Pharmaceutical composition for preventing or treating corneal diseases comprising corneal endothelial cells induced to differentiate

Abstract

According to a composition according to an aspect, when corneal endothelial cells and stem cells that are induced to differentiate are administered together, damaged corneal endothelial cells are improved, thus being effective in the prevention or treatment of corneal diseases, such as improvement of corneal opacity, a reduction in corneal thickness, a reduction in intra-corneal inflammation, and alleviation of corneal fibrosis.

Description

Pharmaceutical composition for preventing or treating corneal diseases comprising differentiated corneal endothelial cells

[0001] The present invention relates to a pharmaceutical composition for preventing or treating corneal diseases, comprising differentiated corneal endothelial cells.

[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. These ion pumps, embedded within the corneal endothelium, remove excess water from the corneal stroma, maintaining corneal transparency. If the corneal hydration function is not functioning properly, the cornea can swell and become cloudy, leading to vision loss.

[0004] Meanwhile, corneal endothelial cells (CECs) have limited proliferative capacity in adults. When corneal endothelial cells are damaged, they repair themselves through cell expansion and migration rather than mitosis. As a result, the cornea swells to several times its normal thickness, becomes opaque, and loses its function. The damage is permanent.

[0005] Accordingly, the inventors of the present invention have developed a composition capable of preventing or treating corneal diseases by injecting stem cell-derived corneal endothelial cells and stem cells to improve damaged corneal endothelial cells. This composition can help replace or repair damaged corneal endothelial cells, thereby restoring corneal transparency. It is also effective in reducing corneal edema by restoring the corneal endothelium's water-regulating function.

[0006] One aspect is to provide a pharmaceutical composition for preventing or treating corneal diseases, comprising differentiated corneal endothelial cells (CECs) and stem cells.

[0007] Another aspect provides a combination formulation for preventing or treating corneal diseases, comprising a first formulation comprising differentiated corneal endothelial cells; and a second formulation comprising stem cells.

[0008] Another aspect is to provide a cell therapy for preventing or treating corneal diseases comprising differentiated corneal endothelial cells and stem cells.

[0009] Another aspect provides the use of a composition comprising differentiated corneal endothelial cells (CECs) and stem cells for preventing or treating corneal diseases.

[0010] Another aspect provides a method for preventing or treating a corneal disease, comprising administering to a subject in need thereof a composition comprising an effective amount of differentiated corneal endothelial cells (CECs) and stem cells.

[0011] Another aspect provides the use of a composition comprising differentiated corneal endothelial cells (CECs) and stem cells for use in the manufacture of a medicament for preventing or treating corneal diseases.

[0012] One aspect provides a pharmaceutical composition for preventing or treating corneal diseases, comprising differentiated corneal endothelial cells (CECs) and stem cells.

[0013] As used herein, the term "corneal endothelial cell (CEC)" 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 part 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.

[0014] In one specific example, the induced differentiation corneal endothelial cells may be induced differentiation from embryonic stem cells (ESCs), adult stem cells, or human induced pluripotent stem cells (iPSCs). For example, the induced differentiation corneal endothelial cells may be induced differentiation from mesenchymal stem cells (MSCs), or may be induced differentiation from any one selected from the group consisting of stem cells derived from bone marrow, blood, umbilical cord blood, umbilical cord, adipose tissue, liver, skin, gastrointestinal tract, placenta, and uterus, but are not limited thereto.

[0015] The term "stem cell" in this specification 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.

[0016] In one specific example, the stem cell may be an embryonic stem cell (ESC), an adult stem cell, or an induced pluripotent stem cell (iPSC).

[0017] 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.

[0018] In one specific example, the adult stem cells may be, but are not limited to, mesenchymal stem cells (MSCs).

[0019] In one specific example, the adult stem cells may be selected from the group consisting of, but not limited to, stem cells derived from bone marrow, blood, umbilical cord blood, umbilical cord, adipose tissue, liver, skin, gastrointestinal tract, placenta, and uterus.

[0020] In this specification, the term "differentiation" may mean a phenomenon in which structures or functions become specialized while cells divide and proliferate and grow, that is, cells, tissues, etc. of a living organism change in form or function to perform their respective assigned tasks.

[0021] In this specification, the term "induction of differentiation from stem cells into corneal endothelial cells" may include not only cases where complete differentiation is induced from stem cells, including iPSCs, into corneal endothelial cells, but also cases where differentiation is induced into cells expressing corneal endothelial cell-specific markers.

[0022] The term "culture medium for inducing differentiation of stem cells into corneal endothelial cells" used herein refers to any medium that can induce differentiation of stem cells into corneal endothelial cells by reducing the characteristics of stem cells as pluripotent cells and increasing the characteristics specific to corneal endothelial cells during the process of inducing differentiation of stem cells into corneal endothelial cells. For example, the culture medium may be one that reduces the expression of a pluripotent stem cell-specific marker or increases the expression of a corneal endothelial cell-specific marker.

[0023] In one specific example, the pharmaceutical composition for preventing or treating corneal disease may be characterized by satisfying one or more of the following characteristics.

[0024] - Improves corneal opacity;

[0025] - Reduces corneal thickness;

[0026] - Improves corneal pumping function and intercellular adhesion;

[0027] - Reduces the expression of inflammatory cytokines;

[0028] - Reduces apoptosis in the cornea;

[0029] - Reduces corneal fibrosis; and

[0030] - Alleviates allograft rejection.

[0031] In one specific example, the composition for preventing or treating corneal disease may contain the differentiated corneal endothelial cells and stem cells in a ratio of 0.5 to 2: 0.5 to 2. For example, the ratio of the above-mentioned differentiation-induced corneal endothelial cells and stem cells is 0.5: 0.5, 0.5: 0.6, 0.5: 0.7, 0.5: 0.8, 0.5: 0.9, 0.5: 1.0, 0.5: 1.1, 0.5: 1.2, 0.5: 1.3, 0.5: 1.4, 0.5: 1.5, 0.5: 1.6, 0.5: 1.7, 0.5: 1.8, 0.5: 1.9, 0.5: 2.0, 0.6: 0.5, 0.6: 0.6, 0.6: 0.7, 0.6: 0.8, 0.6: 0.9, 0.6: 1.0, 0.6: 1.1, 0.6: 1.2, 0.6: 1.3, 0.6: 1.4, 0.6: 1.5, 0.6: 1.6, 0.6: 1.7, 0.6: 1.8, 0.6: 1.9, 0.6: 2.0, 0.7: 0.5, 0.7: 0.6, 0.7: 0.7, 0.7: 0.8, 0.7: 0.9, 0.7: 1.0, 0.7: 1.1, 0.7: 1.2, 0.7: 1.3, 0.7: 1.4, 0.7: 1.5, 0.7: 1.6, 0.7: 1.7, 0.7: 1.8, 0.7: 1.9, 0.7: 2.0, 0.8: 0.5, 0.8: 0.6, 0.8: 0.7, 0.8: 0.8, 0.8: 0.9, 0.8: 1.0, 0.8: 1.1, 0.8: 1.2, 0.8: 1.3, 0.8: 1.4, 0.8: 1.5, 0.8: 1.6, 0.8: 1.7, 0.8: 1.8, 0.8: 1.9, 0.8: 2.0, 0.9: 0.5, 0.9: 0.6, 0.9: 0.7, 0.9: 0.8, 0.9: 0.9, 0.9: 1.0, 0.9: 1.1, 0.9: 1.2, 0.9: 1.3, 0.9: 1.4, 0.9: 1.5, 0.9: 1.6, 0.9: 1.7, 0.9: 1.8, 0.9: 1.9, 0.9: 2.0, 1.0: 0.5, 1.0: 0.6, 1.0: 0.7, 1.0: 0.8, 1.0: 0.9, 1.0: 1.0, 1.0: 1.1, 1.0: 1.2, 1.0: 1.3, 1.0: 1.4, 1.0: 1.5, 1.0: 1.6, 1.0: 1.7, 1.0: 1.8, 1.0: 1.9, 1.0: 2.0, 1.1: 0.5, 1.1: 0.6, 1.1: 0.7, 1.1: 0.8, 1.1: 0.9, 1.1: 1.0, 1.1: 1.1, 1.1: 1.2, 1.1: 1.3, 1.1: 1.4, 1.1: 1.5, 1.1: 1.6, 1.1: 1.7, 1.1: 1.8, 1.1: 1.9, 1.1: 2.0, 1.2: 0.5, 1.2: 0.6, 1.2: 0.7, 1.2: 0.8, 1.2: 0.9, 1.2: 1.0, 1.2: 1.1, 1.2: 1.2, 1.2: 1.3, 1.2: 1.4, 1.2: 1.5, 1.2: 1.6, 1.2: 1.7, 1.2: 1.8, 1.2: 1.9, 1.2: 2.0, 1.3: 0.5, 1.3: 0.6, 1.3: 0.7, 1.3: 0.8, 1.3: 0.9, 1.3: 1.0, 1.3: 1.1, 1.3: 1.2, 1.3: 1.3, 1.3: 1.4, 1.3: 1.5, 1.3: 1.6, 1.3: 1.7, 1.3: 1.8, 1.3: 1.9, 1.3: 2.0, 1.4: 0.5, 1.4: 0.6, 1.4: 0.7, 1.4: 0.8, 1.4: 0.9, 1.4: 1.0, 1.4: 1.1, 1.4: 1.2, 1.4: 1.3, 1.4: 1.4, 1.4: 1.5, 1.4: 1.6, 1.4: 1.7, 1.4: 1.8, 1.4: 1.9, 1.4: 2.0, 1.5: 0.5, 1.5: 0.6, 1.5: 0.7, 1.5: 0.8, 1.5: 0.9, 1.5: 1.0, 1.5: 1.1, 1.5: 1.2, 1.5: 1.3, 1.5: 1.4, 1.5: 1.5, 1.5: 1.6, 1.5: 1.7, 1.5: 1.8, 1.5: 1.9, 1.5: 2.0, 1.6: 0.5, 1.6: 0.6, 1.6: 0.7, 1.6: 0.8, 1.6: 0.9, 1.6: 1.0, 1.6: 1.1, 1.6: 1.2, 1.6: 1.3, 1.6: 1.4, 1.6: 1.5, 1.6: 1.6, 1.6: 1.7, 1.6: 1.8, 1.6: 1.9, 1.6: 2.0, 1.7: 0.5, 1.7: 0.6, 1.7: 0.7, 1.7: 0.8, 1.7: 0.9, 1.7: 1.0, 1.7: 1.1, 1.7: 1.2, 1.7: 1.3, 1.7: 1.4, 1.7: 1.5, 1.7: 1.6, 1.7: 1.7, 1.7: 1.8, 1.7: 1.9, 1.7: 2.0, 1.8: 0.5, 1.8: 0.6, 1.8: 0.7, 1.8: 0.8, 1.8: 0.9, 1.8: 1.0, 1.8: 1.1, 1.8: 1.2, 1.8: 1.3, 1.8: 1.4, 1.8: 1.5, 1.8: 1.6, 1.8: 1.7, 1.8: 1.8, 1.8: 1.9, 1.8: 2.0, 1.9: 0.5, 1.9: 0.6, 1.9: 0.7, 1.9: 0.8, 1.9: 0.9, 1.9: 1.0, 1.9: 1.1, 1.9: 1.2, 1.9: 1.3, 1.9: 1.4, 1.9: It may be, but is not limited to, 1.5, 1.9: 1.6, 1.9: 1.7, 1.9: 1.8, 1.9: 1.9, 1.9: 2.0, 2.0: 0.5, 2.0: 0.6, 2.0: 0.7, 2.0: 0.8, 2.0: 0.9, 2.0: 1.0, 2.0: 1.1, 2.0: 1.2, 2.0: 1.3, 2.0: 1.4, 2.0: 1.5, 2.0: 1.6, 2.0: 1.7, 2.0: 1.8, 2.0: 1.9 or 2.0: 2.0.

[0032] In one specific example, the composition for preventing or treating corneal disease comprises 1Х10 of the differentiated corneal endothelial cells. 4 Dog or 1Х10 7 It may contain a dog. For example, the composition for preventing or treating corneal disease may contain 1Х10 of the induced corneal endothelial cells. 4 Dog, 2Х10 4 Dog, 3Х10 4 Dog, 4Х10 4 Dog, 5Х104 Dog, 6Х10 4 Dog, 7Х10 4 Dog, 8Х10 4 Dog, 9Х10 4 Dog, 1Х10 5 Dog, 2Х10 5 Dog, 3Х10 5 Dog, 4Х10 5 Dog, 5Х10 5 Dog, 6Х10 5 Dog, 7Х10 5 Dog, 8Х10 5 Dog, 9Х10 5 Dog, 1Х10 6 Dog, 2Х10 6 Dog, 3Х10 6 Dog, 4Х10 6 Dog, 5Х10 6 Dog, 6Х10 6 Dog, 7Х10 6 Dog, 8Х10 6 Dog, 9Х10 6 dog or 1Х10 7 May include, but is not limited to, dogs.

[0033] In one specific example, the composition for preventing or treating corneal disease comprises 1Х10 of the stem cells. 4 Dog or 1Х10 7 It may contain a dog. For example, the composition for preventing or treating corneal disease may contain 1Х10 of the stem cells. 4 Dog, 2Х10 4 Dog, 3Х10 4 Dog, 4Х10 4 Dog, 5Х10 4 Dog, 6Х10 4 Dog, 7Х10 4 Dog, 8Х10 4 Dog, 9Х10 4 Dog, 1Х10 5 Dog, 2Х10 5 Dog, 3Х10 5 Dog, 4Х10 5 Dog, 5Х10 5 Dog, 6Х10 5 Dog, 7Х10 5 Dog, 8Х105 Dog, 9Х10 5 Dog, 1Х10 6 Dog, 2Х10 6 Dog, 3Х10 6 Dog, 4Х10 6 Dog, 5Х10 6 Dog, 6Х10 6 Dog, 7Х10 6 Dog, 8Х10 6 Dog, 9Х10 6 dog or 1Х10 7 May include, but is not limited to, dogs.

[0034] In the present invention, the term "corneal disease" means any disease or damage in which corneal cells are damaged and the cornea cannot maintain transparency, and specifically, the corneal disease may be at least one selected from the group consisting of Corneal Endothelial Decompensation, Fuchs' Dystrophy, Corneal Endothelial Cell Dystrophy, Corneal Edema, Posterior Polymorphous Corneal Dystrophy (PPCD), and Corneal Transplant Rejection, but is not limited thereto.

[0035] As used herein, the term "treatment" may refer to the healing of corneal diseases and the like in a shorter period of time compared to natural healing. Such treatment may include improvement and / or alleviation of the corneal disease. Furthermore, such treatment may refer to the healing and / or recovery of symptoms resulting from the corneal disease.

[0036] As used herein, the term "prevention" 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 onset of a corneal disease by administering a composition according to the present invention.

[0037] The above pharmaceutical composition may contain, in addition to the above-described active ingredient, a pharmaceutically acceptable carrier, excipient or diluent for administration.

[0038] In the present invention, the term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the injected compound. The type of the carrier usable in the present invention is not particularly limited, and any pharmaceutically acceptable carrier commonly used in the art may be used. Non-limiting examples of the carrier include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and the like. 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.

[0039] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the degree of the disease, the drug form, and the time, but can be appropriately selected by a person skilled in the art.

[0040] The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and generally 1 to 2 million cells per eye can be administered once or several times daily. However, for the purposes of the present invention, it is preferable that a specific therapeutically effective amount for a specific patient be applied differently depending on various factors including the type and degree of response to be achieved, the specific composition including whether other agents are used in some cases, the patient's age, weight, general health, sex and diet, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, drugs used together or concurrently with the specific composition, and similar factors well known in the medical field.

[0041] The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. It can be administered singly or in multiple doses. Taking all of the above factors into account, it is important to administer the amount that achieves maximum effect with the minimum amount possible without causing side effects, a determination readily made by those skilled in the art.

[0042] The term "administration" used in the present invention means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and since the pharmaceutical composition including the differentiated corneal endothelial cells (CECs) and stem cells of the present invention is effective in preventing or treating corneal diseases or conjunctival diseases, the route of administration of the composition may be through the eye.

[0043]

[0044] Another aspect provides a combination formulation for preventing or treating corneal diseases, comprising a first formulation comprising differentiated corneal endothelial cells (CECs) and a second formulation comprising stem cells.

[0045] In one specific example, the first formulation and the second formulation may be for direct administration to the eye.

[0046] In one specific example, the first preparation and the second preparation may be administered concurrently, sequentially, or in reverse order.

[0047] As used herein, the terms "combination," "combined treatment," or "in combination" refer to any form of simultaneous or concurrent treatment using at least two separate therapeutic agents. The components of the combination therapy may be administered simultaneously, sequentially, in reverse order, or in any order. The components may be administered in different dosages, at different frequencies, or via different routes, as appropriate.

[0048] The above combination administration may mean administering simultaneously, sequentially or separately, and in any order, a first preparation comprising differentiated corneal endothelial cells (CECs); and a second preparation comprising stem cells.

[0049] The term "administered simultaneously" as used herein is not particularly limited and means that the components of the combination therapy are administered substantially simultaneously, for example, as a mixture or in an immediately subsequent sequence.

[0050] The term "administered sequentially" as used herein is not particularly limited and means that the components of the combination therapy are administered not simultaneously, but one after another or in clusters with a specific time interval between administrations. The time intervals may be the same or different between the administrations of each of the components of the combination therapy, and may be selected, for example, from the range of 2 minutes to 96 hours, 1 day to 7 days, or 1 week, 2 weeks, or 3 weeks. Typically, the time interval between administrations may range from several minutes to several hours, for example, from 2 minutes to 72 hours, from 30 minutes to 24 hours, or from 1 to 12 hours. Additional examples include time intervals ranging from 24 to 96 hours, from 12 to 36 hours, from 8 to 24 hours, and from 6 to 12 hours.

[0051] For example, the combination therapy may be administered simultaneously with a first agent containing differentiated corneal endothelial cells (CECs) and a second agent containing stem cells, or may be administered after administering the first agent containing differentiated corneal endothelial cells (CECs). The combination therapy according to the present invention may be defined as providing a synergistic effect if the efficacy, as measured by, for example, the degree of response, the rate of response, or the time until disease progression, is therapeutically superior to the efficacy obtained by administering one or the other of the components of the combination therapy at a conventional dosage. For example, the efficacy of the combination therapy is synergistic if the efficacy is therapeutically superior to the efficacy obtained by using each of the components alone. In particular, a synergistic effect is considered to exist if the usual dose of a material containing differentiated corneal endothelial cells (CECs) and stem cells can be reduced without compromising one or more of the following data: degree of response, rate of response, and time to disease progression, and in particular without compromising duration of response, and with a reduction and / or fewer problematic side effects than would occur when either component is used at its usual dose.

[0052] As used herein, the term "administration" refers to introducing a given substance into a subject in an appropriate manner, and "subject" refers to any living organism, including humans, rats, mice, and livestock, that may have corneal disease. A specific example may be a mammal, including humans.

[0053] The term "combination formulation" as used herein refers to a combination designed to be used together with two or more drugs or therapeutic agents for the purpose of preventing or treating two corneal diseases. For example, the combination formulation may refer to a combination designed to be administered simultaneously, sequentially, or separately, and in any order, a first formulation comprising differentiated corneal endothelial cells (CECs); and a second formulation comprising stem cells. Specifically, the formulation may be in a dosage form selected from the group consisting of eye drops, injections, eye ointments, ocular inserts, drops, gels, suspensions, and emulsions, but is not limited thereto.

[0054] In one specific example, the first formulation comprises 1X10 corneal endothelial cells (CECs) that have been induced to differentiate 4 Dog or 1Х10 7 It may contain a dog. For example, the first preparation may contain 1Х10 of the induced corneal endothelial cells. 4 Dog, 2Х10 4 Dog, 3Х10 4 Dog, 4Х10 4 Dog, 5Х10 4 Dog, 6Х10 4 Dog, 7Х10 4 Dog, 8Х10 4 Dog, 9Х10 4 Dog, 1Х10 5 Dog, 2Х10 5 Dog, 3Х10 5 Dog, 4Х10 5 Dog, 5Х10 5 Dog, 6Х10 5 Dog, 7Х10 5 Dog, 8Х10 5 Dog, 9Х10 5 Dog, 1Х10 6 Dog, 2Х10 6 Dog, 3Х10 6 Dog, 4Х10 6 Dog, 5Х10 6 Dog, 6Х10 6Dog, 7Х10 6 Dog, 8Х10 6 Dog, 9Х10 6 dog or 1Х10 7 May include, but is not limited to, dogs.

[0055] In one specific example, the second formulation comprises 1Х10 of the stem cells 4 Dog or 1Х10 7 It may contain a dog. For example, the second preparation may contain 1Х10 of the stem cells. 4 Dog, 2Х10 4 Dog, 3Х10 4 Dog, 4Х10 4 Dog, 5Х10 4 Dog, 6Х10 4 Dog, 7Х10 4 Dog, 8Х10 4 Dog, 9Х10 4 Dog, 1Х10 5 Dog, 2Х10 5 Dog, 3Х10 5 Dog, 4Х10 5 Dog, 5Х10 5 Dog, 6Х10 5 Dog, 7Х10 5 Dog, 8Х10 5 Dog, 9Х10 5 Dog, 1Х10 6 Dog, 2Х10 6 Dog, 3Х10 6 Dog, 4Х10 6 Dog, 5Х10 6 Dog, 6Х10 6 Dog, 7Х10 6 Dog, 8Х10 6 Dog, 9Х10 6 dog or 1Х10 7 May include, but is not limited to, dogs.

[0056] Another aspect provides a cell therapy for preventing or treating corneal diseases comprising differentiated corneal endothelial cells (CECs) and stem cells.

[0057] The term "cell therapy" as used herein may refer to a therapeutic method that uses living cells to treat a patient's disease or damaged tissue. Specifically, it may refer to a therapeutic method that involves culturing or manipulating cells in vitro and then administering them to a patient to promote tissue regeneration, immune regulation, or restoration of specific cell functions.

[0058] In one specific example, the cell therapeutic agent may be a cell therapeutic agent for preventing or treating corneal disease, characterized in that it satisfies one or more of the following characteristics.

[0059] - Improves corneal opacity;

[0060] - Reduces corneal thickness;

[0061] - Improves corneal pumping function and intercellular adhesion;

[0062] - Reduces the expression of inflammatory cytokines;

[0063] - Reduces apoptosis in the cornea;

[0064] - Reduces corneal fibrosis; and

[0065] - Alleviates allograft rejection.

[0066] In one specific example, the stem cells may be embryonic stem cells (ESCs), adult stem cells, or human induced pluripotent stem cells (iPSCs), and the cell therapeutic agent may be used to prevent or treat corneal diseases.

[0067] In one specific example, the corneal endothelial cells induced to differentiate may be a cell therapeutic agent for preventing or treating corneal diseases, which are induced to differentiate from embryonic stem cells (ESCs), adult stem cells, or human induced pluripotent stem cells (iPSCs).

[0068] In one specific example, the cell therapy agent comprises 1Х10 of the differentiated corneal endothelial cells. 4 Dog or 1Х10 7 may include, but is not limited to, dogs. For example, the cell therapy may comprise 1X10 of the induced corneal endothelial cells. 4 Dog, 2Х10 4 Dog, 3Х10 4 Dog, 4Х10 4 Dog, 5Х10 4 Dog, 6Х10 4 Dog, 7Х10 4 Dog, 8Х10 4 Dog, 9Х10 4 Dog, 1Х10 5 Dog, 2Х10 5 Dog, 3Х10 5 Dog, 4Х10 5 Dog, 5Х10 5 Dog, 6Х10 5 Dog, 7Х10 5 Dog, 8Х10 5 Dog, 9Х10 5 Dog, 1Х10 6 Dog, 2Х10 6 Dog, 3Х10 6 Dog, 4Х10 6 Dog, 5Х10 6 Dog, 6Х10 6 Dog, 7Х10 6 Dog, 8Х10 6 Dog, 9Х10 6 dog or 1Х10 7 May include, but is not limited to, dogs.

[0069] In one specific example, the cell therapy agent comprises 1Х10 of the stem cells 4 Dog or 1Х10 7 May include, but is not limited to, dogs. For example, the cell therapy may comprise 1Х10 4 Dog, 2Х10 4 Dog, 3Х10 4 Dog, 4Х10 4 Dog, 5Х10 4 Dog, 6Х10 4 Dog, 7Х10 4 Dog, 8Х10 4 Dog, 9Х10 4 Dog, 1Х10 5 Dog, 2Х10 5 Dog, 3Х10 5 Dog, 4Х10 5 Dog, 5Х10 5 Dog, 6Х10 5 Dog, 7Х10 5 Dog, 8Х10 5 Dog, 9Х10 5 Dog, 1Х10 6 Dog, 2Х10 6 Dog, 3Х10 6 Dog, 4Х10 6 Dog, 5Х10 6 Dog, 6Х10 6 Dog, 7Х10 6 Dog, 8Х10 6 Dog, 9Х10 6 dog or 1Х10 7 May include, but is not limited to, dogs.

[0070] In one specific example, the cell therapeutic agent may contain the differentiation-induced corneal endothelial cells and stem cells in a ratio of 0.5 to 2: 0.5 to 2, for example, in a ratio of 1.0: 0.5, 1.0: 0.6, 1.0: 0.7, 1.0: 0.8, 1.0: 0.9, 1.0: 1.0, 1.0: 1.1, 1.0: 1.2, 1.0: 1.3, 1.0: 1.4, 1.0: 1.5. More specifically, 0.5: 0.5, 0.5: 0.6, 0.5: 0.7, 0.5: 0.8, 0.5: 0.9, 0.5: 1.0, 0.5: 1.1, 0.5: 1.2, 0.5: 1.3, 0.5: 1.4, 0.5: 1.5, 0.5: 1.6, 0.5: 1.7, 0.5: 1.8, 0.5: 1.9, 0.5: 2.0, 0.6: 0.5, 0.6: 0.6, 0.6: 0.7, 0.6: 0.8, 0.6: 0.9, 0.6: 1.0, 0.6: 1.1, 0.6: 1.2, 0.6: 1.3, 0.6: 1.4, 0.6: 1.5, 0.6: 1.6, 0.6: 1.7, 0.6: 1.8, 0.6: 1.9, 0.6: 2.0, 0.7: 0.5, 0.7: 0.6, 0.7: 0.7, 0.7: 0.8, 0.7: 0.9, 0.7: 1.0, 0.7: 1.1, 0.7: 1.2, 0.7: 1.3, 0.7: 1.4, 0.7: 1.5, 0.7: 1.6, 0.7: 1.7, 0.7: 1.8, 0.7: 1.9, 0.7: 2.0, 0.8: 0.5, 0.8: 0.6, 0.8: 0.7, 0.8: 0.8, 0.8: 0.9, 0.8: 1.0, 0.8: 1.1, 0.8: 1.2, 0.8: 1.3, 0.8: 1.4, 0.8: 1.5, 0.8: 1.6, 0.8: 1.7, 0.8: 1.8, 0.8: 1.9, 0.8: 2.0, 0.9: 0.5, 0.9: 0.6, 0.9: 0.7, 0.9: 0.8, 0.9: 0.9, 0.9: 1.0, 0.9: 1.1, 0.9: 1.2, 0.9: 1.3, 0.9: 1.4, 0.9: 1.5, 0.9: 1.6, 0.9: 1.7, 0.9: 1.8, 0.9: 1.9, 0.9: 2.0, 1.0: 0.5, 1.0: 0.6, 1.0: 0.7, 1.0: 0.8, 1.0: 0.9, 1.0: 1.0, 1.0: 1.1, 1.0: 1.2, 1.0: 1.3, 1.0: 1.4, 1.0: 1.5, 1.0: 1.6, 1.0: 1.7, 1.0: 1.8, 1.0: 1.9, 1.0: 2.0, 1.1: 0.5, 1.1: 0.6, 1.1: 0.7, 1.1: 0.8, 1.1: 0.9, 1.1: 1.0, 1.1: 1.1, 1.1: 1.2, 1.1: 1.3, 1.1: 1.4, 1.1: 1.5, 1.1: 1.6, 1.1: 1.7, 1.1: 1.8, 1.1: 1.9, 1.1: 2.0, 1.2: 0.5, 1.2: 0.6, 1.2: 0.7, 1.2: 0.8, 1.2: 0.9, 1.2: 1.0, 1.2: 1.1, 1.2: 1.2, 1.2: 1.3, 1.2: 1.4, 1.2: 1.5, 1.2: 1.6, 1.2: 1.7, 1.2: 1.8, 1.2: 1.9, 1.2: 2.0, 1.3: 0.5, 1.3: 0.6, 1.3: 0.7, 1.3: 0.8, 1.3: 0.9, 1.3: 1.0, 1.3: 1.1, 1.3: 1.2, 1.3: 1.3, 1.3: 1.4, 1.3: 1.5, 1.3: 1.6, 1.3: 1.7, 1.3: 1.8, 1.3: 1.9, 1.3: 2.0, 1.4: 0.5, 1.4: 0.6, 1.4: 0.7, 1.4: 0.8, 1.4: 0.9, 1.4: 1.0, 1.4: 1.1, 1.4: 1.2, 1.4: 1.3, 1.4: 1.4, 1.4: 1.5, 1.4: 1.6, 1.4: 1.7, 1.4: 1.8, 1.4: 1.9, 1.4: 2.0, 1.5: 0.5, 1.5: 0.6, 1.5: 0.7, 1.5: 0.8, 1.5: 0.9, 1.5: 1.0, 1.5: 1.1, 1.5: 1.2, 1.5: 1.3, 1.5: 1.4, 1.5: 1.5, 1.5: 1.6, 1.5: 1.7, 1.5: 1.8, 1.5: 1.9, 1.5: 2.0, 1.6: 0.5, 1.6: 0.6, 1.6: 0.7, 1.6: 0.8, 1.6: 0.9, 1.6: 1.0, 1.6: 1.1, 1.6: 1.2, 1.6: 1.3, 1.6: 1.4, 1.6: 1.5, 1.6: 1.6, 1.6: 1.7, 1.6: 1.8, 1.6: 1.9, 1.6: 2.0, 1.7: 0.5, 1.7: 0.6, 1.7: 0.7, 1.7: 0.8, 1.7: 0.9, 1.7: 1.0, 1.7: 1.1, 1.7: 1.2, 1.7: 1.3, 1.7: 1.4, 1.7: 1.5, 1.7: 1.6, 1.7: 1.7, 1.7: 1.8, 1.7: 1.9, 1.7: 2.0, 1.8: 0.5, 1.8: 0.6, 1.8: 0.7, 1.8: 0.8, 1.8: 0.9, 1.8: 1.0, 1.8: 1.1, 1.8: 1.2, 1.8: 1.3, 1.8: 1.4, 1.8: 1.5, 1.8: 1.6, 1.8: 1.7, 1.8: 1.8, 1.8: 1.9, 1.8: 2.0, 1.9: 0.5, 1.9: 0.6, 1.9: 0.7, 1.9: 0.8, 1.9: 0.9, 1.9: 1.0, 1.9: 1.1, 1.9: 1.2, 1.9: 1.3, 1.9: 1.4, 1.9: 1.5, 1.9: 1.6, 1.9: 1.7, 1.9: 1.8, 1.9: 1.9, 1.9: 2.0, 2.0: 0.5, 2.0: 0.6, 2.0: 0.7, 2.0: 0.8, 2.0: 0.9, 2.0: 1.0, 2.0: 1.1, 2.0: 1.2, 2.0: 1.3, 2.0: 1.4, This may include, but is not limited to, a ratio of 2.0: 1.5, 2.0: 1.6, 2.0: 1.7, 2.0: 1.8, 2.0: 1.9 or 2.0: 2.0.

[0071] Another aspect provides the use of a composition comprising differentiated corneal endothelial cells (CECs) and stem cells for preventing or treating corneal diseases.

[0072] Another aspect provides a method for preventing or treating a corneal disease, comprising administering to a subject in need thereof a composition comprising an effective amount of differentiated corneal endothelial cells (CECs) and stem cells.

[0073] Another aspect provides the use of a composition comprising differentiated corneal endothelial cells (CECs) and stem cells for use in the manufacture of a medicament for preventing or treating corneal diseases.

[0074] The terms and methods described for the above inventions apply equally to each invention.

[0075] According to the composition according to the aspect, when the differentiation-induced corneal endothelial cells and stem cells are administered together, it is effective in preventing or treating corneal diseases such as improving corneal opacity, reducing corneal thickness, reducing corneal inflammation, and alleviating corneal fibrosis by improving damaged corneal endothelial cells.

[0076] Figure 1 is a schematic diagram of the corneal endothelial-like cell differentiation process using induced pluripotent stem cells.

[0077] Figure 2 illustrates the process of transplanting corneal endothelial cells induced in an animal model of corneal endothelial dysfunction according to one aspect.

[0078] Figure 3 shows photographs of corneal transparency observed using a micro-operating microscope after 1, 2, 3, 4, 5, and 6 weeks of transplantation of umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) according to each experimental group in an animal model of corneal endothelial failure according to the daily aspect.

[0079] Figure 4 is a graph showing the results of evaluating and scoring corneal opacity by experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial insufficiency according to each experimental group.

[0080] Figure 5 is a photograph showing the optical coherence tomography results for each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial insufficiency according to the daily aspect.

[0081] Figure 6 is a graph showing the results of measuring corneal thickness by experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial insufficiency according to each experimental group.

[0082] Figure 7 shows photographs of corneas obtained from each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial insufficiency according to the daily pattern, and performing H&E staining.

[0083] Figure 8 shows photographs of corneas obtained from each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial insufficiency according to the daily pattern, and confirming the expression of Na+K+-ATPase, ZO-1, and N-cadherin.

[0084] Figure 9 is a graph showing the results of confirming the number of cells expressing Na+K+-ATPase, ZO-1, and N-cadherin in the corneal tissue of the corneal endothelial dysfunction animal model experimental group according to the daily aspect.

[0085] Figure 10 shows photographs of corneas obtained from each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial dysfunction according to the daily pattern, and checking for the expression of TNF-α and IL-1β.

[0086] Figure 11 is a graph showing the number of cells expressing TNF-α and IL-1β in corneal tissue of an animal model experimental group of corneal endothelial dysfunction according to the daily aspect.

[0087] Figure 12 is a photograph showing the results of transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial dysfunction according to each experimental group, and after 6 weeks, obtaining corneas from each experimental group and confirming the expression of TNF-α, IL-1, and IL-6 using Western blotting.

[0088] Figure 13 shows photographs of corneas obtained from each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial failure according to the daily pattern, and confirming cell death through TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling).

[0089] Figure 14 is a graph showing the number of cells in which cleaved DNA is detected in corneal tissue of an animal model experimental group of corneal endothelial dysfunction according to the daily aspect.

[0090] Figure 15 shows photographs of corneas obtained from each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial insufficiency according to the daily pattern, and checking for the expression of α-SMA (α-smooth muscle actin).

[0091] Figure 16 is a graph showing the expression of α-SMA in corneal tissue of an animal model experimental group of corneal endothelial dysfunction according to the daily aspect.

[0092] Figure 17 is a graph showing the expression of CXCL9, CXCL10, and CXCL11 in an animal model of corneal endothelial dysfunction according to the daily pattern.

[0093] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited by these examples.

[0094] Example 1. Differentiation of stem cells into corneal endothelial cells

[0095] Human induced pluripotent stem cells (hiPSCs) were provided by Ipcell Co., Ltd., and umbilical cord-derived mesenchymal stem cells (UC-MSCs) were provided by the Cell Therapy Center, Asan Medical Center (Asan Institute for Life Sciences, Seoul).

[0096] Human induced pluripotent stem cells (hiPSCs) or umbilical cord-derived mesenchymal stem cells (UC-MSCs) were cultured using E8 medium (Life Technologies). The medium was then replaced with Human Endothelial Serum-Free Medium (Human Endothelial-SFM) supplemented with supplements to induce differentiation into corneal endothelial cells. When the differentiated cells reached approximately 80-90% confluency, they were subcultured. The differentiation process of corneal endothelial-like cells using iPS cells is illustrated in Figure 1.

[0097] Figure 1 is a schematic diagram of the corneal endothelial-like cell differentiation process using induced pluripotent stem cells.

[0098] Meanwhile, specific culture medium components used to induce corneal endothelial cells from human induced pluripotent stem cells (hiPSCs) or umbilical cord-derived mesenchymal stem cells (UC-MSCs) are shown in Table 1.

[0099]

[0100]

[0101] Example 2. Confirmation of survival rate, morphological changes, and functional maintenance of induced corneal endothelial cells.

[0102] Stem cell-derived corneal endothelial cells differentiated using the method of Example 1 and stored in liquid nitrogen were thawed, and the survival and morphological changes of the stem cell-derived corneal endothelial cells were analyzed. In addition, the intrinsic functions and phenotypic changes of the induced corneal endothelial cells according to passage culture were confirmed.

[0103] Specifically, the survival of the stem cell-derived corneal endothelial-like cells was confirmed through cell counting using trypan blue, the morphological changes of the stem cell-derived corneal endothelial-like cells were confirmed through a microscope, and the unique functions and unique phenotypic changes of the stem cell-derived corneal endothelial cells according to passage culture were confirmed through WB (Western Blot), qPCR, and ICC (Immunocytochemistry).

[0104] Through the above experiment, it was confirmed that most corneal endothelial cells differentiated by the method of Example 1 survived even when stored in liquid nitrogen and thawed, and it was confirmed that they exhibited hexagonal shapes and close cell junctions, which are representative characteristics of corneal endothelial cells. In addition, it was confirmed that the corneal endothelial cells derived from stem cells differentiated by the method of Example 1 maintained their cell-specific functions and unique phenotypes even when subcultured.

[0105]

[0106] Example 3. Transplantation of induced corneal endothelial cells into an animal model of corneal endothelial dysfunction

[0107] 3.1 Creation of an animal model of corneal endothelial dysfunction

[0108] A 3-month-old New Zealand White rabbit (JA BIO) was purchased. The endothelial and stromal layers of the rabbit eye were removed using a reverse sinskey hook to create an animal model of corneal endothelial damage.

[0109] 3.2 Transplantation of induced corneal endothelial cells

[0110] Each cell was transplanted by direct injection into the anterior chamber of the eye of an animal model of corneal endothelial insufficiency produced by the method of Example 3.1.

[0111] Specifically, umbilical cord-derived mesenchymal stem cells (MSCs); corneal endothelial cells induced to differentiate using induced pluripotent stem cells (iPSC-CECs); or corneal endothelial cells induced to differentiate using induced pluripotent stem cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) were transplanted into the anterior chamber of the corneal endothelial defect animal model produced by the method of Example 3.1.

[0112] The above umbilical cord-derived mesenchymal stem cells (MSCs) are 1Х10 6 Dog, the above differentiation-induced corneal endothelial cells (iPSC-CEC) were 1Х10 6 Dog, the above differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) were 2Х10 6 The capacity of the dog was transplanted. When the above differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) were transplanted together, they were transplanted at a ratio of 1:1.

[0113] Figure 2 illustrates the process of transplanting corneal endothelial cells induced in an animal model of corneal endothelial dysfunction according to the daily aspect.

[0114] After transplantation of corneal endothelial cells, human mitochondrial DNA expression was confirmed using PCR in corneal tissue of an animal model of corneal endothelial insufficiency, and the expression of ZO-1, ATP1A1, etc. was confirmed by immunohistochemical staining. This indicates that the transplanted cells survive in the eyes of an animal model of corneal endothelial insufficiency and maintain the unique characteristics of corneal endothelial cells.

[0115]

[0116] Experimental Example 1. Improvement of corneal opacity following induced corneal endothelial cell transplantation.

[0117] Each cell was transplanted into the anterior chamber of the eye of an animal model of corneal endothelial insufficiency produced by the method of Example 3.1 by the method of Example 3.2, and after 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks, corneal transparency was observed using a microsurgical microscope.

[0118] Specifically, the corneal endothelial dysfunction animal model of Example 3 was divided into a control group (group not performing cell transplantation), a group transplanted with only umbilical cord-derived mesenchymal stem cells (MSCs) (group 1: MSC), a group transplanted with only corneal endothelial cells (iPSC-CECs) differentiated using induced pluripotent stem cells (group 2: iPSC-CEC), and a group transplanted with both corneal endothelial cells (iPSC-CECs) differentiated using induced pluripotent stem cells and umbilical cord-derived mesenchymal stem cells (MSCs) (group 3: iPSC-CEC+MSC), and corneal transparency was observed, and corneal opacity was evaluated for each group and scored from 0 to 4 (0: no corneal clouding 1: iris details observable 2: pupillary border part observable, but iris details observable 3: pupillary border not discernible 4: entire corneal opacity).

[0119] Figure 3 shows photographs of corneal transparency observed using a microsurgical microscope after 1, 2, 3, 4, 5, and 6 weeks of transplantation of umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) according to each experimental group in an animal model of corneal endothelial insufficiency.

[0120] Figure 4 is a graph showing the results of evaluating and scoring corneal opacity by experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of the corneal endothelial insufficiency animal model (0: no corneal opacity 1: iris details observable 2: pupillary border part observable, but iris details observable 3: pupillary border unidentifiable 4: total corneal opacity).

[0121] As shown in Figures 3 and 4, it was confirmed that when differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) were transplanted, corneal opacity in the corneal endothelial defect animal model was significantly improved compared to the control group.

[0122] This means that transplantation of differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) according to one embodiment of the present invention is effective in preventing and treating corneal endothelial dysfunction.

[0123]

[0124] Experimental Example 2. Improvement of Corneal Thickness Following Induced Corneal Endothelial Cell Transplantation

[0125] Corneal endothelial cells induced by the method of Example 3.2 were transplanted into the anterior chamber of the eye of an animal model of corneal endothelial insufficiency produced by the method of Example 3.1, and optical coherence tomography (OCT) was performed after 6 weeks.

[0126] Specifically, the corneal endothelial dysfunction animal model of Example 3 was divided into a control group (group not performing cell transplantation), a group transplanted with only umbilical cord-derived mesenchymal stem cells (MSCs) (group 1: MSCs), a group transplanted with only corneal endothelial cells (iPSC-CECs) differentiated using induced pluripotent stem cells (group 2: iPSC-CECs), and a group transplanted with both corneal endothelial cells differentiated using induced pluripotent stem cells (iPSC-CECs) and umbilical cord-derived mesenchymal stem cells (MSCs) (group 3: iPSC-CEC+MSCs), and optical coherence tomography (OCT) was performed, and the results are shown in Figs. 6 and 7.

[0127] Figure 5 is a photograph showing the optical coherence tomography results for each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of the corneal endothelial insufficiency animal model according to each experimental group.

[0128] Figure 6 is a graph showing the results of measuring corneal thickness by experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of the eye of an animal model of corneal endothelial insufficiency according to each experimental group.

[0129] As shown in Figures 5 and 6, when differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) were transplanted together, the corneal thickness of the corneal endothelial defect animal model was confirmed to be significantly thinner compared to the control group.

[0130] This means that transplantation of differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) according to one embodiment of the present invention is effective in preventing and treating corneal endothelial dysfunction.

[0131]

[0132] Experimental Example 3. Improvement of corneal morphology and cell infiltration following induced corneal endothelial cell transplantation.

[0133] Corneal endothelial cells induced by the method of Example 3.2 were transplanted into the anterior chamber of the corneal endothelial insufficiency animal model produced by the method of Example 3.1, and after 6 weeks, the cornea of ​​the corneal endothelial insufficiency animal model was stained with H&E to check the morphology of the cornea and the presence of cell infiltration, and the results are shown in Fig. 7.

[0134] Figure 7 shows photographs of corneas obtained from each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of the eye of an animal model of corneal endothelial insufficiency, and performing H&E staining.

[0135] As shown in Figure 7, when differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) were transplanted together, it was confirmed that inflammatory cell infiltration was reduced and the structure of endothelial cells was significantly improved compared to other groups, resulting in a similar appearance to normal.

[0136] This means that transplantation of differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) according to one embodiment of the present invention is effective in preventing and treating corneal endothelial dysfunction.

[0137]

[0138] Experimental Example 4. Confirmation of improved corneal pump function and intercellular adhesion following induced corneal endothelial cell transplantation.

[0139] Corneal endothelial cells induced by the method of Example 3.2 were transplanted into the anterior chamber of the corneal endothelial insufficiency animal model produced by the method of Example 3.1, and after 6 weeks, the cornea of ​​the corneal endothelial insufficiency animal model was subjected to immunofluorescence staining and observed under a fluorescence microscope.

[0140] Specifically, corneas were collected from the corneal endothelial dysfunction animal model of Example 3, including the control group (group without cell transplantation), the group transplanted with only umbilical cord-derived mesenchymal stem cells (MSCs) (group 1: MSCs), the group transplanted with only corneal endothelial cells (iPSC-CECs) differentiated using induced pluripotent stem cells (group 2: iPSC-CECs), and the group transplanted with both corneal endothelial cells (iPSC-CECs) differentiated using induced pluripotent stem cells and umbilical cord-derived mesenchymal stem cells (MSCs) (group 3: iPSC-CEC+MSCs). The collected corneas were fixed with 4% paraformaldehyde for 2 to 3 days, washed with PBST buffer, permeabilized with 0.5% Triton X-100, and blocked with PBST containing 3% bovine serum albumin (Sigma-Aldrich). Samples were incubated overnight at 4°C with anti-human anti-ZO-1 (1:250; Invitrogen), anti-Na+ / K+ATPase (1:200; Abcam), anti-IL-1β (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-N-cadherin (1:200; Cell Signaling) antibodies as primary antibodies, followed by incubation with IgG Alexa Fluor 488-conjugated and IgG Alexa Fluor 647-conjugated secondary antibodies (1:500; Abcam, Cambridge, UK). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 10 min, and the fluorescence signals were detected by a laser scanning confocal microscope (Olympus, Tokyo, Japan) or a fluorescence microscope (EVOS, Life Technologies), and the results are shown in Fig. 8. In addition, the number of cells expressing Na+K+-ATPase, ZO-1, and N-cadherin was detected using an image analysis system in the photographs obtained using the above-described fluorescent microscope, and this is shown in Fig. 9.

[0141] Figure 8 shows photographs of corneas obtained from each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial insufficiency according to the daily pattern, and confirming the expression of Na+K+-ATPase, ZO-1, and N-cadherin.

[0142] Figure 9 is a graph showing the results of confirming the number of cells expressing Na+K+-ATPase, ZO-1, and N-cadherin in the corneal tissue of the corneal endothelial dysfunction animal model experimental group according to the daily aspect.

[0143] As shown in Figures 8 and 9, when differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) were transplanted together, the number of cells expressing Na+K+-ATPase, ZO-1, and N-cadherin was confirmed to significantly increase compared to the control group.

[0144] This means that the corneal pump function and cell tight junction formation are improved, and that transplantation of differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) according to one embodiment of the present invention is effective in preventing and treating corneal endothelial dysfunction.

[0145]

[0146] Experimental Example 5. Confirmation of reduction in corneal inflammation following induced corneal endothelial cell transplantation.

[0147] To confirm the reduction of corneal inflammation following induced corneal endothelial cell transplantation, the experiment was conducted in the same manner as Experimental Example 4, except that anti-human anti-TNF-α (1:250; Life Technologies) antibody and anti-IL-1β antibody (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) were used as the primary antibodies, and the results are shown in Figs. 10 and 11.

[0148] Figure 10 shows photographs of corneas obtained from each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial dysfunction according to the daily pattern, and checking for the expression of TNF-α and IL-1β.

[0149] Figure 11 is a graph showing the number of cells expressing TNF-α and IL-1β in corneal tissue of an animal model experimental group of corneal endothelial dysfunction according to the daily aspect.

[0150] Meanwhile, to confirm the effect of reducing corneal inflammation when umbilical cord-derived mesenchymal stem cells (MSCs) and / or differentiated corneal endothelial cells (iPSC-CECs) were administered to an animal model of corneal endothelial dysfunction according to the daily pattern, the expression of TNF-α, IL-1, and IL-6 was confirmed through Western blot.

[0151] Specifically, corneal samples collected from rabbit models were washed in cold PBS and incubated in a lysis buffer containing phosphatase and protease inhibitors. The tissues, digested by homogenization or sonication, were centrifuged, and the supernatant was separated. Proteins were adsorbed onto PVDF membranes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membranes were then blocked in TBST buffer containing bovine serum albumin. The previously prepared membranes were incubated overnight at 4°C with IL-1, IL-6, TNF-α, and β-actin. The membranes were then washed and incubated with HRP-linked secondary antibodies. The bands were identified using a chemiluminescence imaging system, and the results are shown in Figure 12.

[0152] Figure 12 is a photograph showing the results of transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial dysfunction according to each experimental group, and after 6 weeks, obtaining corneas from each experimental group and confirming the expression of TNF-α, IL-1, and IL-6 using Western blotting.

[0153] As shown in Figures 10 to 12, when differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) were transplanted together, the number of cells expressing TNF-α, IL-1β, and IL-6 was confirmed to significantly increase compared to the control group.

[0154] TNF-α, IL-1β, and IL-6 are all cytokines associated with promoting inflammatory responses, and the above experimental results indicate that corneal inflammation in an animal model of corneal endothelial failure was improved. Therefore, according to one embodiment of the present invention, transplantation of differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) is effective in preventing and treating corneal endothelial failure.

[0155]

[0156] Experimental Example 6. Inhibition of Apoptosis in Corneal Cells Following Induced Corneal Endothelial Cell Transplantation

[0157] Corneal endothelial cells induced by the method of Example 3.2 were transplanted into the anterior chamber of the corneal endothelial insufficiency animal model produced by the method of Example 3.1, and after 6 weeks, the cornea of ​​the corneal endothelial insufficiency animal model was obtained and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining was performed.

[0158] Specifically, the pottery tissues sectioned from paraffin blocks at 5 μm in size were heated at 60°C for approximately 30 minutes and dewaxed by washing with xylene and ethanol. Afterwards, the cells were stained with DAPI, 4', 6-diamidino-2-phenylindole (Roche, Inc., Mannheim, Germany) and fluorescence photographs were taken using a confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany). The distribution and number of TUNEL-positive cells were confirmed, and the results are shown in Figs. 13 and 14.

[0159] Figure 13 shows photographs of corneas obtained from each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial failure according to the daily pattern, and confirming cell death through TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining.

[0160] Figure 14 is a graph showing the number of cells in which cleaved DNA is detected in corneal tissue of an animal model experimental group of corneal endothelial dysfunction according to the daily aspect.

[0161] As shown in Figures 13 and 14, when differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) were transplanted together, the number of cells in which cleaved DNA was detected was significantly reduced compared to the control group.

[0162] This means that the phenomenon of apoptosis in the cornea has been improved, and that transplantation of differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) according to one embodiment of the present invention is effective in preventing and treating corneal endothelial dysfunction.

[0163]

[0164] Experimental Example 7. Confirmation of Alleviation of Corneal Fibrosis Following Induced Corneal Endothelial Cell Transplantation

[0165] To confirm the alleviation of corneal fibrosis following induced corneal endothelial cell transplantation, the experiment was conducted in the same manner as Experimental Example 4, except that anti-human anti-α-SMA (1:250; Life Technologies) antibody was used as the primary antibody, and the results are shown in Figs. 15 and 16.

[0166] Figure 15 shows photographs of corneas obtained from each experimental group 6 weeks after transplanting umbilical cord-derived mesenchymal stem cells (MSCs), differentiated corneal endothelial cells (iPSC-CECs), and differentiated corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSCs) into the anterior chamber of an animal model of corneal endothelial insufficiency according to the daily pattern, and checking for the expression of α-SMA (α-smooth muscle actin).

[0167] Figure 16 is a graph showing the expression of α-SMA in corneal tissue of an animal model experimental group of corneal endothelial dysfunction according to the daily aspect.

[0168] As shown in FIGS. 15 and 16, when transplanting corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) induced to differentiate according to one embodiment of the present invention, it was confirmed that the expression level of α-SMA (α-smooth muscle actin) was significantly reduced compared to the control group.

[0169] This means that corneal fibrosis in the corneal endothelial failure animal model experimental group was alleviated, and that transplantation of differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) according to one embodiment of the present invention is effective in preventing and treating corneal endothelial failure.

[0170]

[0171] Experimental Example 8. Confirmation of mitigation of allograft rejection following induced corneal endothelial cell transplantation.

[0172] To determine whether corneal endothelial cell and / or stem cell transplantation induced in an animal model of corneal endothelial dysfunction according to the daily pattern alleviates allograft rejection, ELISA was performed.

[0173] Specifically, reagents, standards, and samples (water-resistant fluid) were prepared (CXCL-9, 10, and 11, R and D systems). 150 μL of Assay Diluent was added to each well, followed by 100 μL of standards, controls, and water-resistant fluid (75 μL), and incubated at room temperature for 2 hours. Afterwards, each well was washed four times, 200 μL of Conjugate was added, and incubated for 2 hours. Afterwards, each well was washed four times, 200 μL of Substrate Solution was added, and incubated for 30 minutes in a light-protected state. After adding 50 μL of Stop Solution, measurements were taken at 450 nm within 30 minutes, and the wavelength was corrected to 540 nm or 570 nm.

[0174] Figure 17 is a graph showing the expression of CXCL9, CXCL10, and CXCL11 in an animal model of corneal endothelial dysfunction according to the daily pattern.

[0175] As shown in Figure 17, when differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) were transplanted together, it was confirmed that the expression levels of CXCL9, CXCL10, and CXCL11 were reduced compared to the control group.

[0176] This means that the allograft rejection reaction was alleviated in the corneal endothelial failure animal model experimental group, and that transplantation of differentiation-induced corneal endothelial cells and umbilical cord-derived mesenchymal stem cells (iPSC-CEC+MSC) according to one embodiment of the present invention is effective in preventing and treating corneal endothelial failure.

Claims

1. A pharmaceutical composition for preventing or treating corneal disease comprising differentiated corneal endothelial cells (CECs) and stem cells.

2. A pharmaceutical composition for preventing or treating corneal disease, wherein the composition comprises the differentiation-induced corneal endothelial cells and stem cells in a ratio of 0.5 to 2.0: 0.5 to 2.0 according to claim 1.

3. In claim 1, the composition comprises 1Х10 corneal endothelial cells induced to differentiate 4 Dog 1X10 7 A pharmaceutical composition for preventing or treating corneal diseases, comprising a dog.

4. In claim 1, the composition comprises 1Х10 of the stem cells. 4 Dog 1X10 7 A pharmaceutical composition for preventing or treating corneal diseases, comprising a dog.

5. A pharmaceutical composition for preventing or treating corneal disease, characterized in that the composition satisfies at least one of the following characteristics in claim 1: - Improves corneal opacity; - Reduces corneal thickness; - Improves corneal pumping function and intercellular adhesion; - Reduces the expression of inflammatory cytokines; - Reduces apoptosis in the cornea; - Reduces corneal fibrosis; and - Alleviates allograft rejection.

6. A pharmaceutical composition for preventing or treating corneal disease, wherein the stem cells of claim 1 are embryonic stem cells (ESCs), adult stem cells or human induced pluripotent stem cells (iPSCs).

7. A pharmaceutical composition for preventing or treating corneal disease, wherein the adult stem cells of claim 6 are mesenchymal stem cells (MSCs).

8. A pharmaceutical composition for preventing or treating corneal disease according to claim 6, wherein the adult stem cells are selected from the group consisting of stem cells derived from bone marrow, blood, umbilical cord blood, umbilical cord, adipose tissue, liver, skin, gastrointestinal tract, placenta, and uterus.

9. A pharmaceutical composition for preventing or treating corneal disease according to claim 1, wherein the corneal endothelial cells induced to differentiate are differentiated from embryonic stem cells (ESCs), adult stem cells or human induced pluripotent stem cells (iPSCs).

10. A pharmaceutical composition for preventing or treating corneal disease according to claim 1, wherein the corneal disease is at least one selected from the group consisting of corneal endothelial decompensation, Fuchs' dystrophy, corneal endothelial cell dystrophy, corneal edema, primary posterior polymorphous corneal dystrophy (PPCD), and corneal transplant rejection.

11. A first formulation comprising differentiated corneal endothelial cells (CEC); and A combination preparation for preventing or treating corneal disease, comprising a second preparation containing stem cells.

12. A combination preparation for preventing or treating corneal disease, wherein the first preparation and the second preparation according to claim 11 are for direct administration to the eye.

13. A combination administration preparation for preventing or treating corneal disease, wherein the first preparation and the second preparation in claim 10 are administered simultaneously, sequentially or in reverse order.

14. In claim 11, the first preparation comprises 1X10 corneal endothelial cells (CECs) that have been induced to differentiate. 4 Dog 1X10 7 A combination preparation for the prevention or treatment of corneal diseases, comprising a dog.

15. In claim 11, the second preparation comprises 1X10 of the stem cells. 4 Dog 1X10 7 A combination preparation for the prevention or treatment of corneal diseases, comprising a dog.

16. A cell therapy agent for preventing or treating corneal diseases, comprising differentiated corneal endothelial cells (CECs) and stem cells.

17. Use of a composition comprising differentiated corneal endothelial cells (CECs) and stem cells for preventing or treating corneal diseases.

18. A method for preventing or treating corneal disease, comprising administering to a subject in need thereof a composition comprising an effective amount of differentiated corneal endothelial cells (CECs) and stem cells.

19. Use of a composition comprising differentiated corneal endothelial cells (CECs) and stem cells for use in the manufacture of a medicament for preventing or treating corneal diseases.

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