5,000 cells / mm 2 , which is known as mitotic potential. However, because the mitotic potential is limited, the total cell number decreases with age.

[0004] When corneal endothelial cells (CECs) are damaged, the cornea swells to several times its normal thickness, becomes opaque, and loses its function as the cells repair themselves through cell expansion and migration rather than mitosis. The damage is permanent.

[0005] Currently, the established treatment for corneal endothelial damage is corneal transplantation. It can be repaired by transplanting donor tissue through penetrating keratoplasty (PK) or lamellar keratoplasty.

[0006] However, given the critical shortage of corneal donors, the development of efficient methods to induce stem cell differentiation into corneal endothelial cells is urgently needed to overcome this shortage. Furthermore, existing methods for directly inducing differentiation into corneal endothelial cells are time-consuming, require complex processes, are expensive, and have low purity of differentiated cells, limiting their practicality. Therefore, the development of novel methods capable of efficiently isolating and differentiating high-purity corneal endothelial cells is urgently needed.

[0007] Accordingly, the inventors of the present invention have developed a method for directly inducing differentiation of stem cells into corneal endothelial cells and producing high-purity corneal endothelial cells. Corneal endothelial cells differentiated through the above method exhibit gene expression patterns similar to those of human-derived corneal endothelial cells and can be utilized as an alternative source for corneal endothelial cell transplantation. In particular, the inventors of the present invention intend to provide a method for increasing the efficiency of corneal endothelial cell differentiation by introducing a wash-out method into the above manufacturing method, thereby improving not only the purity but also the function and viability of the ultimately obtained corneal endothelial cells.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] Korean Patent No. 10-2618788

[0011] One aspect provides a method for producing a high-purity corneal endothelial cell population, comprising the steps of: inducing differentiation of stem cells into corneal endothelial cells in a medium containing a ROCK (Rho-associated protein kinase) inhibitor; washing out the surface of the culture vessel with a washing solution to remove non-adherent cells on the surface of the culture vessel when the ratio of cells adhering to the surface of the culture vessel reaches a target attachment ratio; and obtaining the adherent cells remaining on the surface of the culture vessel.

[0012] Another aspect is to provide corneal endothelial cells manufactured by the above manufacturing method.

[0013] Another aspect is to provide a pharmaceutical composition for preventing or treating corneal endothelial diseases comprising the corneal endothelial cells.

[0014] Another aspect provides a use of the corneal endothelial cells for preventing or treating corneal endothelial disease.

[0015] Another aspect provides the use of said corneal endothelial cells for the manufacture of a pharmaceutical preparation for the prevention or treatment of corneal endothelial disease.

[0016] Another aspect provides a method for preventing or treating corneal endothelial disease comprising administering an effective amount of the corneal endothelial cells to a subject in need thereof.

[0017] A method for producing a high-purity corneal endothelial cell population is provided, comprising: a step of inducing differentiation of stem cells into corneal endothelial cells in a medium containing a ROCK (Rho-associated protein kinase) inhibitor; a step of washing out the surface of the culture vessel with a washing solution to remove non-adherent cells on the surface of the culture vessel when the ratio of cells adhering to the surface of the culture vessel reaches a target attachment ratio; and a step of obtaining the adherent cells remaining on the surface of the culture vessel.

[0018] The above term "stem cell" may mean a totipotent cell that can differentiate into all types of cells or a pluripotent cell that can differentiate into multiple types of cells, and a stem cell is an undifferentiated cell that can differentiate into a cell of a specific tissue.

[0019] In one specific example, the stem cells may be one or more selected from the group consisting of embryonic stem cells (ESCs), adult stem cells, and human induced pluripotent stem cells (iPSCs).

[0020] The embryonic stem cells above may be cultured in vitro by extracting the inner cell mass from a blastocyst embryo just before the fertilized egg is implanted in the mother's uterus, and the adult stem cells may be undifferentiated cells that exist in extremely small quantities in each tissue of the body and may be cells that replace dead cells or damaged tissues. The induced pluripotent stem cells (iPSCs) above may be cells that have been induced to have pluripotency like embryonic stem cells by injecting differentiation-related genes into differentiated somatic cells to return them to the cell stage before differentiation.

[0021] The term "corneal endothelial cell (CEC)" above may refer to a cell that constitutes the corneal endothelium. Specifically, the corneal endothelial cell is a single layer of cells located at the innermost layer of the cornea and plays an important role in maintaining the transparency and water balance of the eye. Corneal endothelial cells use ion pumps, particularly Na+ / K+ ATPase, to move water from the stroma to the aqueous humor to control excessive water inflow into the cornea and maintain corneal transparency.

[0022] The above term "ROCK (Rho-associated protein kinase) inhibitor" refers to a substance that inhibits Rho kinase (ROCK), and ROCK inhibitor can be used interchangeably with ROCK inhibitor.

[0023] In one specific example, the ROCK inhibitor is fasudil, N-(4-pyridinyl)-4β-[(R)-1-aminoethyl]cyclohexane-1αcarbamide (Y-27632), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepine (H-1152), 4β-[(1R)-1-aminoethyl]-N-(4-pyridyl)benzene-1zecarbamide (Wf-536), N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4PER(R)-1-aminoethyl]cyclohexane-1rohecarbamide (Y-30141), It may be at least one selected from the group consisting of N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide (GSK269962A), and N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A).

[0024] The above term "differentiation" means a phenomenon in which cells become specialized in structure or function while growing through division and proliferation, that is, cells and tissues of living organisms change in form or function to perform their respective assigned tasks.

[0025] In one specific example, the stem cells can be directly induced to differentiate into corneal endothelial cells without going through neural crest cells (NCC).

[0026] In one embodiment, the stem cells may be induced pluripotent stem cells.

[0027] The above term "neural crest cell (NCC)" may refer to a cell that is generated at the boundary between the neural tube and the epidermal ectoderm during the formation of the central nervous system during embryonic development and migrates to various regions within the body through epithelial-mesenchymal transition (EMT).

[0028] In one specific example, the corneal endothelial cells may be differentiated in a medium that directly induces differentiation of the stem cells into corneal endothelial cells, and specifically, the medium may include the ROCK inhibitor and may additionally include a component suitable for inducing direct differentiation of the stem cells into corneal endothelial cells.

[0029] In one specific example, the medium that directly induces differentiation of the stem cells into corneal endothelial cells may include at least one selected from the group consisting of a ROCK inhibitor, a TGF-beta inhibitor, an insulin-transferin-selenium (ITS), ascorbic acid, calcium chloride (CaCl2), and epidermal growth factor (EGF).

[0030] In one embodiment, the medium that directly induces differentiation of the stem cells into corneal endothelial cells may include human EGF, insulin-transferrin-selenium, CaCl2, 2-phosphate ascorbic acid, SB431542, ROCK inhibitor H-1152, and fasudil.

[0031] The above term "culture vessel" generally refers to a cell culture plate used for cell culture or biochemical experiments, but is not particularly limited thereto, and may include, for example, a dish, a flask, a multi-well plate, etc.

[0032] In one specific example, the surface of the culture vessel may be modified with an extracellular matrix (ECM).

[0033] The term "coating" above may refer to forming a new layer of a certain thickness by forming a film with a specific substance on the target surface. The target surface and the modifying substance may be coated through various chemical bonds, such as ionic bonds, covalent bonds, and hydrogen bonds. When modifying the surface of a culture vessel with an extracellular matrix, the extracellular matrix may form a sealed layer that completely surrounds the surface of the culture vessel, or it may form a partially sealed layer.

[0034] In one specific example, the degree of adhesion to the culture vessel surface may vary depending on the degree of differentiation of the stem cells into corneal endothelial cells. Specifically, the degree of adhesion to the culture vessel surface may increase as the stem cells differentiate more into corneal endothelial cells. This is because, during the differentiation process, the expression of cell adhesion proteins (e.g., N-cadherin, fibronectin, laminin, etc.) and extracellular matrix proteins increases, thereby strengthening the interaction between the cells and the culture vessel surface. In addition, undifferentiated cells have relatively low adhesiveness and are likely to easily fall off the culture vessel, whereas, as differentiation progresses, cell-to-cell bonds are strengthened and the expression of adhesive proteins increases, allowing for more stable attachment to the culture vessel. This characteristic can be an important indicator for evaluating the maturity of corneal endothelial cells.

[0035] In one specific example, the extracellular matrix may include at least one selected from the group consisting of vitronectin, collagen, fibronectin, laminin, gelatin, matrigel, hyaluronic acid, polylysine, and heparan sulfate proteoglycan, and preferably may be vitronectin.

[0036] In one specific example, the washing may be performed 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

[0037] In one specific example, the washing may be performed 1 to 5 times, preferably 2 times.

[0038] In one specific example, the washing may be performed with a washing liquid.

[0039] The term "washing solution" refers to a solution used to remove impurities, residual substances, proteins, foreign substances, etc. from cells or their surfaces during cell culture, tissue processing, and experimental processes. A washing solution is a solution designed to effectively remove impurities without damaging cells, while maintaining the structural stability of cells by controlling osmotic pressure and pH.

[0040] In one specific example, the cleaning solution may be a buffer.

[0041] The above term "buffer" refers to a solution designed to maintain cell stability by minimizing pH changes, and can contribute to preventing cell damage caused by external stimuli during cell washing and creating an optimal cell culture environment.

[0042] In one specific example, the washing solution may include at least one selected from the group consisting of phosphate-buffered saline (PBS), distilled water, normal saline, HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer solution, Bicine (N,N-Bis(2-hydroxyethyl) glycine) buffer solution, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, TES (NTris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffer solution, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) buffer solution, and mixtures thereof, and preferably may be phosphate-buffered saline.

[0043] In one embodiment, the washing may be performed at Endo Day 3, 10, and 17, when the time of first replacing the medium that directly induces differentiation of the stem cells into corneal endothelial cells is defined as Endo Day 0.

[0044] In one specific example, the washing may be performed at a point in time when the ratio of cells attached to the culture vessel surface reaches a target attachment rate.

[0045] In one specific example, the target attachment rate may mean the ratio of the cell density attached to the surface of the culture vessel to the cell seeding density.

[0046] The above term "seeding" refers to the process of dividing cells at a certain density into a culture vessel (plate, dish, flask, etc.) during the cell culture process to induce attachment and proliferation.

[0047] In one specific example, the cell seeding density is 1×10 3 5×10 4 cells / cm 2 , 2×10 3 5×10 4 cells / cm 2 , 3×10 3 5×10 4 cells / cm 2 , 4×10 3 5×10 4 cells / cm 2 , 1×10 3 4×10 4 cells / cm 2 , 1×10 3 3×10 4 cells / cm 2 , 1×10 3 2×10 4 cells / cm 2 It could be.

[0048] In one specific example, the target attachment rate may be a ratio of the cell density attached to the surface of a culture vessel to the cell seeding density, and may be calculated by the following mathematical formula 1.

[0049] [Mathematical Formula 1]

[0050] Target attachment rate (%) = (attached cell density (cells / cm²)) / (cell seeding density (cells / cm²)) × 100

[0051] In one specific example, the target adhesion rate may be 40% to 60%, 45% to 60%, 50% to 60%, 40% to 55% or 40% to 50%, and preferably 50%.

[0052] If the target attachment rate falls below 40%, cells may be removed before they fully attach, delaying their differentiation into corneal endothelial cells. Furthermore, low seeding density may impede cell growth and differentiation. This reduces the purity and culture efficiency of the resulting cells, requiring additional culture time to obtain high-purity corneal endothelial cells and potentially reducing overall productivity.

[0053] On the other hand, if the target attachment rate exceeds 60%, attachment of undifferentiated cells that should be filtered out may occur, which may ultimately make it difficult to secure corneal endothelial cells of uniform quality and may reduce the functionality of corneal endothelial cells.

[0054] In one specific example, after the step of removing non-adherent cells on the surface of the culture vessel, the step of subculturing the adherent cells on the surface of the culture vessel may be further included.

[0055] In one embodiment, the step of subculturing the adherent cells on the surface of the culture vessel may be performed whenever the cell density within the culture vessel increases to a certain level or higher, resulting in a shortage of nutrients and space. A person skilled in the art can appropriately control the timing of subculturing by considering the cell growth rate and culture conditions. Specifically, the subculturing may be performed when the cell confluence within the culture vessel reaches 90% or higher.

[0056]

[0057] Another aspect provides corneal endothelial cells manufactured by the above manufacturing method.

[0058] In one specific example, the corneal endothelial cells may have reduced expression of stem cell-specific genes.

[0059] The above "stem cell-specific genes" are genes that regulate the self-renewal and pluripotency (or multipotency) of stem cells, and play a key role in the maintenance of the undifferentiated state and differentiation process of stem cells.

[0060] In one specific example, the stem cell-specific gene may be one or more selected from the group consisting of NANOG, OCT4, PODXL, ESRG, and CNMD.

[0061] In one embodiment, corneal endothelial cells manufactured by the manufacturing method may have reduced expression of one or more genes selected from the group consisting of NANOG, OCT4, PODXL, ESRG, and CNMD.

[0062] In one specific example, the corneal endothelial cells may have increased expression of corneal endothelial cell-specific genes.

[0063] The above "corneal endothelial cell-specific genes" are genes that play a crucial role in the development, maintenance of function, and homeostasis of corneal endothelial cells. These genes are specifically expressed in corneal endothelial cells and are involved in cell survival, ion and water regulation, maintenance of cell adhesion, and proliferation.

[0064] In one specific example, the corneal endothelial cell-specific gene may be one or more selected from the group consisting of TJP1, CDH2, ATP1A1, ALCAM, PRDX6, COL8A1, and SLC25A11.

[0065] In one embodiment, corneal endothelial cells manufactured by the above manufacturing method may have increased expression of one or more genes selected from the group consisting of TJP1, CDH2, ATP1A1, ALCAM, PRDX6, COL8A1, and SLC25A11.

[0066] In one specific example, the corneal endothelial cells may have reduced expression of stem cell-specific genes and increased expression of corneal endothelial cell-specific genes.

[0067] In one specific example, the stem cell-specific gene may include at least one selected from the group consisting of NANOG, OCT4, PODXL, ESRG, and CNMD, and the corneal endothelial cell-specific gene may include at least one selected from the group consisting of TJP1, CDH2, ATP1A1, ALCAM, PRDX6, COL8A1, and SLC25A11.

[0068] In one specific example, the corneal endothelial cells may have reduced expression of a gene associated with abnormal cell proliferation, for example, the gene associated with abnormal cell proliferation may be WNT6 or MIR205HG.

[0069] In one specific example, the corneal endothelial cells may have a reduced expression level of one or more genes selected from the group consisting of WNT6 and MIR205HG.

[0070] In one embodiment, it was confirmed that by performing washing during the process of directly inducing differentiation of stem cells into corneal endothelial cells, the survival rate of the corneal endothelial cells ultimately obtained increased.

[0071] In one embodiment, when the corneal endothelial cells were transplanted into a rabbit model in which corneal endothelial cell damage or deficiency was induced, it was confirmed that the corneal endothelial cells stably engrafted without being attacked by the host immune system and were maintained within the host cornea without an immune rejection response.

[0072] In one embodiment, it was confirmed that the corneal endothelial cells exhibit high activity in intercellular signal transmission, energy metabolism, protein synthesis, structural stability, and maintenance of cell function.

[0073]

[0074] Another aspect provides a pharmaceutical composition for preventing or treating corneal endothelial diseases comprising the corneal endothelial cells.

[0075] The above "Corneal Endothelial Disease" is a group of diseases caused by the decline or loss of corneal endothelial cells, which can lead to corneal edema, clouding, and decreased vision. The disease is divided into congenital and acquired causes, with representative examples being Fuchs corneal endothelial dystrophy and congenital endothelial dystrophy. It can also be secondary to cataract surgery, ocular trauma, glaucoma, inflammation, drug toxicity, etc. Initially, it starts with mild vision loss, but as it progresses, the cornea can become severely swollen and lose its transparency, causing serious vision impairment. Depending on the severity of the symptoms, treatments include osmotic agents, therapeutic contact lenses, and corneal endothelial transplantation (DMEK, DSAEK). Early detection and appropriate management of the disease are important for maintaining corneal transparency and vision.

[0076] In one specific example, the corneal endothelial disease may be at least one selected from the group consisting of Fuchs' Endothelial Dystrophy (FED), Congenital Hereditary Endothelial Dystrophy (CHED), Posterior Polymorphous Corneal Dystrophy (PPCD), Bullous Keratopathy (BK), Pseudophakic Bullous Keratopathy (PBK) after cataract surgery, Secondary Corneal Edema, Traumatic Endothelial Damage, Inflammatory Endothelial Damage, and Graft Failure after Corneal Transplantation.

[0077] The term "prevention" above refers to a method of partially or completely delaying or preventing the onset or recurrence of a disease, disorder, or its associated symptoms, preventing the acquisition or reacquisition of a disease or disorder, or reducing the risk of acquiring a disease or disorder. For example, the term "prevention" refers to any action that inhibits or delays the development of corneal endothelial disease by administering a composition according to the present invention.

[0078] The term "treatment" above may refer to the healing of corneal endothelial disorders, etc., in a shorter period of time compared to natural healing. The treatment may include improvement and / or alleviation of corneal endothelial disorders. Furthermore, the treatment may refer to the healing and / or recovery of symptoms resulting from the corneal endothelial disorders.

[0079] In one specific example, the pharmaceutical composition may contain corneal endothelial cells produced by the production method according to the present invention as an active ingredient.

[0080] The above pharmaceutical composition may contain, in addition to the above-described active ingredient, a pharmaceutically acceptable carrier, excipient or diluent for administration.

[0081] The above "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the injected compound. The type of carrier usable in the present invention is not particularly limited, and any pharmaceutically acceptable carrier commonly used in the art may be used.

[0082] Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, etc. These may be used alone or in combination of two or more. The carrier may include a non-naturally occurring carrier. In addition, if necessary, other conventional additives such as antioxidants, buffers, and / or bacteriostatic agents may be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added and used in the form of injectable formulations such as aqueous solutions, suspensions, and emulsions.

[0083] In one embodiment, the pharmaceutical composition may also be formulated as an ophthalmic composition, such as an eye drop or ophthalmic ointment. These forms may include any ophthalmic preparation for topical administration to the eye used in ophthalmology. Eye drops are prepared by dissolving the active ingredient in a sterile aqueous solution, such as saline or a buffer. Eye drops may be provided as a powder composition to be dissolved prior to use, or may be formulated with a powder composition to be dissolved prior to use. Ophthalmic ointments may be prepared by mixing the active ingredient into an ointment base.

[0084] The above pharmaceutical composition can be administered in a pharmaceutically effective amount.

[0085] The above term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer at a reasonable benefit / risk ratio applicable to medical use, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, activity of the drug, weight, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field. At this time, the content of the effective ingredient included in the pharmaceutical composition may be 0.0001 wt % to 10 wt %, specifically 0.001 wt % to 1 wt %, based on the total weight of the composition.

[0086] In one embodiment, the corneal endothelial cells were administered to the anterior chamber of the cornea in a rabbit model in which corneal endothelial cell damage or deficiency was induced, and the effect of regenerating the damaged corneal endothelium was confirmed. In addition, corneal edema was also significantly alleviated, and a reduction in corneal thickness and restoration of transparency were confirmed (see Example 6).

[0087] In one specific example, the pharmaceutical composition may further include corneal endothelial cell-derived exosomes (EVs) and functional substances, and the pharmaceutical composition may further include a pharmaceutical composition for preventing or treating corneal endothelial diseases other than the corneal endothelial cells.

[0088] The above pharmaceutical composition may be provided mixed with a pharmaceutical composition for preventing or treating other corneal endothelial diseases, and the other pharmaceutical composition for preventing or treating corneal endothelial diseases may be a conventionally known pharmaceutical composition or a newly developed pharmaceutical composition.

[0089] When the above pharmaceutical composition further comprises a pharmaceutical composition for the prevention or treatment of other corneal endothelial diseases, it is important to mix the two in an amount that can achieve the maximum effect with the minimum amount without causing side effects, which can be easily determined by a person skilled in the art.

[0090] When the pharmaceutical composition further comprises a pharmaceutical composition for the prevention or treatment of another corneal endothelial disease, a synergistic effect may be exhibited in which the effect of preventing or treating the corneal endothelial disease, such as the effect of regenerating damaged corneal endothelial cells or the effect of increasing corneal endothelial thickness and corneal endothelial cell layer thickness, is more significant than when the pharmaceutical composition comprises only the corneal endothelial cells as an active ingredient.

[0091] In one specific embodiment, the pharmaceutical composition may be administered alone or in combination with other pharmaceutical compositions for the prevention or treatment of retinal diseases. Specifically, the pharmaceutical composition may be administered in combination with other known pharmaceutical compositions that have a preventive or therapeutic effect on retinal diseases, and may be administered simultaneously, separately, or sequentially, or in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that achieves maximum effect with the minimum amount without causing side effects, and this can be readily determined by those skilled in the art.

[0092] The above “combination administration” means administering two or more drugs simultaneously or during the same treatment period.

[0093] When the pharmaceutical composition is administered in combination with another pharmaceutical composition for the prevention or treatment of corneal endothelial diseases, a synergistic effect may be exhibited in which the effect of preventing or treating corneal endothelial diseases is more pronounced than when the pharmaceutical composition is administered alone, such as the effect of regenerating damaged corneal endothelial cells or the effect of increasing corneal endothelial thickness and corneal endothelial cell layer cell thickness.

[0094] In one embodiment, the corneal endothelial cells can be administered to the subject in any amount or number, e.g., an effective amount, that results in a detectable therapeutic benefit to the subject. The cells can be administered to the subject in an absolute or relative number of cells, specifically the corneal endothelial cells can be administered in a number of about, at least about, or at most about 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10, 5 x 10 10 , or 1 x 10 11 Can be administered to a subject as a dog, or said corneal endothelial cell-derived exosomes are about, at least about, or at most about 1 x 10 5 , 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 , 5 x 10 8 , 1 x 10 9 , 5 x 10 9 , 1 x 10 10 , 5 x 10 10 , or 1 x 10 11 It can be administered to individuals as a dog.

[0095] In one specific example, the dosage of the pharmaceutical composition may be 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. However, the dosage may be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The number of administrations may be once or twice or more within the range of clinically acceptable side effects, and the administration site may be one or two or more sites. For animals other than humans, the same dosage per kg as for humans may be administered, or the dosage may be converted to the above dosage based on the volume ratio (e.g., average value) of the organs (heart, etc.) of the target animal and humans.

[0096] The pharmaceutical composition may be administered orally or parenterally, and when the pharmaceutical composition is administered parenterally, it may be administered by external application to the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, subretinal injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection, percutaneous injection, intranasal injection, intraenteric injection, local injection, sublingual injection, intrarectal injection, ventricular injection, intracranial microinjection, intracontrast injection, intrathecal injection, spinal facet joint injection, or intrathoracic injection, and specifically, it may be administered into the anterior chamber of the eye.

[0097] A pharmaceutical composition according to one specific example comprises the corneal endothelial cells as an active ingredient, and the pharmaceutical composition can be used for the purpose of preventing or treating various corneal endothelial diseases by performing a role of replacing or protecting damaged corneal endothelial cells.

[0098] According to the method for producing a high-purity corneal endothelial cell population according to a daily aspect, not only can the efficiency of corneal endothelial cell differentiation from stem cells be increased, but also the function, survival rate, and purity of corneal endothelial cells directly differentiated from stem cells can be improved.

[0099] Figure 1 is a graph comparing the PE-CD166 fluorescence signal intensity over time (D14, D21, D28) under No Wash-Out and Wash-Out conditions.

[0100] Figure 2 is a graph showing changes in the expression of OCT4, NANOG, PODXL, CDH2, and COL8A1 genes over time (D7, D10, D14, D21, D28) according to the differentiation process from iPSCs.

[0101] Figure 3 is a heatmap analyzing the expression levels of CEC-specific gene markers in the ACE1, ACE2, ACE2W14, ACE2W21, ACE2W.DM14, primary hCEC-1, and primary hCEC-2 groups.

[0102] Figure 4 is a heatmap analyzing the low-quality CEC-specific gene expression levels of ACE1, ACE2, ACE2W14, ACE2W21, ACE2W.DM14, primary hCEC-1, and primary hCEC-2 groups.

[0103] Figures 5a and 5b are UMAP DimPlot graphs showing the gene cluster labels of the ACE1, ACE2, ACE2W14, ACE2W21, ACE2W.DM14, primary hCEC-1, and primary hCEC-2 groups.

[0104] Figure 6 is a UMAP that compares and analyzes the expression patterns of representative CEC-specific genes, TJP1, CDH2, ATP1A1, ALCAM, PRDX6, and SLC25A11, in the ACE1, ACE2, ACE2W14, ACE2W21, ACE2W.DM14, primary hCEC-1, and primary hCEC-2 groups.

[0105] Figure 7 is a UMAP comparing and analyzing the expression patterns of iPSC-specific genes NANOG, OCT4, ESRG, and CNMD in the ACE1, ACE2, ACE2W14, ACE2W21, ACE2W.DM14, primary hCEC-1, and primary hCEC-2 groups.

[0106] Figure 8 is a violin plot graph comparing and analyzing the expression patterns of CEC-specific genes ALCAM, CDH2, PRDX6, and COL8A1 in the ACE2, ACE2W14, and ACE2W21 groups.

[0107] Figure 9 is a violin plot graph comparing the expression patterns of CEC-specific genes ALCAM, TJP1, ATP1A1, PRDX6, CDH2, and COL8A1 between the ACE1 and ACE2W groups.

[0108] Figure 10 is a graph of the expression levels of WNT6 and MIR205HG genes in the ACE1, ACE2, ACE2W, primary hCEC-1, and primary hCEC-2 groups.

[0109] Figure 11 shows the results of the Cellular Component analysis of the ACE2W21 group.

[0110] Figure 12 shows the results of molecular function analysis of the ACE2W21 group.

[0111] Figure 13 is an image (left) and graph (right) showing the changes in corneal transparency and thickness over time in the group transplanted with ACE2W (Group 1) and the control group (Group 2) without ACE2W transplantation in a corneal endothelial decompensation (CED) model, in which the corneal endothelial cells of a rabbit were selectively removed by scraping them in a circular pattern. Figure 14 is an image showing the results of an immunohistochemical analysis to detect a human-specific marker (STEM121) and a CEC marker (N-cadherin) in the animal cornea at 1 and 16 weeks after transplantation of ACE2W in the corneal endothelial decompensation (CED) model.

[0112] Figure 15 is an immunofluorescent tissue section image showing the absence of immunoreactivity of CD4, CD8 T cells and CD11b macrophages / neutrophils in the ACE2W transplant group.

[0113] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0114]

[0115] Example 1. Method for producing high-purity CEC from stem cells

[0116] Human induced pluripotent stem cells (hiPSCs) were provided by Ipcell Co., Ltd. To differentiate hiPSCs directly into neural crest cells (CECs) without undergoing differentiation into NCCs, differentiation was initiated by seeding hiPSCs on vitronectin-modified plates. After culturing iPSCs in E8 medium (Life Technologies) for 4–5 days, the cell seeding density was 5,000 cells / cm. 2 The CEC differentiation step was performed by setting it to .

[0117] CEC differentiation medium contained 10 ng / ml human EGF, 1% insulin-transferrin-selenium, and 0.2 mg / mL CaCl 2, Human endothelial-SFM medium (primary CEC differentiation medium) containing 0.02 mg / mL 2-phosphate ascorbic acid, 1 μM SB431542, 2.5 μM ROCK inhibitor H-1152, and 10 μM fasudil was used.

[0118] At this time, the day on which the differentiation medium was first replaced was defined as Endo Day 0, and culture was continued thereafter by replacing it with fresh differentiation medium every day.

[0119] On Endo Day 3, cell density reached approximately 90% confluence, at which point the first subculture was performed. After subculture, the cell seeding density was maintained at 5,000 cells / cm² to continue CEC differentiation.

[0120] During the CEC differentiation process, Wash-Out was performed at Endo Days 3, 10, and 17. Wash-Out is a cell separation method that utilizes the difference in adhesive strength between differentiated CECs and relatively less differentiated stem cells, and utilizes the characteristic that differentiated CECs adhere more quickly to the surface of a vitronectin-coated culture vessel than immature CECs or stem cells.

[0121] When approximately 50% of the cells were attached to the bottom of the culture dish, the remaining 50% of floating cells were removed by washing once with PBS.

[0122] For cell seeding, cells attached to the culture vessel surface were recovered from the vessel, collected into a cell pellet, and gently loosened to form a uniform pellet. During this process, a cell strainer with 20 μm pores was used to prevent cell cluster formation and maintain single-cell status. Afterwards, cell counts were accurately measured using Trypan Blue staining, and culture was continued at a seeding density of 5,000 cells / cm².

[0123] Specifically, considering that approximately 50% of cells would be removed by the Wash-Out, the number of cells to be added to the culture vessel at the initial cell seeding stage during subculture was set to 10,000 cells / cm², and culture was continued in a new CEC differentiation medium.

[0124] After cell seeding, whether approximately 50% of the cells were attached to the bottom of the culture vessel was confirmed by observing the ratio of attached cells to non-attached floating cells using a microscope for 3 to 25 minutes after the start of culture and qualitatively evaluating this.

[0125] To ensure experimental accuracy, variables that could affect cell attachment time were minimized. Cells were gently shaken to ensure even distribution on the surface of the culture vessel, and the incubator's position and height were aligned to maintain consistent conditions. Furthermore, to prevent differences in attachment rates due to time differences, only two culture vessels were handled at a time.

[0126] The above experimental results confirmed that cells less differentiated into CECs tended to have slower attachment rates. While it took more than 20 minutes for Endo Day 3 cells to reach 50% attachment, this time was shortened to less than 5 minutes for Endo Day 17 cells. Furthermore, a CEC-specific hexagonal morphology began to be observed from Endo Day 10 onwards, and it was confirmed that stable culture was possible even with medium replacement every other day.

[0127]

[0128] Comparative Example 1. Method for differentiating stem cells into neural crest cells (NCCs) and then into CECs.

[0129] Human induced pluripotent stem cells (hiPSCs) were provided by Ipcell Co., Ltd. The hiPSCs were cultured in E8 medium on vitronectin-modified plates under serum-free, feeder-free conditions.

[0130] To differentiate hiPSCs into neural crest cells (NCCs), hiPSCs were plated at low density on the surface of vitronectin-modified plates. After undifferentiated hiPSCs reached approximately 30-40% confluency, the first medium was added to the plates to induce differentiation into neural crest cells.

[0131] More specifically, the medium was first replaced with neural crest cell induction E8 medium (Life Technologies) containing 10 μM retinoic acid (Sigma-Aldrich), 5 μM CHIR99021 (Biogems Korea) and cultured for 1 day. Then, the medium was again replaced with E8 medium containing 5 μM retinoic acid, 10 μM SB431542 (Selleckchem), 5 μM CHIR99021, and 500 nM LDN193189 (Selleckchem) and cultured for 1 day. After that, the medium was again replaced with E8 medium containing 5 μM retinoic acid, 10 μM SB431542, 5 μM CHIR99021, and 100 nM BGJ398 (Selleckchem) and cultured for 1 day. Thereafter, differentiation into neural crest cells was induced by replacing the medium daily with neurobasal medium with neural induction supplements (Life Technologies) for 10 to 14 days.

[0132] And for differentiation of the above-derived NCC into corneal endothelial cells (CEC), iPSC-derived NCC were dissociated using TrypLE™ Express (Life Technologies). Then, they were seeded at a low cell density (20-30%) in vitronectin-modified 35 mm plates. Then, differentiation into corneal endothelial cells was induced for 14 to 28 days using human endothelial-SFM medium containing 10 ng / ml hEGF (Life Technologies), 1% insulin-transferrin-selenium (Life Technologies), 0.2 mg / mL CaCl2 (Sigma-Aldrich), 0.02 mg / mL 2-phosphate ascorbic acid, 1 μM SB431542 (Selleckchem), 2.5 μM ROCK inhibitor H-1152 (Tocris, Abington, UK), and 10 μM Fasudil (Stemcell Technologies). And when the cells differentiated into CECs reached a cell density of about 80 to 90%, they were subcultured to induce differentiation.

[0133]

[0134] Comparative Example 2. Direct differentiation method from stem cells to CECs

[0135] Human induced pluripotent stem cells (hiPSCs) were provided by Ipcell Co., Ltd. To differentiate hiPSCs directly into CECs without undergoing differentiation into neural crest cells (NCCs), hiPSCs were plated at low density on the surface of vitronectin-modified plates. After undifferentiated hiPSCs reached approximately 30-40% cell density, a culture medium suitable for direct CEC differentiation was added to the plates to induce differentiation.

[0136] Specifically, iPSCs were first cultured for 4–5 days using E8 medium (Life Technologies). Subsequently, they were seeded at a low cell density (20–30%) onto 35 mm plates modified with vitronectin. Afterwards, differentiation into CECs was induced for 10 days in human endothelial-SFM medium containing 10 ng / ml human Epidermal growth factor (hEGF) (Life Technologies), 1% insulin-transferrin-selenium (Life Technologies), 0.2 mg / mL CaCl2 (Sigma-Aldrich), 0.02 mg / mL 2-phosphate ascorbic acid, 1 μM SB431542 (Selleckchem), 2.5 μM ROCK inhibitor H-1152 (Tocris, Abington, UK), and 10 μM Fasudil (Stemcell Technologies) at 37°C with daily medium replacement. From the 14th day of differentiation induction, differentiation into CECs was induced by replacing the medium every other day.

[0137]

[0138] Comparative Example 3. Culture of human primary corneal endothelial cells (hCEC)

[0139] Human primary CECs were isolated from two different donor corneas provided by Eversight (Chicago, IL, USA), cultured in vitro, and then passaged twice. The cells were seeded on a VTN-coated culture dish and cultured in DMEM supplemented with 5% fetal bovine serum (FBS; Gibco), 1% Insulin-Transferrin-Selenium (ITS; Life Technologies), 10 μM Y27632, 0.02 mg / mL ascorbic acid, and 0.02 mg / mL CaCl. 2,The cells were cultured in DMEM / F12 medium (Gibco) supplemented with 10 ng / mL hEGF and 1% antibiotic-antimycotic (Gibco). The medium was changed every 2–3 days, and the cells were used as controls (hCEC-1, hCEC-2 groups) in experiments using single-cell RNA sequencing and immunostaining.

[0140]

[0141] Experimental Example 1. FACS Analysis Using CD166 Antibody

[0142] CECs obtained by performing wash-out in the process of directly inducing differentiation of stem cells into CECs by the method according to Example 1 and CECs directly induced to differentiate without wash-out by the method according to Comparative Example 2 were subjected to FACS (Florescence Activated Cell Sorting) analysis using CD166 antibody.

[0143] The results of FACS analysis using the CD166 antibody are shown in Figure 1.

[0144] Figure 1 is a graph comparing the PE-CD166 fluorescence signal intensity over time (D14, D21, D28) under No Wash-Out and Wash-Out conditions.

[0145] As shown in Fig. 1, the CECs obtained by performing Wash-Out according to the method of Example 1 maintained a high CD166 positive cell ratio of 97% and 98% at 14 days (D14) and 1 day (D21), respectively, as a result of FACS analysis using CD166 antibody, and also showed a relatively high level of 74% at 28 days (D28).

[0146] On the other hand, CECs directly induced to differentiate without wash-out by the method according to Comparative Example 2 showed a CD166 positive cell ratio of 92% at 14 days, but the expression rate decreased to 62% and 56% at 21 and 28 days, respectively, over time.

[0147] This means that during the process of direct CEC differentiation induction, Wash-Out filters out undifferentiated stem cells, preventing them from continuing to proliferate within the cell population and obtaining a more pure CEC cell population.

[0148]

[0149] Experimental Example 2. Evaluation of the Efficacy of the Wash-Out Method

[0150] To evaluate the effect of wash-out on obtaining high-purity CEC, RT-qPCR analysis was performed to compare the expression levels of five genes: OCT4, NANOG, PODXL, CDH2, and COL8A1, and the results are shown in Fig. 2.

[0151] Specifically, the sequence of the RT-qPCR analysis is as follows.

[0152] First, total RNA from the iPSCs and iPSC-derived CECs was extracted using TRIzol reagent (Invitrogen), and complementary DNA was synthesized using a kit (Superscript III; Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, CA, USA) on a Step One ABI Real-Time PCR System (Applied Biosystems).

[0153] During differentiation of iPSCs into CECs, the expression levels of iPSC-specific genes OCT4, NANOG, and PODXL and CEC-specific genes CDH2 and COL8A1 were measured at different time points (D7, D10, D14, D21, and D28), and mRNA levels were normalized to GAPDH.

[0154] Figure 2 is a graph showing changes in the expression of OCT4, NANOG, PODXL, CDH2, and COL8A1 genes over time (D7, D10, D14, D21, D28) according to the differentiation process from iPSCs.

[0155] As shown in Fig. 2, the expression of OCT4, NANOG, and PODXL, which are iPSC-specific genes, decreased in the Wash-Out treatment group (D7W, D10W, D14W, D21W, D28W), confirming that the Wash-Out method contributes to suppressing stem cell characteristics and effectively inducing differentiation into CECs. On the other hand, the expression of CDH2 and COL8A1, which are CEC-specific genes, was maintained at a high level in the Wash-Out treatment group (D7W, D10W, D14W, D21W, D28W), confirming that the Wash-Out method contributes to enhancing the endothelial cell characteristics of CECs and securing highly pure mature CECs. In particular, a significant difference was found in the expression of iPSC markers between the Wash-Out group and the Non-Wash-Out group after differentiation D14, and it was confirmed that the CDH2 expression level in the Wash-Out group (D21W) significantly increased at D21.

[0156] This means that Wash-Out promotes CEC differentiation by removing immature cells and enhances endothelial cell characteristics, contributing to securing high-purity CECs.

[0157]

[0158] Experimental Example 3. Comparison of Gene Expression Between CEC Groups Using Single Cell Sequencing

[0159] Single Cell Sequencing was performed to compare the gene expression patterns of CECs (ACE2W group) obtained by performing wash-out in the process of directly inducing differentiation into CECs by the method of Example 1, CECs differentiated from iPSCs via NCCs by the method of Comparative Example 1 (ACE1 group), CECs directly differentiated from iPSCs by the method of Comparative Example 2 (ACE2 group), and two types of primary hCECs (hCEC-1, hCEC-2 groups) obtained by the method of Comparative Example 3.

[0160] Specifically, single-cell RNA-sequencing was performed using the 10X Genomics platform. The five group RNA libraries were prepared using Chromium Next GEM Single Cell 3p RNA Library v3.1 (10X Genomics), and 3' digital gene expression profiling of 5,000–20,000 individual cells per sample was performed. The samples were analyzed using Cell Ranger 7.0.1, and cell population analysis was performed using the Seurat4.3.0 R package.

[0161] A total of 36,601 genes and 89,192 cells were analyzed through the single cell RNA-sequencing.

[0162]

[0163] 3.1 Heat Map Analysis

[0164] The results of the single cell RNA-sequencing experiment of the above five groups (ACE1, ACE2, ACE2W, hCEC-1, hCEC-2) were visualized using a heat map and shown in Figures 3 and 4.

[0165] Figure 3 is a heatmap analyzing the expression levels of CEC-specific gene markers in the ACE1, ACE2, ACE2W14, ACE2W21, ACE2W.DM14, primary hCEC-1, and primary hCEC-2 groups.

[0166] Figure 4 is a heatmap analyzing the low-quality CEC-specific gene expression levels of ACE1, ACE2, ACE2W14, ACE2W21, ACE2W.DM14, primary hCEC-1, and primary hCEC-2 groups.

[0167] In this specification, low-quality CECs (corneal endothelial cells) refer to CECs that have low maturity and functional completeness, are insufficiently differentiated, have damaged morphological characteristics, or have a tendency toward fibroblast-like transformation (EMT), and exhibit gene expression characteristics that are different from those of normal mature CECs.

[0168] In this specification, genes that are selectively or excessively expressed in the low-quality CECs are termed "low-quality CEC-specific genes," and have an expression profile that reflects differentiation immaturity, functional incompleteness, or an abnormal corneal environment. Since the low-quality CEC-specific genes have low or almost no expression in mature and functionally complete CECs, the expression level of the genes can be used as an indicator for evaluating the quality of CECs. Specifically, the low-quality CEC-specific genes include CD9, ITGA5, THBS2, NT5E, MME, VMO1, etc.

[0169] As shown in Fig. 3, it was confirmed that the expression patterns of several CEC-specific genes in the ACE2W group were more similar to those in the Primary hCEC group than in the ACE2 group.

[0170] Additionally, as shown in Fig. 4, various low-quality CEC-specific genes were confirmed to be expressed more highly in the Primary hCEC group compared to the ACE1, ACE2, and ACE2W groups.

[0171] This means that the ACE2W group has gene expression characteristics more similar to the Primary hCEC group than the ACE2 group, and can be evaluated as a CEC with higher quality due to lower expression of low-quality CEC-specific gene markers.

[0172]

[0173] 3.2 UMAP (Uniform Manifold Approximation and Projection) analysis

[0174] Among the above five groups (ACE1, ACE2, ACE2W, hCEC-1, hCEC-2), the ACE2W group was subdivided based on the number of differentiation days and the type of medium. Specifically, the CECs were divided into the ACE2W14 group (CECs obtained on the 14th day of differentiation induction by applying wash-out according to Example 1), the ACE2W21 group (CECs obtained on the 21st day), and the ACE2W.DM14 group (CECs obtained using CTS-DMEM on the 14th day of differentiation induction). Afterwards, single cell RNA-sequencing was performed for each group, and the results were visualized using UMAP and are shown in Figures 5 to 7.

[0175] Figures 5a and 5b are UMAP DimPlot graphs showing the gene cluster labels of the ACE1, ACE2, ACE2W14, ACE2W21, ACE2W.DM14, primary hCEC-1, and primary hCEC-2 groups.

[0176] As shown in Figures 5a and 5b, UMAP DimPlot containing gene cluster labels was analyzed, and the distribution of gene clusters divided by group was confirmed. As a result, the overall pattern of the types and distribution of gene clusters constituting each cell group was confirmed. Specifically, compared to the control groups hCEC1 and hCEC2, the washed-out ACE2W groups (ACE2W14, ACE2W21, and ACE2W.DM14) showed more similar distribution characteristics, and among them, the ACE2W.DM14 cell group showed the highest similarity.

[0177] Figure 6 is a UMAP that compares and analyzes the expression patterns of representative CEC-specific genes, TJP1, CDH2, ATP1A1, ALCAM, PRDX6, and SLC25A11, in the ACE1, ACE2, ACE2W14, ACE2W21, ACE2W.DM14, primary hCEC-1, and primary hCEC-2 groups.

[0178] As shown in Fig. 6, gene expression analysis confirmed that representative CEC-specific genes (TJP1, CDH2, ATP1A1, ALCAM, PRDX6, SLC25A11) were clearly expressed in most cells within each ACE cell group, and showed levels similar to those of the control hCECs. Specifically, the ACE2W.DM14 group using CTS-DMEM formed the most similar cluster to the hCEC1 and hCEC2 groups, confirming that the characteristics of mature CECs were further enhanced. These results indicate that the treatment with CTS-DMEM and Wash-Out is effective in enhancing CEC maturation.

[0179] Figure 7 is a UMAP comparing and analyzing the expression patterns of iPSC-specific genes NANOG, OCT4, ESRG, and CNMD in the ACE1, ACE2, ACE2W14, ACE2W21, ACE2W.DM14, primary hCEC-1, and primary hCEC-2 groups.

[0180] As shown in Figure 7, the expression of iPSC-specific genes NANOG, OCT4, ESRG, and CNMD showed a distinct difference between the ACE2 and ACE2W cell groups. Specifically, some iPSC gene-expressing cells were observed in the ACE2 cell group, whereas they were rarely observed in the ACE2W cell group. These results indicate that the wash-out process effectively removed undifferentiated iPSCs. In addition, the above genes were hardly expressed in the control hCEC1 and hCEC2 groups, and the ACE2W.DM14 group using CTS-DMEM showed the most similar expression results to the hCEC1 and hCEC2 groups.

[0181]

[0182] 3.3 Comparison of violin plots between CEC groups

[0183] To analyze the expression patterns of CEC-specific genes ALCAM, CDH2, PRDX6, and COL8A1, violin plots were used to compare the expression between the ACE2, ACE2W14, and ACE2W21 groups, and the results are shown in Figure 8.

[0184] Figure 8 is a violin plot graph comparing and analyzing the expression patterns of CEC-specific genes ALCAM, CDH2, PRDX6, and COL8A1 in the ACE2, ACE2W14, and ACE2W21 groups.

[0185] As shown in Fig. 8, ALCAM and CDH2 showed high expression in the ACE2W14 and ACE2W21 groups, and relatively low expression in the ACE2 group. In addition, PRDX6 showed similar expression levels in the ACE2W14 and ACE2W21 groups, and showed overall higher expression than the ACE2 group. COL8A1 was barely expressed in the ACE2 group, but showed increased expression in the ACE2W14 and ACE2W21 groups. This means that the wash-out treatment and differentiation period increased the number of cells expressing CEC-specific genes and also increased the overall expression intensity of the genes, thereby affecting the enhancement of CEC characteristics.

[0186] Meanwhile, to analyze the expression patterns of CEC-specific genes ALCAM, TJP1, ATP1A1, PRDX6, CDH2, and COL8A1, a violin plot was used to compare the expression between the ACE1 and ACE2W groups, and the results are shown in Figure 9.

[0187] Figure 9 is a violin plot graph comparing the expression patterns of CEC-specific genes ALCAM, TJP1, ATP1A1, PRDX6, CDH2, and COL8A1 between the ACE1 and ACE2W groups.

[0188] As shown in Figure 9, the ACE2W group showed higher expression levels of all CEC-specific genes, ALCAM, TJP1, ATP1A1, PRDX6, CDH2, and COL8A1, compared to the ACE1 group. In particular, ALCAM and TJP1 showed significantly higher expression levels in the ACE2W group, confirming that Wash-Out treatment is effective in removing undifferentiated cells and enhancing the characteristics of mature CECs.

[0189] Furthermore, increased expression of genes such as PRDX6 and COL8A1 revealed enhanced oxidative stress regulation and ECM (extracellular matrix)-related functions in endothelial cells. In summary, the above experimental results suggest that wash-out treatment yields endothelial cells with more mature and functionally enhanced ACE2W group cells compared to ACE1 group cells.

[0190]

[0191] 3.4 Comparison of WNT6 and MIR205HG expression levels between CEC groups

[0192] The results of comparing the expression levels of genes related to cancer progression and abnormal cell states, such as WNT6 and MIR205HG, in the five groups (ACE1, ACE2, ACE2W, hCEC-1, hCEC-2) above are shown in Fig. 10. ACE2W in this experimental example used CEC (ACE2W21) obtained on the 21st day of differentiation induction by applying Wash-out according to the method of Example 1.

[0193] Figure 10 is a graph showing the expression levels of the WNT6 and MIR205HG genes in the ACE1, ACE2, ACE2W, primary hCEC-1, and primary hCEC-2 groups. ACE2W described in Figure 10 refers to ACE2W21.

[0194] As shown in Figure 10, the expression of WNT6 and MIR205HG was high in the ACE2 group, while it was confirmed that the expression was significantly low in the ACE1, ACE2W groups and the hCEC-1, hCEC-2 groups.

[0195] This suggests that the wash-out process, which directly induces differentiation of CECs and yields them, is effective in improving cell quality and function by reducing the expression of specific genes. In particular, by confirming that the wash-out process suppressed the expression of WNT6 and MIR205HG, genes associated with tumor progression or stem cell renewal, we were able to determine that the ACE2W group can be evaluated as more stable and high-quality CECs than the ACE2 group.

[0196]

[0197] Experimental Example 4. Functional Annotation Analysis

[0198] Based on the results of the single cell RNA-sequencing experiment of Experimental Example 3, functional annotation analysis was performed to analyze the gene expression pattern of the ACE2W21 group, and the results are shown in Figures 11 and 12.

[0199] Figure 11 shows the results of the Cellular Component analysis of the ACE2W21 group.

[0200] Figure 12 shows the results of molecular function analysis of the ACE2W21 group.

[0201] As shown in Figure 11, Cellular Component analysis revealed that cytoplasm, extracellular vesicles, ribosomal subunits, and mitochondria-related components were identified as major components. This suggests that the ACE2W21 group is involved in cell-to-cell interaction and secretion, protein synthesis, and energy metabolism.

[0202] Additionally, as shown in Fig. 12, in the molecular function analysis, protein binding, structural molecule activity, electron transfer activity, and primary active transmembrane transporter activity were observed at high frequencies. This indicates that the metabolic and transport capabilities were improved in the ACE2W21 group similar to those in the hCEC group, implying that intracellular stability and energy efficiency were optimized.

[0203] In particular, the markedly elevated levels of oxidoreductase activity and NADH dehydrogenase activity suggest that the ACE2W21 group contributes to energy production and metabolic processes through high mitochondrial metabolic activity. These characteristics play a crucial role in enhancing cell survival and function.

[0204] In conclusion, functional annotation analysis confirmed that the ACE2W21 group exhibits high activity in intercellular signaling, energy metabolism, protein synthesis, structural stability, and maintenance of cellular function.

[0205]

[0206] Experimental Example 5. Confirmation of the therapeutic effect of iPSC-derived CECs in the DM (Descemet's Membrane) detachment model.

[0207]

[0208] 5.1 Measurement of corneal opacity and corneal thickness

[0209] The CEC obtained by the method of Example 1 was applied to the anterior chamber of the corneal endothelial decompensation (CED) rabbit model (ID 111, ID 112) in which the corneal endothelial cells were selectively removed by scraping the corneal endothelial cells in a circular pattern, 1Х10 6 iPSC-CECs were transplanted in an amount of 150 μL. In this example, a rabbit model of corneal endothelial dysfunction was used, which was created by selectively removing endothelial cells by scraping the corneal endothelial cells of a New Zealand white rabbit (Zabio) in a circular pattern. The model was divided into a group that received ACE2W transplantation (Group 1) and a control group that did not receive ACE2W transplantation (Group 2), and the two groups were observed on day 3 (D3) and day 7 (D7) to measure corneal opacity. The results are shown in Fig. 13. The ACE2W in this experimental example used CECs (ACE2W21) obtained on day 21 of differentiation induction by applying Wash-out according to Example 1.

[0210] Figure 13 is an image (left) and graph (right) showing the changes in corneal transparency and thickness over time in the group transplanted with ACE2W (Group 1) and the control group (Group 2) that did not transplant ACE2W in an endothelial dysfunction model in which the endothelial cells of a rabbit were selectively removed by scraping them in a circular pattern. The left image of Figure 13 shows changes in corneal transparency, and the right image shows the results of AS-OCT (Anterior Segment Optical Coherence Tomography) that measured corneal thickness.

[0211] The corneal opacity was calculated as a score from 0 to 4, and the criteria for judging corneal opacity are as shown in Table 1 below.

[0212] Score Corneal Opacity Degree 0 None 1 Mild opacity, iris texture clearly visible 2 Moderate opacity, iris texture unclear 3 Severe opacity, pupil faintly visible 4 Severe opacity, pupil not visible

[0213] As shown in the left image of Fig. 13, it was confirmed that corneal opacity was significantly reduced in the group transplanted with ACE2W (Group 1) compared to the control group without ACE2W transplantation (Group 2). In the control group without ACE2W transplantation (Group 2), ID 111 recorded a opacity score of 3 on the 3rd day and 4 on the 7th day, indicating that the opacity remained high, while ID 112 recorded a score of 4 on the 3rd day and 4 on the 7th day, confirming that corneal transparency did not improve.

[0214] On the other hand, in the group transplanted with ACE2W (group 1), ID 111 recovered to normal transparency with a turbidity score of 2 on day 3 and 0 on day 7, and ID 112 showed a significant decrease in turbidity with a score of 3 on day 3 and 1 on day 7. In addition, in group 1, which received the CEC transplant, it was confirmed that corneal opacity improved proportionally over time after the CEC transplant.

[0215] This suggests that transplantation of CECs differentiated from iPSCs effectively replaces damaged CECs and contributes to rapid recovery of CEC function.

[0216] Meanwhile, the subjects were divided into the normal group, the CEC scraping group, and the group in which ACE2W was transplanted after CEC scraping, and the corneal thickness of each group was measured on the 7th day. Corneal thickness was measured noninvasively using the optical coherence tomography (OCT) technique.

[0217] As shown in the right graph in Figure 13, the normal group maintained a stable corneal thickness of approximately 400 μm on average. In contrast, the CEC scraping group experienced a significant increase in corneal thickness to approximately 1,200 μm seven days after scraping due to corneal edema. This indicates that functional loss due to CEC loss caused the edema.

[0218] Meanwhile, the group that underwent ACE2W transplantation after CEC scraping showed a significant reduction in corneal thickness, reaching approximately 400 μm on day 7, compared to the CEC scraping group. This suggests that ACE2W transplantation is effective in promoting the recovery of damaged CECs and alleviating edema.

[0219]

[0220] 5.2 IHC analysis (immunohistochemical analysis)

[0221] Immunohistochemical analysis (IHC) was performed to evaluate the survival of transplanted iPSC-derived CECs and the occurrence of immune rejection. To this end, cells from the ACE2W group were transplanted into the corneal endothelial anterior chamber of rabbits, and corneal tissues were collected 1 and 16 weeks later and immunofluorescence staining was performed. During this process, double staining for the human-specific marker STEM121 and the CEC marker N-cadherin was performed, and the survival and tissue distribution of the transplanted cells were analyzed. The ACE2W in this experimental example was CEC (ACE2W21) obtained on day 21 of differentiation induction by applying wash-out according to Example 1.

[0222] Figure 14 is an image of the results of immunohistochemical analysis to detect human-specific markers (STEM121) and CEC markers (N-cadherin) in animal corneas at 1 and 16 weeks after transplantation of ACE2W into a corneal endothelial dysfunction model.

[0223] As shown in Figure 14, IHC analysis confirmed that STEM121 and N-cadherin were simultaneously expressed in the posterior layer of the animal cornea at 1 and 16 weeks post-transplantation. This indicates that the transplanted iPSC-derived CECs engrafted and survived long-term. Furthermore, double staining confirmed that the transplanted cells were stably maintained within the host tissue, indicating successful cell engraftment.

[0224] Meanwhile, to evaluate the immunogenicity of the transplanted cells, immunofluorescence staining for immune cell markers CD4, CD8 (T cell markers) and CD11b (macrophage / neutrophil marker) was performed, and the results are shown in Fig. 15.

[0225] Figure 15 is an immunofluorescent tissue section image showing the absence of immunoreactivity of CD4, CD8 T cells and CD11b macrophages / neutrophils in the ACE2W transplant group.

[0226] As shown in Figure 15, no immunoreactivity to CD4, CD8, or CD11b was observed in the group transplanted with cells from the ACE2W group. This indicates that the transplanted cells survived without being attacked by the host immune system and without immune rejection.

[0227] In summary, this experiment confirmed that iPSC-derived CECs can successfully engraft into the posterior corneal layer of rabbits and be stably maintained for a long period of time without immune rejection.

Claims

1. A step of inducing differentiation of stem cells into corneal endothelial cells in a medium containing a ROCK (Rho-associated protein kinase) inhibitor; A step of washing out the surface of the culture vessel with a washing solution to remove non-adherent cells on the surface of the culture vessel at a point when the ratio of cells attached to the surface of the culture vessel reaches the target attachment ratio; and A method for producing a high-purity corneal endothelial cell population, comprising the step of obtaining adherent cells remaining on the surface of the culture vessel.

2. A method for producing a high-purity corneal endothelial cell population according to claim 1, wherein the washing is performed 1 to 5 times.

3. A method for producing a high-purity corneal endothelial cell population according to claim 2, wherein the washing solution comprises at least one selected from the group consisting of phosphate-buffered saline (PBS), distilled water, normal saline, HBSS (Hank's balanced salt solution), TBS (Tris buffered saline), TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) buffer solution, Bicine (N,N-Bis(2-hydroxyethyl) glycine) buffer solution, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution, TES (NTris(hydroxymethyl)methyl-2-aminoethanesulfonic acid) buffer solution, PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid) buffer solution, and mixtures thereof.

4. A method for producing a high-purity corneal endothelial cell population according to claim 1, wherein the target attachment rate is a ratio of the cell density attached to the surface of a culture vessel to the cell seeding density, and is determined by the following mathematical formula 1. [Mathematical Formula 1] Target attachment rate (%) = (attached cell density (cells / cm²)) / (cell seeding density (cells / cm²)) × 100 5. A method for producing a high-purity corneal endothelial cell population, wherein the target attachment rate in claim 1 is 40% to 60%.

6. A method for producing a high-purity corneal endothelial cell population, further comprising a step of subculturing adherent cells on the surface of the culture vessel after the step of removing non-adherent cells on the surface of the culture vessel in claim 1.

7. A method for producing a high-purity corneal endothelial cell population according to claim 1, wherein the surface of the culture vessel is modified with an extracellular matrix (ECM).

8. A method for producing a high-purity corneal endothelial cell population according to claim 7, wherein the extracellular matrix comprises at least one selected from the group consisting of vitronectin, collagen, fibronectin, laminin, gelatin, matrigel, hyaluronic acid, polylysine, and heparan sulfate proteoglycan.

9. A method for producing a high-purity corneal endothelial cell population according to claim 1, wherein the stem cells include at least one selected from the group consisting of embryonic stem cells (ESCs), adult stem cells, and human induced pluripotent stem cells (iPSCs).

10. In claim 1, the ROCK inhibitor is fasudil, N-(4-pyridinyl)-4β-[(R)-1-aminoethyl]cyclohexane-1 α-carbamide (Y-27632), (2S)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]hexahydro-1H-1,4-diazepine (H1152), 4β-[(1R)-1-aminoethyl]-N-(4-pyridyl)benzene-1 zepinecarbamide (Wf-536), N-(1H-pyrrolo[2,3-b]pyridin-4-yl)-4PER(R)-1-aminoethyl]cyclohexane-1 α-carbamide (Y-30141), N-(3-{[2- A method for producing a high-purity corneal endothelial cell population, comprising at least one selected from the group consisting of (4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide (GSK269962A), and N-(6-fluoro-1H-indazol-5-yl)-6-methyl-2-oxo-4-[4-(trifluoromethyl)phenyl]-3,4-dihydro-1H-pyridine-5-carboxamide (GSK429286A).

11. Corneal endothelial cells manufactured by the method of claim 1.

12. In claim 11, the corneal endothelial cell has a reduced expression level of one or more genes selected from the group consisting of WNT6 and MIR205HG.

13. In claim 11, the corneal endothelial cell has reduced expression of stem cell-specific genes and increased expression of corneal endothelial cell-specific genes.

14. A corneal endothelial cell according to claim 13, wherein the stem cell-specific gene comprises at least one selected from the group consisting of NANOG, OCT4, PODXL, ESRG, and CNMD, and the corneal endothelial cell-specific gene comprises at least one selected from the group consisting of TJP1, CDH2, ATP1A1, ALCAM, PRDX6, COL8A1, and SLC25A11.

15. A pharmaceutical composition for preventing or treating corneal endothelial disease, comprising the corneal endothelial cells of claim 11.

16. A pharmaceutical composition for preventing or treating corneal endothelial disorders according to claim 15, wherein the corneal endothelial disease comprises at least one selected from the group consisting of Fuchs' Endothelial Dystrophy (FED), Congenital Hereditary Endothelial Dystrophy (CHED), Posterior Polymorphous Corneal Dystrophy (PPCD), Bullous Keratopathy (BK), Pseudophakic Bullous Keratopathy (PBK), Secondary Corneal Edema, Traumatic Endothelial Damage, Inflammatory Endothelial Damage, and Graft Failure after Corneal Transplantation.

17. Use of corneal endothelial cells of claim 11 for preventing or treating corneal endothelial disease 18. Use of corneal endothelial cells of claim 11 for the manufacture of a pharmaceutical preparation for the prevention or treatment of corneal endothelial disease.

19. A method for preventing or treating corneal endothelial disease, comprising administering an effective amount of corneal endothelial cells of claim 11 to a subject in need thereof.

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