Use of corneal cell-derived mitochondria for treating corneal damage

Abstract

The present invention relates to the use of corneal cell-derived mitochondria for preventing or treating corneal damage or corneal endothelial cell diseases. Mitochondria isolated from corneal endothelial cells differentiated from induced pluripotent stem cells (iPSCs), of the present invention, were confirmed to alleviate inflammatory responses of corneal endothelial cells in an inflammatory environment, restore the integrity and function of corneal endothelial cells, inhibit inflammatory responses, particularly inflammation caused by physical damage, upon delivery into xenogeneic primary corneal endothelial cells, and promote the regeneration of corneal endothelial cells. Therefore, mitochondria having these effects can overcome the oncogenic limitations of conventional stem cell therapeutic agents, and are widely applicable and exhibit excellent cost efficiency due to their ease of production and fewer regulations.

Description

Use of corneal cell-derived mitochondria for the treatment of corneal damage

[0001] The present invention relates to the use of corneal cell-derived mitochondria for the prevention or treatment of corneal damage or corneal endothelial cell disease.

[0002] The cornea is the outermost structure of the eyeball and is one of the primary organs responsible for refracting light. It consists of a total of six layers: the corneal epithelium, Bowman's layer, corneal stroma, Dua's layer, Descemet's membrane, and corneal endothelium. The corneal epithelium is composed of 5 to 6 cell layers; base cells are generated from peripheral stem cells, migrate to the center, and slough off after 7 days. Bowman's layer consists of cellless collagen fibers and is colorless and transparent. However, it cannot regenerate and leaves scars in the event of surgery or trauma. The corneal stroma accounts for 90% of the corneal thickness, and the direction and size of its cells are uniform. Descemet's membrane is composed of 3 to 4 layers of cells and is presumed to be the basement membrane of the endothelium. The corneal endothelium consists of a single layer of cells; the composition of these cells is fixed, regeneration is extremely limited, and the number of cells decreases with age. When the number of cells decreases, surrounding cells enlarge to fill the space. The corneal endothelium is a single-layer structure of hexagonal cells located on the posterior surface of the cornea and contains embedded physiological ion pumps. Corneal endothelial cells (CECs) maintain mechanical strength by controlling hydration through the pumping of water from the stroma into the aqueous humor. At birth, the average density of human corneal endothelial cells is approximately 5,000 cells / mm³. 2It is known that... However, because mitotic potential is limited, the total number of cells decreases with age. When corneal endothelial cells are damaged, the cornea repairs itself through cell expansion and migration rather than mitosis; consequently, the cornea swells to several times its normal thickness, becomes opaque, and loses its function. The damage is permanent. Currently, the established treatment for corneal endothelial cell damage is corneal transplantation. Recovery can be achieved through the transplantation of donor tissue via penetrating keratoplasty (PK) or lamellar keratoplasty. However, due to the severe shortage of corneal donors, the development of technology to overcome this is an urgent priority.

[0003] Meanwhile, new research on stem cell differentiation, such as the induction of induced pluripotent stem cells (iPSCs) and mesenchymal stem cells into corneal endothelial cells, has recently been conducted for clinical treatment. A study reported in 2016 described the derivation of corneal endothelial-like cells (CECs) from induced pluripotent stem cells (iPSCs) derived from BJ human foreskin fibroblasts (Zhao and Afshari. 2016). Similarly, another study devised a procedure for induced pluripotent stem cells (iPSCs) derived from adult fibroblasts to induce corneal endothelial-like cells (CECs) (Wagoner et al. 2018). Although research related to these stem cells has been actively conducted, their use is limited due to the risks associated with direct stem cell injection, such as the potential to induce inflammation or differentiate into other cells, particularly tumors. Therefore, research has recently been moving toward utilizing stem cell-derived substances to achieve the therapeutic effects of stem cells while limiting the risks.

[0004] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of corneal damage.

[0005] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of corneal endothelial cell disease.

[0006] In addition, the present invention provides a composition for corneal endothelial cell transplantation.

[0007] In addition, the present invention provides a method for producing mitochondria derived from corneal endothelial cells or corneal endothelial-like cells.

[0008] In addition, the present invention provides a method for treating corneal damage.

[0009] In addition, the present invention provides a method for treating corneal endothelial cell disease.

[0010] In addition, the present invention provides a use for the prevention or treatment of corneal damage.

[0011] In addition, the present invention provides a use for the prevention or treatment of corneal endothelial cell disease.

[0012] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of corneal damage comprising mitochondria derived from corneal endothelial cells or corneal endothelial-like cells as an active ingredient.

[0013] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of corneal endothelial cell diseases comprising mitochondria derived from corneal endothelial cells or corneal endothelial-like cells as an active ingredient.

[0014] In addition, the present invention provides a composition for corneal endothelial cell transplantation comprising mitochondria derived from corneal endothelial cells or corneal endothelial-like cells.

[0015] In addition, the present invention provides a method for producing mitochondria derived from corneal endothelial cells or corneal endothelial-like cells.

[0016] In addition, the present invention provides a method for treating corneal damage comprising the step of administering mitochondria derived from corneal endothelial cells or corneal endothelial-like cells to a damaged cornea.

[0017] In addition, the present invention provides a method for treating corneal endothelial disease comprising the step of administering mitochondria derived from corneal endothelial cells or corneal endothelial-like cells to an individual suffering from corneal endothelial disease.

[0018] In addition, the present invention provides mitochondria derived from corneal endothelial cells or corneal endothelial-like cells for the prevention or treatment of corneal damage.

[0019] In addition, the present invention provides the use of mitochondria derived from corneal endothelial cells or corneal endothelial-like cells for the prevention or treatment of corneal endothelial cell diseases.

[0020] It was confirmed that mitochondria isolated from corneal endothelial cells differentiated from induced pluripotent stem cells (iPSCs) of the present invention alleviate the inflammatory response of corneal endothelial cells in an inflammatory environment, restore the integrity and function of corneal endothelial cells, and, when delivered into heterologous primary corneal endothelial cells, suppress inflammatory responses, particularly inflammation caused by physical damage, and promote the regeneration of corneal endothelial cells. Therefore, mitochondria possessing such effects can overcome the tumorigenic limitations of conventional stem cell therapies, and due to their ease of production and minimal regulatory requirements, they offer excellent benefits for broad application and cost-effectiveness.

[0021] Figures 1a and 1b are a schematic diagram of the process of differentiating induced pluripotent stem cells (iPSCs) into corneal endothelial cells and a cell image (a) of the corresponding period, and Figure (b) confirming the expression of representative corneal endothelial cell markers by immunofluorescence staining.

[0022] Figures 2a and 2b show the confirmation of mitochondria (iPSC-CEC Mt) (a) isolated from corneal endothelial cells differentiated from induced pluripotent stem cells by Western blot analysis (Figure (b)).

[0023] Figure 3 shows the changes in the mitochondrial network induced by iPSC-CEC Mt in an inflammatory environment / condition in which induced pluripotent stem cell-derived corneal endothelial cells (iPSC-CEC) were treated with LPS and IFN-γ:

[0024] Complete media: Badge-treated group (control group);

[0025] Inflammatory condition (I): group treated with LPS (20 μg / ml) and IFN-γ (30 ng / ml); and

[0026] Inflammatory condition + iPSC-CEC Mt 2.5μg / ml: Groups treated with LPS (20μg / ml), IFN-γ (30ng / ml) and iPSC-CEC Mt (2.5μg / ml).

[0027] Figure 4 shows the antioxidant effect of iPSC-CEC Mt on iPSC-CECs in an inflammatory environment / condition, confirmed by the analysis of the degree of ROS generation and mitochondrial morphology:

[0028] Complete media: Badge-treated group (control group);

[0029] Inflammatory condition (I): group treated with LPS (20 μg / ml) and IFN-γ (30 ng / ml); and

[0030] Inflammatory condition + iPSC-CEC Mt 2.5μg / ml: Groups treated with LPS (20μg / ml), IFN-γ (30ng / ml) and iPSC-CEC Mt (2.5μg / ml).

[0031] Figure 5 is a figure confirming the anti-inflammatory effect of iPSC-CEC Mt on iPSC-CECs in an inflammatory environment / condition through the expression of related markers:

[0032] Complete media: Badge-treated group (control group);

[0033] Inflammatory condition (I): Group treated with LPS (20 μg / ml) and IFN-γ (30 ng / ml);

[0034] I + iPSC-CEC Mt 2.5μg / ml: group treated with LPS (20μg / ml), IFN-γ (30ng / ml) and iPSC-CEC Mt (2.5μg / ml); and

[0035] I + iPSC-CEC Mt 5μg / ml: Groups treated with LPS (20μg / ml), IFN-γ (30ng / ml) and iPSC-CEC Mt (5μg / ml).

[0036] Figures 6a and 6b confirm the effect of iPSC-CEC Mt on restoring cell integrity / function of iPSC-CECs in an inflammatory environment / conditions through cell motility analysis (a) and the expression of cell survival markers and growth markers (b):

[0037] Complete media: Group treated with badge (control group);

[0038] Inflammatory condition (I): Group treated with LPS (20 μg / ml) and IFN-γ (30 ng / ml);

[0039] iPSC-CEC Mt 2.5μg / ml: Group treated with 2.5μg / ml of iPSC-CEC Mt;

[0040] iPSC-CEC Mt 5μg / ml: group treated with 5μg / ml of iPSC-CEC Mt; and

[0041] I + iPSC-CEC Mt 2.5μg / ml: Groups treated with LPS (20μg / ml), IFN-γ (30ng / ml) and iPSC-CEC Mt (2.5μg / ml).

[0042] Figure 7 confirms the recovery of cell morphology of iPSC-CECs by iPSC-CEC Mt in an inflammatory environment / condition:

[0043] Complete media: Group treated with badge (control group);

[0044] Inflammatory condition: Group treated with LPS (20 μg / ml) and IFN-γ (30 ng / ml);

[0045] iPSC-CEC Mt 2.5μg / ml: group treated with 2.5μg / ml of iPSC-CEC Mt; and

[0046] iPSC-CEC Mt 5μg / ml: Group treated with 5μg / ml of iPSC-CEC Mt.

[0047] Figure 8 shows the changes in the expression of corneal endothelial cell markers induced by iPSC-CEC Mt in iPSC-CECs under inflammatory environments / conditions:

[0048] Complete: Group treated with badge (control group);

[0049] Inflammatory condition (I): group treated with LPS (20 μg / ml) and IFN-γ (30 ng / ml); and

[0050] I + iPSC-CEC Mt 2.5: Group treated with LPS (20 μg / ml), IFN-γ (30 ng / ml) and iPSC-CEC Mt (2.5 μg / ml).

[0051] Figures 9a and 9b show the delivery effect of human-derived iPSC-CEC Mt into primary corneal endothelial cells (RCECs) of heterologous rabbits:

[0052] a: Co-localization of RCEC endogenous mitochondria and iPSC-CEC Mt;

[0053] b: Transfer of iPSC-CEC Mt into RCEC over time;

[0054] RCEC Mitochondria: Endogenous mitochondria of RCEC; and

[0055] Exogenous Mt. from iPSC-CECs: iPSC-CEC Mt.

[0056] Figure 10 illustrates the induction of an inflammatory environment / conditions in primary corneal endothelial cells (RCECs) of heterologous rabbits and the verification of mitochondrial network restoration and antioxidant effects by iPSC-CEC Mt:

[0057] Complete media: Group treated with complete media containing FBS;

[0058] Basal media: Group treated with basal media excluding FBS (Serum starvation); and

[0059] I + iPSC-CEC Mt 2.5: Group treated with LPS (20 μg / ml), IFN-γ (30 ng / ml) and iPSC-CEC Mt (2.5 μg / ml).

[0060] Figure 11 shows the results of inducing an inflammatory environment / condition in primary corneal endothelial cells (RCECs) of heterologous rabbits and analyzing the anti-inflammatory effect induced by iPSC-CEC Mt through changes in the mRNA expression of IL-6 and TNF-α:

[0061] Control: Group treated with basic media excluding FBS (Serum starvation);

[0062] Inflammatory condition (I): Group treated with LPS (20 μg / ml) and IFN-γ (30 ng / ml);

[0063] I + iPSC-CEC Mt 2.5μg / ml: group treated with LPS (20μg / ml), IFN-γ (30ng / ml) and iPSC-CEC Mt (2.5μg / ml); and

[0064] I + iPSC-CEC Mt 5μg / ml: Groups treated with LPS (20μg / ml), IFN-γ (30ng / ml) and iPSC-CEC Mt (5μg / ml).

[0065] Figure 12 shows the results of inducing an inflammatory environment / condition or serum starvation in primary corneal endothelial cells (RCECs) of heterologous rabbits and confirming the effect of iPSC-CEC Mt on cell integrity / function recovery through cell motility analysis:

[0066] Complete media: Group treated with complete media containing FBS;

[0067] Serum starvation: Group treated with basic media excluding FBS;

[0068] Control: Control group treated only with the corresponding medium;

[0069] iPSC-CEC Mt 2.5μg / ml: group treated with 2.5μg / ml of iPSC-CEC Mt; and

[0070] iPSC-CEC Mt 5μg / ml: Group treated with 5μg / ml of iPSC-CEC Mt.

[0071] Figures 13a and b show the results of transplanting iPSC-CEC Mt into a rabbit corneal endothelial edema model and confirming ocular recovery:

[0072] a: Corneal endothelial edema and turbidity;

[0073] b: corneal thickness;

[0074] Normal: Normal group;

[0075] Corneal endothelial dystrophy (CED): Corneal endothelial edema induction group;

[0076] untreated: untreated group;

[0077] Reagent C: Group that processed the vehicle; and

[0078] iPSC-CEC Mt 45μg / ml: Group treated with 45μg / ml of iPSC-CEC Mt.

[0079] Figure 14 shows an H&E stained image of a rabbit corneal endothelial edema model transplanted with iPSC-CEC Mt:

[0080] Normal: Normal group;

[0081] CED: Corneal endothelial edema induction group;

[0082] CED + Reagent C: Group treated with vehicle and induced corneal endothelial edema; and

[0083] CED + iPSC-CEC Mt: Group treated with 45 μg / ml of iPSC-CEC Mt after inducing corneal endothelial edema.

[0084] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments thereof. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited by this. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the description have been omitted, and similar parts throughout the specification have been given similar reference numerals.

[0085] Throughout the specification, when it is stated that a part is "connected (connected, in contact, combined)" with another part, this includes not only cases where they are "directly connected," but also cases where they are "indirectly connected" with other members interposed between them. Furthermore, when it is stated that a part "includes" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but rather allows for the inclusion of additional components.

[0086] The terms used herein are merely for describing specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as “comprising” or “having” are intended to indicate the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0087] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0088]

[0089] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of corneal damage comprising mitochondria derived from corneal endothelial cells or corneal endothelial-like cells as an active ingredient.

[0090] In one embodiment, the corneal endothelial cells or corneal endothelial-like cells may be derived from embryonic stem cells (ESCs), adult stem cells (ASCs), or induced pluripotent stem cells (iPSCs), and it is most preferable that the corneal endothelial cells or corneal endothelial-like cells are derived from induced pluripotent stem cells.

[0091] In one embodiment, the induced pluripotent stem cells may be derived from animals including humans, such as monkeys, chimpanzees, pigs, horses, cattle, sheep, dogs, cats, mice, rats, rabbits, etc.

[0092] In one embodiment, the adult stem cells may be mesenchymal stem cells (MSCs), and the mesenchymal stem cells may be umbilical cord blood-derived mesenchymal stem cells (UCB-MSC), umbilical cord-derived mesenchymal stem cells (UC-MSC), adipose-derived mesenchymal stem cells (AD-MSC), or bone marrow-derived mesenchymal stem cells (BM-MSC).

[0093] In one embodiment, the mitochondria derived from the corneal endothelial cell or corneal endothelial-like cell may be isolated from a culture, lysate, or lysate of the cell.

[0094] In one embodiment, corneal damage may be physical or chemical damage, and it is more preferable that it be physical damage to corneal endothelial cells.

[0095] In one embodiment, the composition may have the effect of alleviating corneal endothelial edema, regenerating corneal endothelial cells, restoring the integrity or function of corneal endothelial cells, increasing the mobility or proliferation of corneal endothelial cells, improving corneal opacity, or restoring corneal thickness in relation to corneal damage.

[0096] In one embodiment, the composition can reduce mitochondrial fission or ROS generation in corneal endothelial cells.

[0097] In one embodiment, the composition can increase mitochondrial fusion in corneal endothelial cells.

[0098] In one embodiment, the composition can reduce the expression of IL-6 (Interleukin 6) or TNF-α (tumor necrosis factor-α) in corneal endothelial cells.

[0099] In one embodiment, the composition can increase the expression of MFN-2 (Mitofusin-2), ZO-1 (Zonula Occludens-1), N-cadherin, CD166, ATP1A1 (Sodium / potassium-transporting ATPase subunit alpha-1) or SLC4A11 (Solute Carrier Family 4 Member 11) in corneal endothelial cells.

[0100] In one embodiment, the composition can reduce the phosphorylation of p38 or NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) in corneal endothelial cells.

[0101] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of corneal endothelial cell diseases comprising mitochondria derived from corneal endothelial cells or corneal endothelial-like cells as an active ingredient.

[0102] In one embodiment, the corneal endothelial cell disease may be corneal endothelial cell inflammation, corneal endothelial cell edema, corneal endothelial cell damage, or a decrease in the number of corneal endothelial cells, and it is more preferable that the disease be related to inflammation caused by a decrease in the number of corneal endothelial cells.

[0103] In one embodiment, the corneal endothelial disease may be corneal endothelial insufficiency, corneal endothelial dystrophy (CED), bullous keratopathy, Fuchs endothelial corneal dystrophy (FECD), corneal endothelial dystrophy (CED), or corneal endotheliitis.

[0104] In one embodiment, the corneal endothelial cell disease may be caused by one or more selected from ocular trauma, sequelae of ocular surgery, viral infection, ocular inflammation, aging, diabetes, oxidative stress, oxygen deficiency due to contact lens wear, increased intraocular pressure, and lysosome accumulation.

[0105] In one embodiment, the composition can suppress the occurrence of inflammation in corneal endothelial cells, particularly the inflammatory response that occurs as the number of corneal endothelial cells decreases.

[0106] In one embodiment, the pharmaceutical composition may be for intravitreal injection, subretinal injection, and ocular local administration in the form of eye drops, and the pharmaceutical composition may be any one formulation selected from the group consisting of topical skin preparations, injections, infusions, sprays, liquids, ointments, suspensions, syrups, emulsions, powders, granules, tablets, sustained-release preparations, eye drops, capsules, intraocular implants, intraocular injections, contact lens cleaning agents, and contact lens lubricants.

[0107] The term "corneal endothelial dystrophy (CED)" used in the present invention is also referred to as "corneal dystrophy" and may be a group of rare genetic diseases in which substances accumulate abnormally in the cornea, including one or more selected from granular corneal dystrophy, lattice corneal dystrophy, and maculopylate corneal dystrophy. In this case, the vision may become blurred due to the substances.

[0108] The term "Fuchs Endothelial Corneal Dystrophy (FECD)" used in this invention is also known as Fuchs dystrophy and is a disease in which damage occurs as the number of corneal endothelial cells decreases without any particular reason, causing edema in the corneal stroma or epithelium, which can lead to a decline in vision.

[0109] The term "Bullous keratopathy (BK)" used in the present invention may refer to an eye disorder in which the cornea swells up like a blister.

[0110] The term "corneal endotheliitis" used in the present invention is characterized by causing edema of the corneal stroma without accompanying infiltration of the stroma, is caused by infection or delayed hypersensitivity reaction, and may appear in the form of discoid keratodermatitis, extensive keratodermatitis, linear keratodermatitis, etc.

[0111] As used in the present invention, the term "expression" generally refers to a cellular process in which a biologically active polypeptide is generated from a DNA sequence and exhibits biological activity in a cell. In this sense, gene expression includes not only transcription and translation processes, but also post-transcriptional and post-translational processes that may affect the biological activity of the gene or gene product. These processes include, but are not limited to, RNA synthesis, processing, and transport, as well as polypeptide synthesis, transport, and post-translational modification of the polypeptide.

[0112] In the present invention, mitochondria are intracellular organelles comprising a double membrane of an inner membrane and an outer membrane made of a phospholipid bilayer, a matrix inside the inner membrane, and an intermembrane space between the inner membrane and the outer membrane, wherein a portion of the inner membrane protrudes inward and forms a 'cristae' structure that is folded in multiple layers, and the average length of the longest portion may be about 10 nm to about 50 µm, about 10 nm to about 30 µm, about 10 nm to about 10 µm, about 50 nm to about 50 µm, about 50 nm to about 30 µm, or about 50 nm to about 10 µm. The mitochondria may include mitochondria contained within corneal endothelial cells, mitochondria secreted from corneal endothelial cells, mitochondria produced by the division of said mitochondria, or all of these.

[0113] In the present invention, the expression can be confirmed by measuring the expression level of a gene or mRNA using a polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, nuclease protection assay (RNase, S1 nuclease assay), in situ hybridization, nucleic acid microarray, Northern blot, or DNA chip method using a nucleic acid sequence, a nucleic acid sequence complementary to the nucleic acid sequence, a primer pair, a probe, or a primer pair and a probe that specifically recognize the nucleic acid sequence and a fragment of the sequence complementary to the nucleic acid sequence, and using an antibody, antibody fragment, aptamer, avidity multimer, or peptidomimetics that specifically recognize the entire length of the corresponding protein or its fragment using Western blot, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay (RIA), Protein expression levels can be confirmed by measuring them using radioimmunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray methods.

[0114] The term "treatment" as used in the present invention, unless otherwise noted, means reversing, alleviating, inhibiting the progression thereof, or preventing exacerbation after onset of a disease or condition to which the term applies, or one or more symptoms of said disease or condition, and the term "treatment" as used in the present invention refers to the act of treating corneal endothelial cell disease. Accordingly, treatment or therapeutic regimen for corneal endothelial cell disease in mammals may include one or more of the following:

[0115] (1) Inhibits the development of corneal endothelial disease, that is, inhibits its development;

[0116] (2) Prevents worsening of corneal endothelial disease after it occurs;

[0117] (3) Alleviates corneal endothelial disease;

[0118] (4) Prevents recurrence of corneal endothelial disease; and

[0119] (5) Palliating the symptoms of corneal endothelial disease.

[0120] As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation, or experiment, preferably a human.

[0121] If the recipient animal can tolerate the administration of the composition or if the administration of the composition to the animal is appropriate, the composition indicates "pharmaceutical or physiologically acceptable." If the administered amount is physiologically significant, the said preparation may be said to have been administered at a "therapeutically effective amount." If the presence of the said preparation causes a physiologically detectable change in the recipient patient, the said preparation is physiologically significant.

[0122] The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, and the patient's condition. Therefore, when used in humans, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations for determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0123] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level may be determined based on factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Considering all of the above factors, it is important to administer an amount that obtains maximum effect with a minimum amount without adverse effects, and this can be easily determined by a person skilled in the art.

[0124] The composition of the present invention may also include carriers, diluents, excipients, or combinations of two or more thereof that are commonly used in biological preparations. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition, and may be used, for example, compounds listed in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the composition into primary formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the field or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0125] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions, each according to conventional methods.

[0126] As used in the present invention, the term "pharmaceuticalally acceptable" means exhibiting a property that is not toxic to cells or humans exposed to the composition.

[0127] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0128] The active ingredient or pharmaceutical composition provided in the present invention may be administered by parenteral administration or by contacting cells, tissues, or body fluids in or separated from the body. Specifically, the active ingredient or pharmaceutical composition may be administered parenterally, for example, directly to the site where inflammation of the corneal endothelial cells has occurred (e.g., by injection). In this case, the active ingredient or pharmaceutical composition may be formulated in the form of an injectable that can be directly administered (e.g., by injection) to the affected area, i.e., the site where inflammation of the corneal endothelial cells has occurred. In this case, to ensure product stability during the prescription and distribution of the injectable, the injectable may be manufactured as a physically or chemically very stable injectable by adjusting the pH using a buffer solution such as an acidic aqueous solution or a phosphate that can be used as an injectable. Specifically, the injectable may contain water for injection. The above-mentioned water for injection is distilled water prepared for dissolving solid injectables or diluting water-soluble injectables, and may be glucose injection, xylitol injection, D-mannitol injection, fructose injection, physiological saline, dextran 40 injection, dextran 70 injection, amino acid injection, Ringer's solution, lactic acid-Ringer's solution, or a phosphate buffer solution or sodium dihydrogen phosphate-citrate buffer solution with a pH range of 3.5 to 7.5.

[0129] The term "administration" as used in the present invention means providing a specific substance to a patient by any appropriate method, and depending on the intended method, it may be administered parenterally (e.g., intravitreal, subretinal, intravenously, subcutaneously, intraperitoneally, or locally as an injectable formulation) or orally, and the dosage varies depending on the patient's weight, age, gender, health condition, diet, time of administration, method of administration, excretion rate, and severity of the disease. It is obvious that the concentration of the active ingredient included in the above pharmaceutical composition can be selected in various ways depending on the target.

[0130] The pharmaceutical composition of the present invention may be formulated into various oral or parenteral administration forms. Oral administration formulations include, for example, tablets, pills, hard and soft capsules, liquids, suspensions, emulsifiers, syrups, granules, etc., and these formulations may additionally include a diluent (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine) and a lubricant (e.g., silica, talc, stearic acid and its magnesium or calcium salt and / or polyethylene glycol) in addition to the active ingredient. Additionally, the tablet may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and, in some cases, may contain disintegrants or boiling mixtures such as starch, agar, alginic acid or its sodium salt, and / or absorbents, coloring agents, flavoring agents, and sweeteners. The formulation may be prepared by conventional mixing, granulation, or coating methods. Furthermore, a representative formulation for parenteral administration is an injectable formulation, and examples of solvents for the injectable formulation include water, Ringer's solution, isotonic saline, or suspensions. The sterile fixing oil of the injectable formulation may be used as a solvent or suspension medium, and any non-irritating fixing oil, including mono- and di-glycerides, may be used for this purpose. Additionally, the injectable formulation may use fatty acids such as oleic acid.

[0131] As used in this invention, the term "prevention" refers to any act of suppressing or delaying the occurrence, exacerbation, and recurrence of corneal endothelial cell disease by administering a pharmaceutical composition according to this invention.

[0132] In one aspect, the present invention relates to a composition for corneal endothelial cell transplantation comprising mitochondria derived from corneal endothelial cells or corneal endothelial-like cells.

[0133] In one embodiment, the corneal endothelial cells or corneal endothelial-like cells may be derived from embryonic stem cells (ESCs), adult stem cells (ASCs), or induced pluripotent stem cells (iPSCs), and it is most preferable that the corneal endothelial cells or corneal endothelial-like cells are derived from induced pluripotent stem cells.

[0134] In one embodiment, the induced pluripotent stem cells may be derived from animals including humans, such as monkeys, chimpanzees, pigs, horses, cattle, sheep, dogs, cats, mice, rats, rabbits, etc.

[0135] The terms “administering,” “introducing,” and “implanting” as used in the present invention are used interchangeably and may refer to the placement of a composition according to one embodiment into an individual by a method or route that results in at least partial localization of the composition according to one embodiment to a desired site. At least a portion of the components of the composition according to one embodiment may be administered by any suitable route to deliver to a desired location within a living individual.

[0136] In one aspect, the present invention relates to a method for producing mitochondria derived from corneal endothelial cells or corneal endothelial-like cells, comprising the steps of: differentiating induced pluripotent stem cells into corneal endothelial cells or corneal endothelial-like cells; and isolating mitochondria from corneal endothelial cells or corneal endothelial-like cells.

[0137] In one aspect, the present invention relates to a method for treating corneal damage comprising the step of administering mitochondria derived from corneal endothelial cells or corneal endothelial-like cells to a damaged cornea.

[0138] In one aspect, the present invention relates to a method for treating corneal endothelial disease comprising the step of administering mitochondria derived from corneal endothelial cells or corneal endothelial-like cells to an individual suffering from corneal endothelial disease.

[0139] In one aspect, the present invention relates to the use of mitochondria derived from corneal endothelial cells or corneal endothelial-like cells for the prevention or treatment of corneal damage.

[0140] In one aspect, the present invention relates to the use of mitochondria derived from corneal endothelial cells or corneal endothelial-like cells for the prevention or treatment of corneal endothelial cell diseases.

[0141] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0142]

[0143] Example 1. Isolation of mitochondria from corneal endothelial cells

[0144] 1-1. Preparation of stem cell-derived and corneal endothelial cell-derived mitochondria

[0145] Induced pluripotent stem cells (iPSCs) were differentiated into cornea endothelial-like cells (iPSC-CECs) for 14-15 days using cornea endothelial cell differentiation medium, and differentiation into cornea endothelial cells was confirmed by analyzing ZO-1, N-cadherin, CD166, SLC4A11, and ATP1A1, which are representative markers of cornea endothelial cells, using Immunocytochemistry (ICC) (Fig. 1). Mitochondria (iPSC-CECs Mt) were isolated from cornea endothelial cells (iPSC-CECs) differentiated from induced pluripotent stem cells using a mitochondria isolation kit (cat no. ab110170, abcam) (Fig. 2a).

[0146]

[0147] 1-2. Mitochondrial Analysis

[0148] The concentration of iPSC-CEC Mt isolated in 1-1 above was confirmed by BCA analysis, and the expression of a complex consisting of five complexes present in the inner membrane of mitochondria in the iPSC-CEC cells, the mitochondria isolated from iPSC-CEC (iPSC-CEC Mt), the supernatant remaining after mitochondria isolation, and iPSC-CEC cells was confirmed using an OXPHOS antibody.

[0149] As a result, it was confirmed that mitochondria were successfully isolated from iPSC-CECs, and the high purity of the isolated mitochondria was confirmed by the low expression of the cytoskeleton Actin (Fig. 2b).

[0150]

[0151] Example 2. Analysis of the therapeutic effect of iPSC-CEC Mt on corneal endothelial cell inflammation

[0152] 2-1. Effects of Mitochondrial Network Restoration

[0153] Inflammation was induced in induced pluripotent stem cell-derived corneal endothelial cells (iPSC-CECs) by treating them with LPS (5, 10, 20, or 30 μg / ml) and IFN-γ (10, 30, or 50 ng / ml) for 3, 24, 48, or 72 hours, and then an in vitro corneal endothelial cell inflammation model was constructed by determining the condition that best mimics the inflammatory environment (treatment with LPS 20 μg / ml and IFN-γ 30 ng / ml for 24 hours). Subsequently, to confirm the effect of iPSC-CEC Mt isolated in Example 1-1, an inflammatory environment was induced in induced pluripotent stem cell-derived corneal endothelial cells (iPSC-CECs) by treating them with LPS (20 μg / ml) and IFN-γ (30 ng / ml), while simultaneously treating the culture medium with 2.5 μg / ml of iPSC-CEC Mt. Immunofluorescence staining was used to analyze the expression of MFN-2 (Mitofusin-2) and changes in mitochondrial morphology 24 hours after inducing an inflammatory environment and administering iPSC-CEC Mt.

[0154] As a result, in the group that induced an inflammatory environment for 24 hours (Inflammatory condition), the production of MFN-2, which is involved in mitochondrial fusion, decreased and mitochondria were found to be fissioning, whereas in the group treated with iPSC-CEC Mt (Inflammatory condition + iPSC-CEC Mt 2.5 μg / ml), the expression of MFN-2 was restored to a level similar to the control group (Complete media), and the shape of the mitochondria was also confirmed to be fused to form a network, just like in the control group (Fig. 3).

[0155]

[0156] 2-2. Antioxidant Effects

[0157] To determine whether the iPSC-CECs Mt isolated in Example 1-1 above has antioxidant and anti-inflammatory effects that inhibit ROS generated in the inflammatory environment of corneal endothelial cells, an inflammatory environment was induced by treating iPSC-CECs with LPS (20 μg / ml) and IFN-γ (30 ng / ml), and at the same time, 2.5 μg / ml of iPSC-CECs Mt was treated, and immunofluorescence staining was performed after 30 minutes, 1, 3, 6, or 24 hours to analyze the morphology of mitochondria and total ROS generation.

[0158] As a result, the increased ROS generation upon inflammation induction (an increase of approximately 1.5 times compared to the control group) was found to decrease to a level similar to the control group (Complete media) upon treatment with iPSC-CECs Mt (Fig. 4). In addition, it was confirmed that the cell hypertrophy and changes in mitochondrial shape observed upon inflammation induction were restored to normal mitochondrial shape upon treatment with iPSC-CECs Mt (Fig. 4).

[0159]

[0160] 2-3. Inhibitory Effect on Inflammatory Marker Expression

[0161] To determine whether iPSC-CECs Mt has anti-inflammatory efficacy in the inflammatory environment of iPSC-CECs, inflammation was induced in iPSC-CECs by treating them with LPS 20 μg / ml and IFN-γ 30 ng / ml for 24 hours, and simultaneously with the induction of inflammation, iPSC-CECs Mt was treated with 2.5 or 5 μg / ml, after which the expression of p-p38 and p-NFκB, inflammatory markers involved in the secretion of inflammatory cytokines, was analyzed by Western blot analysis.

[0162] As a result, it was confirmed that the expression of p-p38, which increased by approximately 1.5 times under an inflammatory condition, recovered to a level similar to the control group upon treatment with iPSC-CECs Mt (Fig. 5). In addition, p-NFκB, whose expression increased under an inflammatory condition, also showed a tendency to decrease upon treatment with iPSC-CECs Mt (Fig. 5).

[0163]

[0164] Through this, it was confirmed that the mitochondria derived from stem cell-derived corneal endothelial cells (iPSC-CECs Mt) of the present invention regulate the expression of inflammation-related factors, induce mitochondrial fusion, and reduce ROS generation, thereby reducing the inflammatory response in corneal endothelial cells.

[0165]

[0166] Example 3. Analysis of the effect of iPSC-CEC Mt on restoring corneal endothelial cell integrity

[0167] To determine whether iPSC-CECs Mt has an effect on restoring the integrity and function of corneal endothelial cells in the environment of physical damage and subsequent inflammation observed in corneal diseases, physical damage and inflammation were induced in corneal endothelial cells, and changes in cell motility and the expression of inflammation-related markers induced by iPSC-CECs Mt were analyzed. Specifically, the physical damage induction group consisted of: control group (physical damage induction + Complete media); iPSC-CECs Mt 2.5 μg / ml treatment group (physical damage induction + iPSC-CECs Mt 2.5 μg / ml); and iPSC-CECs Mt 5 μg / ml treatment group (physical damage induction + iPSC-CECs Mt 5 μg / ml); and the physical damage and inflammation induction (Inflammatory condition) group consisted of: control group (physical damage induction + inflammation induction (LPS 20 μg / ml and IFN-γ 30 ng / ml) + Complete media); Cell groups were divided into an iPSC-CECs Mt 2.5 μg / ml treatment group (physical damage induction + inflammation induction + iPSC-CECs Mt 2.5 μg / ml) and an iPSC-CECs Mt 5 μg / ml treatment group (physical damage induction + inflammation induction + iPSC-CECs Mt 5 μg / ml). To induce physical damage, scratches were made on the cell culture dishes of each group, and the corresponding culture medium was added and cultured for 24 hours. Subsequently, cell migration was analyzed through the recovery of the induced scratches on the dishes, and the expression of the cell survival marker p-AKT and the cell growth marker p-ERK was confirmed by Western blot analysis. In addition, cell recovery was analyzed using a light microscope, and the expression of corneal endothelial cell markers ZO-1, ATP1A1, N-cadherin, CD166, and SLC4A11 was confirmed by immunofluorescence staining.

[0168] Analysis of cell migration revealed that in the groups induced with only physical damage, treatment with iPSC-CECs Mt resulted in scratch recovery to a degree similar to the control group (Fig. 6a). Unlike the group induced with only physical damage, in the group induced with both physical and inflammation, scratch recovery did not occur even after 24 hours, whereas in the group treated with iPSC-CECs Mt, scratch recovery was complete to a degree similar to that of the control group (Fig. 6a). Furthermore, analysis of the expression of related protein markers to confirm cell growth and survival recovery showed that the expression of p-AKT and p-ERK, which was decreased by inflammation induction, increased upon treatment with iPSC-CECs Mt (Fig. 6b). Analysis of cell morphology revealed that fibrosis was observed in control cells 24 hours after inflammation induction, unlike in the complete media group of cells without inflammation, whereas in cells treated with iPSC-CECs Mt, the cell morphology recovered to a degree similar to that of the complete media group (Fig. 7). Furthermore, analysis of corneal endothelial cell marker expression showed that the expression of ZO-1 and N-Cadherin, which decreased upon inflammation induction, was restored by iPSC-CECs Mt treatment, confirming the recovery of the cell barrier; additionally, the expression of ATP1A1 and SLC4A11, which act on water pumps, was also restored (Fig. 8).

[0169] Through this, it was found that the iPSC-CECs Mt of the present invention reduces the inflammatory response, and as a result, the integrity / function of the cells is also restored.

[0170]

[0171] Example 4. Analysis of the anti-inflammatory effects of iPSC-CEC Mt on heterologous primary corneal endothelial cells

[0172] 4-1. Analysis of Xenotransplantation Potential

[0173] To confirm the feasibility of transplanting human-derived iPSC-CECs Mt into primary cells, specifically rabbit corneal endothelial cells (RCECs), mitochondria of RCECs cultured in complete media were stained with Red mitotracker, and the iPSC-CECs Mt stained by incubating with Green mitotracker 500 nM for 30 minutes in a cell culture incubator at 37°C was added to the culture medium of the stained RCECs and confirmed by immunofluorescence staining. In addition, to determine the time required for the transplanted iPSC-CECs Mt to be delivered to RCECs and their retention, iPSC-CECs Mt were stained with mitotracker Red and added to the culture medium of RCECs.

[0174] As a result, it was confirmed that iPSC-CECs Mt and RCEC Mt coexist within RCEC (Fig. 9a). In addition, it was found that iPSC-CECs Mt entered the RCEC cells starting 1 hour after iPSC-CECs Mt treatment (Fig. 9b), confirming that the iPSC-CECs Mt of the present invention can be delivered to heterologous primary cells.

[0175]

[0176] 4-2. Mitochondrial Network Restoration and Antioxidant Effects

[0177] To determine whether iPSC-CECs Mt has an anti-inflammatory effect on primary rabbit corneal endothelial cells, an inflammatory environment was induced by treating with 20 μg / ml of LPS and 30 ng / ml of IFN-γ, and simultaneously, 2.5 μg / ml of iPSC-CECs Mt was added. After 24 hours, the recovery of the mitochondrial network and the amount of ROS generated were analyzed using immunofluorescence staining. At this time, to confirm the exact pattern resulting from inflammation induction, serum starvation was performed using basal media in which FBS was excluded from RCEC culture medium.

[0178] As a result, compared to the basic medium group, the amount of RCEC mitochondria in the inflammation-induced group was reduced and fragmented, and the generation of ROS was increased (Fig. 10). In contrast, in the group treated with iPSC-CECs Mt, mitochondrial fusion increased, indicating that the mitochondrial network was restored, and it was confirmed that the amount of ROS generated was reduced (Fig. 10).

[0179] Through this, it was confirmed that the iPSC-CECs Mt of the present invention in heterologous primary cells have an anti-inflammatory effect through the restoration of mitochondrial networks and the reduction of antioxidant activity.

[0180]

[0181] 4-3. Inhibitory Effect on Inflammatory Marker Expression

[0182] To determine whether iPSC-CECs Mt has an anti-inflammatory effect on primary rabbit corneal endothelial cells, an inflammatory environment was induced by treating with LPS 20 μg / ml and IFN-γ 30 ng / ml, and at the same time, iPSC-CECs Mt was treated with 2.5 or 5 μg / ml. After 24 hours, the expression of IL-6 and TNF-α, which are well-known inflammatory markers, was analyzed by RT-qPCR.

[0183] As a result, the expression of IL-6 and TNF-α, which was increased due to inflammation induction, was found to decrease upon iPSC-CECs Mt treatment (Fig. 11).

[0184]

[0185] Example 5. Analysis of the effect of iPSC-CEC Mt on restoring the integrity of heterologous primary corneal endothelial cells

[0186] To determine whether iPSC-CECs Mt has the effect of restoring the integrity / function of corneal endothelial cells in primary cells, such as rabbit corneal endothelial cells, in the environment of physical damage and subsequent inflammation occurring in corneal diseases, scratching was induced on a cell dish as in Example 3 and an inflammatory environment was induced by treating with LPS 20 μg / ml and IFN-γ 30 ng / ml, and at the same time, iPSC-CECs Mt was treated with 2.5 μg / ml or 5 μg / ml, and cell migration was analyzed after 0, 12, and 24 hours.

[0187] As a result, when treated with basic medium without FBS (Serum starvation), unlike the control group treated only with basic medium (Control), cell migration in the group treated with iPSC-CECs Mt proceeded to a similar extent as the group treated with complete media (Complete media) despite the nutrient-free environment (Fig. 12). In addition, while no significant cell migration occurred even at 24 hours in an inflammatory condition, it was confirmed that cell migration was significantly promoted when treated with iPSC-CEC Mt (Fig. 12).

[0188]

[0189] Example 6. Analysis of the in vivo therapeutic effect of iPSC-CEC Mt on corneal endothelial edema

[0190] 6-1. Establishment of a Corneal Endothelial Edema Model

[0191] To create a corneal endothelial dystrophy (CED) model, physical damage was induced in the eyes of New Zealand White Rabbits using a silicone tip to scrape away corneal endothelial cells while preserving Descemet's membrane (DM) (DM scraping method). After inducing physical damage, the removal of corneal endothelial cells was checked using trypan blue, and corneal opacity due to corneal edema was confirmed on day 3 of damage induction.

[0192]

[0193] 6-2. Analysis of the Effect of iPSC-CEC Mt Transplantation on Alleviating Corneal Endothelial Edema

[0194] iPSC-CEC Mt was implanted into the anterior chamber of the eye at a dose of 45 μg in a corneal endothelial edema model, and the recipient eye was laid down for 3 hours with the recipient eye facing downward. During this time, reagent C, the vehicle used to deliver the iPSC-CEC Mt, was used as a control. The eye was observed 3, 7, 10, and 14 days after implantation, and corneal thickness was analyzed using AS-OCT (anterior segment optical coherence tomography). In addition, the eye was stained with H&E to analyze corneal endothelial cell regeneration in the corneal edema area.

[0195] As a result, on day 7 after iPSC-CEC Mt transplantation, alleviation of corneal endothelial edema and improvement in turbidity due to the recovery of corneal endothelial cells were observed and maintained until Day 14 (Fig. 13a). In addition, it was confirmed that corneal thickness recovered to a level similar to normal by iPSC-CEC Mt transplantation (Fig. 13b). Furthermore, H&E staining results showed that corneal endothelial cells regenerated to a level close to normal by iPSC-CEC Mt transplantation (Fig. 14), confirming that the recovery of corneal edema was due to the regeneration of corneal endothelial cells.

[0196] Through this, it was verified that iPSC-CECs Mt transplantation in a rabbit corneal endothelial edema model promotes corneal endothelial cell regeneration, thereby inducing the recovery of corneal transparency and thickness resulting from the recovery of corneal edema.

Claims

1. A pharmaceutical composition for the prevention or treatment of corneal damage comprising mitochondria derived from corneal endothelial cells or corneal endothelial-like cells as an active ingredient.

2. A pharmaceutical composition for the prevention or treatment of corneal damage according to claim 1, wherein the corneal endothelial cells or corneal endothelial-like cells are derived from embryonic stem cells (ESC), adult stem cells (ASC), or induced pluripotent stem cells (iPSC).

3. A pharmaceutical composition for the prevention or treatment of corneal damage, wherein, in claim 1, the corneal damage is physical or chemical damage.

4. A pharmaceutical composition for the prevention or treatment of corneal damage according to claim 1, having the effects of alleviating corneal endothelial edema, regenerating corneal endothelial cells, restoring the integrity or function of corneal endothelial cells, increasing the mobility or proliferation of corneal endothelial cells, improving corneal opacity, or restoring corneal thickness.

5. A pharmaceutical composition for the prevention or treatment of corneal damage, wherein the composition of claim 1 reduces mitochondrial fission or ROS generation in corneal endothelial cells.

6. A pharmaceutical composition for the prevention or treatment of corneal damage, wherein the composition of claim 1 increases mitochondrial fusion in corneal endothelial cells.

7. A pharmaceutical composition for the prevention or treatment of corneal damage, wherein the composition of claim 1 reduces the expression of IL-6 (Interleukin 6) or TNF-α (tumor necrosis factor-α) in corneal endothelial cells.

8. A pharmaceutical composition for the prevention or treatment of corneal damage according to claim 1, which increases the expression of MFN-2 (Mitofusin-2), ZO-1 (Zonula Occludens-1), N-cadherin, CD166, ATP1A1 (Sodium / potassium-transporting ATPase subunit alpha-1) or SLC4A11 (Solute Carrier Family 4 Member 11) in corneal endothelial cells.

9. A pharmaceutical composition for the prevention or treatment of corneal damage, wherein, in claim 1, the phosphorylation of p38 or NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) in corneal endothelial cells is reduced.

10. A pharmaceutical composition for the prevention or treatment of corneal endothelial cell diseases comprising mitochondria derived from corneal endothelial cells or corneal endothelial-like cells as an active ingredient.

11. A pharmaceutical composition for the prevention or treatment of corneal endothelial disease according to claim 10, wherein the corneal endothelial disease is corneal endothelial inflammation, corneal endothelial edema, corneal endothelial damage, or a decrease in the number of corneal endothelial cells.

12. A pharmaceutical composition for the prevention or treatment of corneal endothelial disease according to claim 10, wherein the corneal endothelial disease is corneal endothelial insufficiency, corneal endothelial dystrophy (CED), bullous keratopathy, Fuchs endothelial corneal dystrophy (FECD), corneal endothelial dystrophy (CED), or corneal endotheliitis.

13. A composition for corneal endothelial cell transplantation comprising mitochondria derived from corneal endothelial cells or corneal endothelial-like cells. 14.1) A step of differentiating induced pluripotent stem cells into corneal endothelial cells or corneal endothelial-like cells; and 2) A method for producing mitochondria derived from corneal endothelial cells or corneal endothelial-like cells, comprising the step of isolating mitochondria from corneal endothelial cells or corneal endothelial-like cells.

15. A method for treating corneal damage comprising the step of administering mitochondria derived from corneal endothelial cells or corneal endothelial-like cells to the damaged cornea.

16. A method for treating corneal endothelial disease comprising the step of administering mitochondria derived from corneal endothelial cells or corneal endothelial-like cells to an individual with corneal endothelial disease.

17. Use of mitochondria derived from corneal endothelial cells or corneal endothelial-like cells for the prevention or treatment of corneal damage.

18. Use of mitochondria derived from corneal endothelial cells or corneal endothelial-like cells for the prevention or treatment of corneal endothelial diseases.

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