Pharmaceutical composition for preventing or treating optic nerve diseases and mitochondrial dysfunction diseases, comprising exosomes isolated from human placenta-derived mesenchymal stem cells

Abstract

Exosomes isolated from human placenta-derived mesenchymal stem cells significantly restore, through the regulation of the expression of LONP1 protein in damaged optic nerve progenitor cells and an optic nerve damage animal model, mitochondrial function by restoring the expression of optic nerve regeneration marker proteins, restoring reduced ATP production and increased ROS levels caused by optic nerve damage, increasing reduced mitophagy expression, and the like. Therefore, the exosomes of the present invention can be used in the prevention, alleviation or treatment of optic nerve diseases and mitochondrial dysfunction diseases.

Description

Pharmaceutical composition for preventing or treating optic nerve disease and mitochondrial dysfunction disease comprising exosomes isolated from human placental-derived mesenchymal stem cells

[0001] The present invention relates to the use of exosomes isolated from human placental-derived mesenchymal stem cells for the prevention or treatment of optic nerve disease and mitochondrial dysfunction.

[0002] Because there are no effective treatments for irreversible damage to the optic nerve, many studies have attempted to enhance the essential regenerative capacity of retinal ganglion cells (RGCs). Macrophage-activating factor and zymosan promote axon regrowth after optic nerve injury. Alterations in the intrinsic regenerative capacity of RGCs can be caused by deficiencies in phosphatase and tensin homolog (PTEN). Combining zymosan injections to induce inflammation, PTEN deficiency, and stimulate intracellular cyclic adenosine monophosphate (cAMP) may aid optic nerve recovery, but clinical applications remain limited.

[0003] For intractable ocular diseases, embryonic stem cells (ESCs), limbal stem cells, retinal pigment epithelial cells, and mesenchymal stem cells (MSCs) are used in cell therapy as part of regenerative medicine. MSCs attract immune cells by releasing immunomodulators, mediators, and chemokines. Furthermore, MSCs exert neuroprotective effects by secreting paracrine components. Numerous studies using animal models of glaucoma have shown that intravitreal injection of MSCs increased the survival of RGCs. In studies using ischemic models, MSC injection increased the number of RGCs and the expression of brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and bFGF (fibroblast growth factor). Several studies have confirmed the therapeutic effects of MSCs in ischemic models, and in addition to bone marrow-derived mesenchymal stem cells, human cord blood, dental pulp, and placenta-derived mesenchymal stem cells have also been reported to have therapeutic effects in terms of inducing regeneration of damaged optic nerve and axon growth (Kwon, H. et al., Hypoxia-Preconditioned Placenta-Derived Mesenchymal Stem Cells Rescue Optic Nerve Axons via Differential Roles of Vascular Endothelial Growth Factor in an Optic Nerve Compression Animal Model. Mol. Neurobiol. 2020, 57, 3362-3375; Labrador-Velandia, S. et al., Mesenchymal stem cell therapy in retinal and optic nerve diseases: An update of clinical trials. World J.Stem Cells 2016, 8, 376-383), but this too has limited clinical application.

[0004] Neurons require significant energy, and mitochondrial homeostasis is essential for neural network formation, intercellular communication, and activity. Furthermore, unique molecular mechanisms regulate mitochondrial distribution.

[0005] Meanwhile, mitophagy, an intracellular degradation mechanism that removes damaged or unnecessary mitochondria, has been reported to play a role in selectively removing damaged mitochondria by surrounding them with membranes, forming autophagosomes, and fusing these with lysosomes. It is known that mitophagy activity is crucial for regulating mitochondrial function in various cells, including neurons, and for maintaining tissue function.

[0006] Recent studies have shown that inhibition of mitophagy activity can lead to the death of motor neurons through the accumulation of damaged mitochondria, which can contribute to neurodegenerative diseases such as Alzheimer's disease. Furthermore, abnormalities in mitophagy activity have been linked to a wide range of human diseases, including neurodegenerative diseases like Parkinson's disease, Alzheimer's disease, and Lou Gehrig's disease, as well as peripheral neuropathy, heart disease, metabolic disorders, and cancer. This has fueled growing interest among researchers in the role of mitophagy in human disease and its potential therapeutic applications.

[0007] Accordingly, the present inventors intend to provide a composition comprising, as an active ingredient, exosomes isolated from human placental-derived mesenchymal stem cells that have a significant effect on optic nerve disease and mitochondrial dysfunction disease.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] (Patent Document 1) Publication No. 10-2023-0089272

[0011] [Non-patent literature]

[0012] (Non-patent Document 1) Hypoxia-Preconditioned Placenta-Derived Mesenchymal Stem Cells Rescue Optic Nerve Axons Via Differential Roles of Vascular Endothelial Growth Factor in an Optic Nerve Compression Animal Model. Kwon, H et al. Mol. Neurobiol. 2020

[0013] One aspect is to provide a pharmaceutical composition for preventing or treating optic nerve disease, comprising placental mesenchymal stem cell-derived exosomes as an active ingredient.

[0014] Another aspect is to provide a health functional food for preventing or improving optic nerve disease, which contains the exosome of the present invention as an active ingredient.

[0015] Another aspect provides a use of a composition comprising the exosome of the present invention as an active ingredient for preventing or treating optic nerve disease.

[0016] Another aspect provides a method for preventing or treating optic nerve disease, comprising administering to a subject in need thereof a composition comprising an effective amount of an exosome of the present invention as an active ingredient.

[0017] Another aspect provides a use of a composition comprising the exosome of the present invention as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of optic nerve disease.

[0018] Another aspect is to provide a composition for preventing or treating a disease caused by mitochondrial dysfunction, comprising the exosome of the present invention as an active ingredient.

[0019] Another aspect is to provide a health functional food for preventing or improving a disease caused by mitochondrial dysfunction, which contains the exosome of the present invention as an active ingredient.

[0020] Another aspect provides a use of a composition comprising the exosome of the present invention as an active ingredient for preventing or treating a disease caused by mitochondrial dysfunction.

[0021] Another aspect provides a method for preventing or treating a disease caused by mitochondrial dysfunction, comprising administering to a subject in need thereof a composition comprising an effective amount of the exosome of the present invention as an active ingredient.

[0022] Another aspect provides a use of a composition comprising the exosome of the present invention as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of a disease caused by mitochondrial dysfunction.

[0023] One aspect provides a pharmaceutical composition for preventing and treating optic nerve disease, comprising placental mesenchymal stem cell-derived exosomes as an active ingredient.

[0024] As used herein, the term “prevention” means any act of inhibiting or delaying the onset of a disease by administering the composition.

[0025] As used herein, the term "treatment" means any form of treatment that provides a benefit to a subject suffering from a disease or at risk of developing a disease, including improving the condition of the subject (e.g., one or more symptoms), delaying the progression of the disease, delaying the onset of a symptom, or slowing the progression of a symptom. Therefore, the terms "treatment" and "prevention" are not intended to mean a cure or complete elimination of a symptom. The term "subject" means a subject in need of treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, dog, cat, horse, or cow.

[0026] The placenta, as mentioned above, is a disc-shaped organ that is created for the fetus during pregnancy, weighing approximately 500 g, measuring 15-20 cm in diameter, and 2-3 cm in thickness. One side of the placenta is in contact with the mother, and the other side is in contact with the fetus, and the space between them contains the mother's blood, providing nutrients to the fetus. The placenta is composed of three layers: the amnion, the chorion, and the decidua. The amnion is a thin, transparent membrane that surrounds the fetus, containing amniotic fluid and containing fetal stem cells. The decidua is a membrane formed by the transformation of uterine epithelial cells in preparation for the implantation of the fertilized egg in the uterus, and contains maternal stem cells. The stem cells contained in the placenta are abundant, proliferate readily, and can differentiate into other cells. The chorion is a membrane located between the amnion and the decidua, which surround the fetus and amniotic fluid. It develops from the fertilized egg and forms part of the egg membrane. Placental stem cells are of fetal or maternal origin (i.e., they may have either fetal or maternal genotype). A placental stem cell population or a cell population containing placental stem cells may include placental stem cells that are exclusively fetal or exclusively maternal, or a mixed placental stem cell population containing both fetal and maternal origin.

[0027] The above "stem cell" is a cell that has the ability to differentiate into various cells through an appropriate environment and stimulation, and has the ability to self-renew, and may be an adult stem cell, a pluripotent stem cell, an induced pluripotent stem cell, or an embryonic stem cell. The stem cell may be derived from the placenta, umbilical cord blood, adipose tissue, muscle, cornea, dental pulp, or bone marrow, but in the present invention, it is preferred that the stem cell be derived from the placenta.

[0028] The above "mesenchymal stem cell (MSC)" may refer to a multipotent stem cell that maintains self-renewal and stemness maintenance and has the ability to differentiate into various mesodermal cells or ectodermal cells such as neural cells, and may include mesenchymal stem cells of mammals, for example, animals including humans. In addition, the mesenchymal stem cell may be an umbilical cord-derived, cord blood-derived, bone marrow-derived, placental-derived, or adipose-derived mesenchymal stem cell, but in the present invention, it is preferably a placental-derived mesenchymal stem cell. The isolation of mesenchymal stem cells can be performed by a method apparent to those skilled in the art, and is disclosed, for example, in the literature by Pittenger et al. (Science 284: 143, 1997) and van et al. (J. Clin. Invest., 58: 699, 1976). The above mesenchymal stem cells may secrete activin A or an active fragment thereof, or may be genetically engineered to secrete the same.

[0029] The above "placental-derived mesenchymal stem cells" refer to cells not derived from the inner cell mass of the blastocyst. Stem cells that can be obtained from the placenta include placental stem cells, pluripotent cells, multipotent cells, and committed progenitor cells. The placental-derived mesenchymal stem cells of the present invention are derived from the chorionic valve of the placenta, and are valuable as a raw material for cell therapy using stem cells because they are derived from a single extra-embryonic mesoderm.

[0030] The above "exosome" or "exosome" refers to a small membrane-structured vesicle that is released by the fusion of a multivesicular body, which is a vesicle during the maturation process of an endosome, with the cell membrane, and may be an extracellular vesicle (EV). The released exosomes contain various proteins, nucleic acids, lipids, etc., and can fuse with other cells to transfer contents. The diameter of the exosome may be about 30 nm to about 500 nm, about 30 nm to about 400 nm, about 30 nm to about 300 nm, about 30 nm to about 200 nm, about 50 nm to about 200 nm, about 50 nm to about 180 nm, or about 75 nm to about 180 nm. The above exosomes can originate from the plasma membrane or multivesicular bodies (MVBs) and be released or secreted outside the cell.

[0031] In one specific example, the placental-derived mesenchymal stem cells of the present invention are placental stem cells cultured under hypoxic conditions, wherein the "hypoxia" may refer to a state of lower oxygen partial pressure than the typical normoxic condition of 21% oxygen partial pressure, and the hypoxic state may have an oxygen concentration of 1 to 5% and a carbon dioxide concentration of 3 to 10%, preferably, the oxygen concentration may be 1 to 4, 1 to 3, or 2 to 3%, and the carbon dioxide concentration may be 3 to 10, 3 to 8, 3 to 7, or 4 to 6%.

[0032] In another specific example, the placental stem cells are exposed to a hypoxic environment for about 10 minutes to 1 hour, and preferably, the placental-derived mesenchymal stem cells can be produced by exposing them to a hypoxic environment for 10 minutes to 50 minutes, 20 minutes to 40 minutes, or 25 minutes to 35 minutes.

[0033] In another specific embodiment, the exosomes of the present invention restore the expression of optic nerve regeneration-related proteins and mitochondrial homeostasis proteins, wherein the optic nerve regeneration-related proteins are Bnip3, Vegf, Neurofilament, NeuN, and Gap43, and the mitochondrial homeostasis proteins are Atp5a, Mfn2, Mul1, and Prdx2, but are not limited thereto.

[0034] However, the restoration of the expression of the optic nerve regeneration-related protein and mitochondrial homeostasis protein is through the regulation of the expression of the LONP1 protein, and preferably, by increasing the expression of the LONP1 protein reduced due to optic nerve damage or mitochondrial dysfunction, the expression of the optic nerve regeneration-related protein and mitochondrial homeostasis protein can be restored. Therefore, the optic nerve disease may be a case where the Lonp1 protein is expressed, but the expression of the protein is reduced compared to a normal individual due to optic nerve damage.

[0035] The above "optic nerve disease" or "optic nerve disorder" may refer to a disease, condition, or disorder that results in vision loss due to damage to the optic nerve or its pathway to the brain. Each optic nerve is separated by a structure within the brain called the optic chiasm, with half of the optic nerve fibers crossing to opposite sides. Due to this anatomical arrangement, damage along the optic nerve pathway can cause specific patterns of vision loss. Furthermore, the optic nerve disease may be caused by trauma, surgery, ischemia, hypoxia, metabolism, infection, drug addiction, immune, inflammatory, genetic factors, tumors, or brain lesions.

[0036] In one specific example, the optic nerve disease is preferably traumatic optic neuropathy, ischemic optic neuropathy, compressive optic neuropathy, toxic optic neuropathy, Leber's hereditary optic neuropathy, optic nerve transection, optic neuritis, neuromyelitis optica, or amblyopia.

[0037] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The carrier is used to mean an excipient, diluent, or auxiliary. The carrier may be selected from the group consisting of, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, saline, a buffer such as PBS, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, or combinations thereof.

[0038] The pharmaceutical composition may be prepared in any dosage form according to conventional methods. The composition may be formulated, for example, as an oral dosage form (e.g., powder, tablet, capsule, syrup, pill, or granule) or a parenteral dosage form (e.g., injection).

[0039] Additionally, the composition may be formulated as a systemic formulation or a topical formulation. The pharmaceutical composition may be administered orally, intravenously, intratumorally, intramuscularly, orally, transdermally, transmucosally, intranasally, intratracheally, subcutaneously, or a combination thereof.

[0040] The pharmaceutical composition may comprise an effective amount of the exosome of the present invention according to one aspect. The term "effective amount" refers to an amount sufficient to exhibit a preventive or therapeutic effect when administered to a subject in need of prevention or treatment. The effective amount can be appropriately selected by a person skilled in the art depending on the cell or subject selected. It can be determined according to factors including the severity of the disease, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, drugs combined with or co-used with the composition used, and other factors well known in the medical field. The effective amount may be about 0.1 μg to about 2 g, about 0.5 μg to about 1 g, about 1 μg to about 500 mg, about 10 μg to about 100 mg, or about 100 μg to about 50 mg per pharmaceutical composition.

[0041] The dosage of the pharmaceutical composition may be, for example, in the range of about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.1 mg / kg to about 1 mg / kg on an adult basis. The administration may be administered once a day, multiple times a day, or once a week, once every two weeks, once every three weeks, or once every four weeks, or once a year.

[0042] Another aspect provides a health functional food for preventing and improving optic nerve disease, which contains placental mesenchymal stem cell-derived exosomes as an active ingredient.

[0043] The above 'placental mesenchymal stem cell-derived exosomes' and 'optic nerve disease' are as described in the above 'pharmaceutical composition for preventing and treating optic nerve disease'.

[0044] The above health functional food can be used alone or in combination with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the composition of the present specification can be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the type of the health functional food. Among the types of health functional foods, the beverage composition can contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame can be used. The health food composition may also contain nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, or combinations thereof. The health functional food composition may also contain fruit pulp for the production of natural fruit juice, fruit juice drinks, vegetable drinks, or combinations thereof.

[0045] The above health functional food is comprised of 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, 0.00001 wt% to 10 wt%, 0.00001 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, It may comprise 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt% of the exosome of the present invention.

[0046] Another aspect provides the use of a composition comprising placental mesenchymal stem cell-derived exosomes as an active ingredient for preventing or treating optic nerve disease.

[0047] Another aspect provides a method for preventing or treating an optic nerve disease, comprising administering to a subject in need thereof a composition comprising an effective amount of placental mesenchymal stem cell-derived exosomes as an active ingredient.

[0048] Another aspect provides the use of a composition comprising placental mesenchymal stem cell-derived exosomes as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of optic nerve disease.

[0049] Another aspect provides a composition for preventing or treating a disease caused by mitochondrial dysfunction, comprising placental mesenchymal stem cell-derived exosomes as an active ingredient.

[0050] The above 'placental mesenchymal stem cell-derived exosome' is as described in the above 'pharmaceutical composition for preventing and treating optic nerve disease'.

[0051] The above mitochondrial dysfunction refers to a breakdown of the process, a decrease in mitochondrial enzymes, an increase in reactive oxygen species, and a change in the number or size of mitochondria, and the above mitochondrial dysfunction may cause ischemic encephalopathy or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, cirrhosis, Fanconi syndrome, atypical phenylketonuria, asthma, diabetes, arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and cancer.

[0052] In one specific example, the placental-derived mesenchymal stem cells of the present invention are preferred for increasing the activity of mitophagy and can increase the expression of mitophagy-related proteins such as Pink1, phosphorylated Parkin, Atg7, p62, Lc3b, Mfn2, and Mul1.

[0053] In another specific example, the disease caused by mitochondrial dysfunction may be at least one selected from the group consisting of ischemic cerebral disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, liver cirrhosis, Fanconi syndrome, atypical phenylketonuria, asthma, diabetes, arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and cancer.

[0054] Another aspect provides a health functional food for preventing or improving diseases caused by mitochondrial dysfunction, which contains placental mesenchymal stem cell-derived exosomes as an active ingredient.

[0055] The above 'placental mesenchymal stem cell-derived exosomes' and 'diseases caused by mitochondrial dysfunction' are as described in the above 'pharmaceutical composition for preventing and treating optic nerve disease' and 'pharmaceutical composition for preventing and treating diseases caused by mitochondrial dysfunction'.

[0056] Another aspect provides the use of a composition comprising placental mesenchymal stem cell-derived exosomes as an active ingredient for preventing or treating a disease caused by mitochondrial dysfunction.

[0057] Another aspect provides a method for preventing or treating a disease caused by mitochondrial dysfunction, comprising administering to a subject in need thereof a composition comprising an effective amount of placental mesenchymal stem cell-derived exosomes as an active ingredient.

[0058] Another aspect provides the use of a composition comprising placental mesenchymal stem cell-derived exosomes as an active ingredient for use in the manufacture of a medicament for the prevention or treatment of a disease caused by mitochondrial dysfunction.

[0059] The terms and methods described for the above inventions apply equally to each invention.

[0060] The exosomes isolated from human placental-derived mesenchymal stem cells of the present invention restore the expression of a nerve regeneration marker protein by regulating the expression of LONP1 protein in damaged optic nerve cells and an animal model of optic nerve damage, restore reduced ATP production and increased ROS levels due to optic nerve damage, and significantly restore mitochondrial function by increasing the expression of reduced mitophagy-related proteins. Therefore, the exosomes of the present invention can be used to prevent, improve, or treat optic nerve damage or mitochondrial dysfunction diseases.

[0061] Figure 1 is a diagram confirming the characteristics and cell recovery effect of exosomes derived from human hypoxia-preconditioned mesenchymal stem cells (hHPPSCs):

[0062] Figure 1a is a diagram showing the expression levels of exosome markers CD9, CD81, and CD63 in EVs derived from hPSCs and hHPPSCs; Figure 1b is a diagram showing the results of BrdU analysis performed 24 hours after exposure of R28 cells to CoCl2 (200 μM) for 9 hours and treatment of EVs in hypoxic cells, and the data are expressed as mean ± SEM; Figure 1c is a diagram showing the fold (mean ± SEM) of ATP production measurement; and Figure 1d is a diagram showing the results of Western blotting using R28 cell lysates treated with CoCl2, and the quantification values ​​(lower panel) of the protein expression values. Significant differences were determined using an unpaired t test (* p < 0.05, ** p < 0.005, *** p < 0.001 vs. control; #p < 0.05, ##p < 0.01, ###p < 0.005 vs. CoCl2; γp < 0.05 vs. PSC_EVs). All experiments were repeated three times.

[0063] Figure 2 shows the results of cluster analysis of differentially expressed proteins (DEPs) by HPPSC_EVs in hypoxia-damaged R28 cells. To identify differentially expressed proteins in the treatment groups (CoCl2, CoCl2+ HPPSC_EVs) and PBS-treated control cells, label-free quantification (LFQ) was used, and MaxQuant version 1.5.8.3 (www.coxdocs.org (accessed on October 13, 2022) was used:

[0064] Figure 2a is a heatmap of differentially expressed proteins (p ≤ 0.05 and log2FC ≥ 1) identified through hierarchical clustering analysis in the lysates of three groups (control, CoCl2, CoCl2+ HPPSC_EVs), with high expression indicated in red and low expression indicated in green; Figure 2b is a Venn diagram showing the number of proteins identified through proteomic analysis of R28 cells treated with each group; and Figure 2c is a diagram confirming the protein-protein interactions of DEPs in R28 cells treated with the experimental groups.

[0065] Figure 3 is a volcano plot showing changes in protein expression in hypoxic, damaged retinal precursor cells treated with HPPSC_EVs. Figures 3a to 3c show differentially expressed proteins (DEPs) in each experimental group in volcano plots, with red dots indicating upregulated proteins and blue dots indicating downregulated proteins. In addition, proteins with significant expression differences are shown.

[0066] Figure 4 illustrates the role of Lonp1 in the restoration process induced by HPPSC_EVs. siRNA targeting Scramble and Lonp1 was transfected into R28 cells. After culture, Lonp1-deficient cells were exposed to CoCl2 (200 μM). After 9 h of culture, hypoxia-induced cells were treated with HPPSC_EVs, and Western blotting was performed. Results are presented as the mean ± SEM. Statistical significance was determined using nonparametric statistical analysis followed by the Mann-Whitney U test and unpaired t test (#p < 0.05, ##p < 0.01 vs. Scramble treatment group, *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001). All experiments were repeated three times, and the KD experiment was performed five times.

[0067] Figure 5 shows the effects of HPPSC_EVs on regulating the regeneration and mitochondrial function of retinal precursor cells: Figure 5a shows the expression levels of neural regeneration-related proteins and the quantified values ​​of Bnip3, Vegf, Neurofilament, NeuN, and Gap43 expression (right panel) by immunoblot analysis in hypoxia-damaged R28 cells; Figure 5b shows the changes in ATP levels by HPPSC_EVs in Lonp1-deficient cells, and Figure 5c shows the fold expression of the mitochondrial marker Atp5a protein (mean ± SEM); Figure 5d shows representative images of damaged R28 cells stained with MitoSox. R28 cells were exposed to CoCl2 (200 μM) for 9 h and then treated with HPPSC_EVs (12 μg / mL), and the intensity was measured using ZEN software; Figure 5e shows the expression of antioxidant enzymes (Pdrx2 and Prdx5) in cells lacking Lonp1 protein. Lonp1 knockdown cells were treated with CoCl2 (200 μM), cultured for 9 h, treated with HPPSC_EVs, and harvested 24 h later for immunoblotting. The results (right panel) show the quantification of protein expression levels. Significant differences were confirmed using an unpaired t test (* p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005, # p < 0.05, ## p < 0.01). KD experiments, ATP, and ROS measurement analyses were repeated three times.

[0068] Fig. 6 shows the effects of HPPSC_EVs on the recovery and regeneration of RGCs in the ONC model; Fig. 6a shows representative confocal microscopy-based fluorescence images through Brn-3a and Tuj1 staining after injection of PSC_EVs and HPPSC_EVs in the retina of an optic nerve compression animal model (original magnification: 400Х); Fig. 6b shows the expression of regeneration-related proteins in the optic nerve tissue using immunoblot analysis; Fig. 6c shows the comparison of the expression of mitochondrial function-related proteins between the retina and optic nerve tissue, with each group using a total of two or three retinas and optic nerves. The results are expressed as the mean ± SEM. Significant differences were determined using an unpaired t test (* p < 0.05, ** p < 0.005, *** p < 0.0005, # p < 0.05, ## p < 0.01 vs. age-matched control).

[0069] The following examples are provided for more detailed explanation. However, these examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples.

[0070]

[0071] Example 1. Isolation of exosomes derived from human hypoxia-preconditioned mesenchymal stem cells (hHPPSCs)

[0072] Human placenta stem cells (hPSCs) were provided by CHA Biotech (Seongnam, Korea). The hPSCs were cultured in minimum essential medium (MEM)-alpha GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific), 1% penicillin / streptomycin (Thermo Fisher Scientific), 25 ng / mL human fibroblast growth factor 4 (Peprotech Inc., Rocky Hill, NJ, USA), and 1 g / mL heparin (Sigma-Aldrich, St. Louis, MO, USA).

[0073] To improve the cell recovery effect of the hPSCs, hPSCs were cultured for 30 minutes under conditions of 2.2% O2 and 5.5% CO2 to produce hHPPSCs. The hPSCs and hHPPSCs were cultured for 48 hours in MEM-alpha GlutaMAX supplemented medium containing 10% exosome-free FBS (Thermo Fisher Scientific). Then, exosomes, which are extracellular vesicles (EVs), were isolated from hHPPSCs (passages 8-10) using the ExoQuick-TC kit (SYSTEM BIOSCIENCES (SBI), Palo Alto, CA, USA), and the isolation was performed according to the manual provided by the company. The exosomes obtained in the final step were dissolved in 100 μL of PBS and quantified using the BCA method. Additionally, the separated EVs were stored at -80°C, and the size of the EVs was measured using a PMX-120 ZetaView Mono Laser (Particle Metrix, Meerbusch, North Rhine-Westphalia, Germany).

[0074]

[0075] Example 2. Cell culture and EVs treatment

[0076] Immortalized R28 retinal precursor cells were cultured in Dulbecco's minimal Eagle's medium (DMEM; Sigma-Aldrich) containing 10% FBS (Thermo Fisher Scientific), 1X minimal essential medium (MEM; Thermo Fisher Scientific) with nonessential amino acids, 100 μg / mL gentamicin (Sigma-Aldrich), and 1% penicillin-streptomycin (Thermo Fisher Scientific). The cells were then cultured in a hypoxic environment using cobalt chloride (CoCl2).

[0077] Specifically, 2 x 10 R28 cells 5 After inoculation with 100 μM of EVs, cells were treated with 200 μM CoCl2 for 9 h, and then treated with 170-180 nm EVs at a concentration of 12 μg / mL in hypoxic R28 cells. Cells were harvested after 24 h and analyzed.

[0078]

[0079] Example 3. Cell proliferation analysis using BrdU (Bromodeoxyuridine)

[0080] The proliferation level of R28 cells was measured using a BrdU ELISA kit (CellBioLabs, Inc., San Diego, CA, USA).

[0081] Specifically, R28 cell culture was performed in triplicate in 96-well plates for each experiment, and the experimental method was performed according to the protocol provided by the manufacturer.

[0082]

[0083] Example 4. Measurement of ATP levels

[0084] To assess ATP production, cell lysates were extracted from damaged R28 cells. ATP levels were determined using an ATP determination kit (Thermo Fisher Scientific). Additionally, ATP production levels were measured using a luminometer (Spectramax iD5; Molecular Devices, San Jose, CA, USA) according to the manufacturer's instructions.

[0085]

[0086] Example 5. Measurement of reactive oxygen species (ROS)

[0087] Mitochondrial superoxide production, a type of reactive oxygen species, in damaged R28 cells was measured using MitoSOX Red (Thermo Fisher Scientific).

[0088] Specifically, the cells seeded on coverslips were treated with MitoSOX Red (5 μM) under light-shielding conditions, incubated at 37°C for 10 min, and then washed. The stained coverslips were mounted on slides using mounting solution. Fluorescence was detected using a Zeiss LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany), and the intensity of the staining was measured using ZEN software (Carl Zeiss).

[0089]

[0090] Example 6. Preparation of siRNA for LONP1 protein

[0091] The following sequence was used for siRNA (Bioneer Corporation, Daejeon) to suppress the expression of Lonp1 protein: siRNA rat Lonp1, 5'-GGG AAC GCU UAA AGG AGC UUG UGG U (SEQ ID NO: 1).

[0092] Additionally, a negative control (Bioneer Corporation) was used as a scramble control. In addition, the siRNA was transfected into R28 cells using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0093]

[0094] Example 7. Immunoblot Analysis

[0095] Immunoblot experiments were performed using the method described in a previous study (Hypoxia-Preconditioned Placenta-Derived Mesenchymal Stem Cells Rescue Optic Nerve Axons Via Differential Roles of Vascular Endothelial Growth Factor in an Optic Nerve Compression Animal Model. Kwon, H et al. Mol. Neurobiol. 2020).

[0096] Specifically, total protein from cells or tissues was extracted using RIPA or PRO-PREP buffer (iNtRON Biotechnology, Gyeonggi, Korea). Protein concentration was determined using the BCA method (Thermo Fisher Scientific). Target proteins were separated by SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Chicago, IL, USA). The membrane contains Anti-Hif-1 (ab179483), Gap43 (ab16053), Ermn (ab90893), Mul1 (ab209263; Abcam, Cambridge, UK), Vegf (GTX102643; GeneTex, Irvine, CA, USA), Thy-1 (SC-53116), Bnip3 (SC-56167), Pink1 (SC-518052), Prdx5 (SC-133072), β-actin (SC-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Neurofilaments (#2837), Lc3b (#2775), Mfn2 (#9482), Prdx2 (#46855; Cell Signaling Technology, Danvers, MA, USA), phospo-Parkin (PA5-114616; Thermo Fisher Scientific), Membranes were incubated with antibodies to Atg7 (10088-2-AP; Proteintech Group, Inc., Rosemont, IL, USA), and P62 (R31056; NSJ Bioreagents, San Diego, CA, USA). All antibodies, except Thy-1 and Prdx5 (1:200 dilution), were used at a 1:1000 dilution. After washing, the membranes were incubated overnight at 4°C with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies at a 1:10,000 dilution.Target bands were visualized using enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA) and detected using ImageQuant LAS 4000 (GE Healthcare).

[0097]

[0098] Example 8. Proteomics analysis of EVs derived from hHPPSCs

[0099] The entire proteomic analysis process was performed using the method described in a previous study (UBA2 activates Wnt / beta-catenin signaling pathway during protection of R28 retinal precursor cells from hypoxia by extracellular vesicles derived from placental mesenchymal stem cells. Koh,K et al. Stem Cell Res. Ther. 2020).

[0100] Specifically, proteomic analysis was performed to determine the broad effects of HPPSCs_EVs on intact cells using R28 cells treated with PBS (control), R28 cells treated with CoCl2, and R28 cells treated with CoCl2 and HPPSCs_EVs. Protein digestion was performed according to the FASP (filter-aided sample preparation) protocol using Ultracel YM-30 centrifugal filters (Merck Millipore, Darmstadt, Germany). Sample analysis was performed using a Q Exactive chromatography system coupled to a Dionex Ultimate 3000 HPLC. TMAnalysis was performed using an LC-MS / MS system consisting of a Hybrid Quadrupole-Orbitrap MS (Thermo Fisher Scientific). Database searching and data processing were performed according to previously reported methods (Interphotoreceptor retinoid-binding protein (IRBP) is downregulated at early stages of diabetic retinopathy. Garcia Arumi, J. et al. Diabetologia 2009). Differentially expressed proteins (DEPs) were filtered with a p-value ≤ 0.05 and log2FC ≥ 1 (fold change) cutoffs. Heatmaps were generated using Perseus software. Protein-protein interactions were analyzed using the String database (https: / string-db.org / , accessed October 13, 2022). Volcano plots were prepared using R version 3.6.1.

[0101]

[0102] Example 9. Building an Optic Nerve Injury Model

[0103] The process of establishing a disease animal model was performed using the method disclosed in a previous study (Hypoxia-Preconditioned Placenta-Derived Mesenchymal Stem Cells Rescue Optic Nerve Axons Via Differential Roles of Vascular Endothelial Growth Factor in an Optic Nerve Compression Animal Model. Kwon, H et al. Mol. Neurobiol. 2020).

[0104] Specifically, the optic nerve of the rat was compressed 2 mm posterior to the globe for 5 seconds using ultra-fine self-closing forceps. Optic nerve compression (ONC) was performed in the left oculus sinister (OS). After that, the canthal incision was sutured, and HPPSC_EVs were injected once into the subtenon of the globe. In addition, the experimental animals were divided as follows: Sham (injection of balanced salt solution (BSS) after optic nerve compression), hPSCs_EVs group (injection of hPSCs_EVs 300 μg / 0.06 mL after optic nerve compression), hHPPSCs_EVs group (injection of hHPPSCs_EVs 300 μg / 0.06 mL after optic nerve compression). Additionally, tissues isolated from the animals 4 weeks after optic nerve compression and injection were used for analysis.

[0105]

[0106] Example 10. Flat-Mounted Retinas and Retinal Ganglion Cell (RGC) Survival Analysis

[0107] Expression of Brn-3a and Tuj1 in the rat retina was analyzed using the flat mount technique, using the method described in a previous study (Hypoxia-Preconditioned Placenta-Derived Mesenchymal Stem Cells Rescue Optic Nerve Axons Via Differential Roles of Vascular Endothelial Growth Factor in an Optic Nerve Compression Animal Model. Kwon, H et al. Mol. Neurobiol. 2020).

[0108]

[0109] Example 11. Statistical Analysis

[0110] All results are expressed as the mean ± standard error of the mean (SEM), and data analysis was performed using GraphPad Prism 9 software (GraphPad Software, Inc., La Jolla, CA, USA). Statistically significant differences are indicated in each figure.

[0111]

[0112] Experimental Example 1. Confirmation of HPPSC_EV characteristics and cell recovery effect

[0113] To confirm the characteristics of HPPSC_EVs, the expression levels of exosome markers CD9, CD81, and CD63 were examined in each EV derived from hPSCs and hHPPSCs isolated in Example 1. As a result, it was confirmed that CD9 was expressed more in hHPPSC_EVs than in PSC_EVs (Fig. 1a).

[0114] Furthermore, to determine whether hHPPSC_EVs improve the recovery of cells damaged under hypoxic conditions, R28 cells were exposed to CoCl2 for 9 hours as in Example 2, and then EVs were treated in hypoxic R28 cells. hHPPSC_EVs significantly increased cell proliferation of damaged R28 cells by 8.7% (Fig. 1b) and significantly restored (1.07-fold) the intracellular ATP level decreased during hypoxia (Fig. 1c).

[0115] In addition, as a result of confirming the expression level of nerve regeneration-related marker proteins affected by CoCl2, it was confirmed that the group treated with hHPPSC_EVs of the present invention recovered to a normal level.

[0116] Specifically, the expression of Hif-1α increased after CoCl2 exposure, but significantly decreased (0.79-fold) after hHPPSC_EVs treatment (Fig. 1d). Conversely, the expression of nerve regeneration-related proteins such as vascular endothelial growth factor (VEGF), Thy-1, Gap43, Ermn, and neurofilament decreased under hypoxic conditions, but after hHPPSC_EVs treatment, the expression levels of VEGF, Thy-1, and neurofilament significantly increased by 1.80-fold, 1.98-fold, and 1.37-fold, respectively (Fig. 1d). In particular, it was confirmed that VEGF expression was significantly increased (1.26-fold) by hHPPSC_EVs of the present invention compared to PSC_EVs (Fig. 1d).

[0117]

[0118] Experimental Example 2. Hierarchical Clustering and Gene Ontology

[0119] Proteomic analysis was performed on R28 cells. Specifically, hierarchical clustering of differentially expressed proteins (DEPs) was performed under three conditions (control, CoCl2, and CoCl2+ HPPSC_EVs) as described in Example 8 (Fig. 2a). Furthermore, the Venn diagram in Fig. 2b shows the expression levels of proteins associated with injury and repair. The control, CoCl2, and CoCl2+ HPPSC_EVs groups expressed 1,887, 1,704, and 2,057 proteins, respectively, and their profiles changed after exposure to CoCl2 (Fig. 2b). In both CoCl2-damaged R28 cells and cells exposed to CoCl2+ HPPSC_EVs, the unique proteins were primarily RNA-binding proteins and their complexes. Additionally, interactions between LONP1, PARK7, HSPD1, VDAC1, 2, and 3 were evident, and these interactions with other proteins involved in mitochondrial function are shown in Fig. 2c.

[0120]

[0121] Experimental Example 3. Confirmation of protein expression changes in hypoxia-damaged retinal precursor cells treated with HPPSC_EVs.

[0122] After treating cells damaged by hypoxic conditions with HPPSC_EVs, changes in protein expression were displayed as a volcano plot, and proteins with up- and down-regulated expression were listed (Fig. 3). LONP1 expression was significantly reduced by CoCl2 (compared to the control group) (Fig. 3a). However, it was confirmed that LONP1 protein expression was restored by HPPSC_EV treatment (Fig. 3b). In addition, HSPA9, a mitochondrial 70 kDa heat shock protein (mtHsp70), was maintained at a high level in the HPPSC_EV or CoCl2 treatment groups (Figs. 3a and 3b). However, HSPA9 expression was confirmed to decrease when HPPSC_EVs were treated in damaged cells (Fig. 3c). In addition, the top 35 up- and down-regulated DEPs in each group are also disclosed in Fig. 3.

[0123]

[0124] Experimental Example 4. Confirmation of the Role of LONP1 and the Pink / Parkin / p62 System during the Recovery of Mitochondrial Function Induced by HPPSC_EVs

[0125] The above experimental examples confirmed that Lonp1 plays a crucial role in mitochondrial quality control in HPPSC_EVs under hypoxic conditions. Furthermore, we investigated how Lonp1-deficient R28 cells were affected by CoCl2 damage.

[0126] As shown in Fig. 4, the expression of mitophagy-related proteins Pink1, phosphorylated Parkin, Atg7, p62, Lc3b, Mfn2, and Mul1 was significantly reduced in R28 cells damaged by CoCl2. In addition, the expression of the above proteins tended to increase after exposure to HPPSC_EVs, and in particular, the levels of p62 and Lc3b were confirmed to be significantly restored (1.21-fold and 1.40-fold, respectively).

[0127] Furthermore, in an environment where Lonp1 expression was suppressed using Lonp1 siRNA, the levels of mitophagy-related proteins such as Pink1, Atg7, and Lc3b significantly increased. However, while CoCl2 treatment suppressed the expression of these proteins in both normal cells and Lonp1-deficient cells, it was confirmed that HPPSC_EVs did not restore the expression of these proteins, unlike scramble-treated cells (Fig. 4).

[0128] Therefore, we confirmed that Lonp1 plays an important role in mitophagy.

[0129]

[0130] Experimental Example 5. Confirmation of the regenerative effect of HPPSC_EVs on hypoxic-damaged retinal precursor cells.

[0131] Bnip3 levels were rapidly increased under hypoxic conditions induced by CoCl2, and HPPSC_EVs significantly reduced the expression level of this protein, regardless of the presence or absence of Lonp1 (Fig. 5A). However, Vegf was significantly restored (1.47-fold) by HPPSC_EVs only in scrambled-treated cells, not in Lonp1-deficient cells. Furthermore, Gap43 was significantly reduced in Lonp1-deficient cells treated with HPPSC_EVs (Fig. 5A). Therefore, based on these results, we confirmed that Vegf is a key target for regeneration.

[0132]

[0133] Experimental Example 6. Confirming the Effect of HPPSC_EVs on Mitochondrial Quality Control

[0134] To determine the effect of HPPSC_EVs on mitochondrial quality control, ATP levels were measured (Fig. 5b). CoCl2-induced hypoxia significantly reduced ATP production (Fig. 5b) and increased reactive oxygen species (ROS) levels (Fig. 5d). However, HPPSC_EVs restored ATP production and ROS levels to normal (Fig. 5d). However, in the absence of Lonp1, HPPSC_EVs were found to have no effect.

[0135] In addition, when the levels of mitochondrial ATP synthase Atp5a and antioxidant protective protein Prdx2 were measured, it was confirmed that HPPSC_EVs significantly restored the expression of Atp5a and Prdx2 reduced by CoCl2 (1.54-fold and 1.33-fold, respectively), but this was only true when Lonp1 was expressed (Fig. 5c and Fig. 5e).

[0136]

[0137] Experimental Example 7. Confirmation of the Effect of HPPSC-EVs in an In Vivo Hypoxic Damage Model

[0138] Experimental Example 7-1. Changes in the expression of neurogenesis markers after HPPSC_EV injection in an optic nerve compression (ONC) animal model.

[0139] To confirm retinal ganglion cell (RGC) regeneration, the number of RGCs in rats stained with Brn-3a and Tuj1 was determined (Fig. 6a). After ONC, PSC_EVs and HPPSC_EVs increased Brn-3a expression levels by 35-fold and 56-fold, and Tuj1 expression levels by 1.0-fold and 1.3-fold, respectively. However, only HPPSC_EVs significantly increased retinal Brn-3a and Tuj1 expression compared to the age-matched sham-operated group after 4 weeks (Fig. 6a). In addition, when we analyzed the changes in the expression levels of Hif-1α, Vegf, Gap43, Ermn, and Neurofilament in the optic nerve of mice 4 weeks after ONC, we found that the levels of Vegf and Gap43 were significantly increased by HPPSC_EVs (1.45-fold and 1.68-fold, respectively), and that it affected the Vegf expression level differently from PSC_EVs (Fig. 6b). Meanwhile, both PSC_EVs and HPPSC_EVs were confirmed to significantly upregulate the Neurofilament level after ONC (20.35-fold and 22.97-fold, respectively) (Fig. 6b).

[0140]

[0141] Experimental Example 7-2. Comparison of Mitochondrial Protein Expression in Retinal and Optic Nerve Tissues After ONC

[0142] Changes in mitochondrial homeostatic protein levels were observed in retinal and optic nerve tissues. One week after ONC, HPPSC_EVs significantly increased the levels of Atp5a, Mfn2, Mul1, and Prdx2 in the retina (Fig. 6c). However, in the 2- and 4-week groups, no differences in other expression levels were observed except for the increase in Atp5a expression. However, unlike in the retina, HPPSC_EVs significantly upregulated Vegf, Mfn2, and Atp5a protein levels in the optic nerve after 4 weeks, and Prdx2 expression in the optic nerve was confirmed to increase from 1 week after HPPSC_EV treatment (Fig. 6c).

[0143] The above results indicate that exosomes isolated from human placental-derived mesenchymal stem cells significantly restore the expression of nerve regeneration marker proteins by regulating the expression of LONP1 protein in damaged optic nerve cells and optic nerve injury animal models, restore reduced ATP production and increased ROS levels due to optic nerve injury, and increase the expression of reduced mitophagy-related proteins, thereby significantly restoring mitochondrial function. Therefore, the exosomes of the present invention have an effect in preventing, improving, or treating optic nerve injury or mitochondrial dysfunction diseases.

Claims

1. A pharmaceutical composition for preventing or treating optic nerve disease, comprising exosomes derived from placental mesenchymal stem cells as an active ingredient.

2. In claim 1, A pharmaceutical composition for preventing or treating optic nerve disease, wherein the above placental mesenchymal stem cells are placental stem cells cultured under hypoxic conditions.

3. In claim 2, A pharmaceutical composition for preventing or treating optic nerve disease, wherein the hypoxic state is one in which the oxygen concentration is 1 to 5% and the carbon dioxide concentration is 3 to 10%.

4. In claim 1, A pharmaceutical composition for preventing or treating optic nerve disease, wherein the exosome restores the expression of optic nerve regeneration-related proteins and mitochondrial homeostasis proteins.

5. In claim 4, A pharmaceutical composition for preventing or treating optic nerve disease, wherein the exosome restores the expression of optic nerve regeneration-related proteins and mitochondrial homeostasis proteins by regulating the expression of LONP1 protein.

6. In claim 1, A pharmaceutical composition for preventing or treating optic nerve disease, wherein the optic nerve disease is caused by reduced expression of the LONP1 protein.

7. In claim 1, A pharmaceutical composition for preventing or treating optic nerve disease, wherein the optic nerve disease is at least one selected from the group consisting of traumatic optic neuropathy, ischemic optic neuropathy, compressive optic neuropathy, toxic optic neuropathy, Leber's hereditary optic neuropathy, optic nerve transection, optic neuritis, neuromyelitis optica, and amblyopia.

8. A health functional food for preventing or improving optic nerve disease, containing placental mesenchymal stem cell-derived exosomes as an active ingredient.

9. A pharmaceutical composition for the prevention or treatment of diseases caused by mitochondrial dysfunction, comprising exosomes derived from placental mesenchymal stem cells as an active ingredient.

10. In claim 9, A pharmaceutical composition for preventing or treating a disease caused by mitochondrial dysfunction, wherein the exosome increases mitophagy activity.

11. In claim 9, A pharmaceutical composition for preventing or treating a disease caused by mitochondrial dysfunction, wherein the disease caused by mitochondrial dysfunction is at least one selected from the group consisting of ischemic cerebral disease or ischemic heart disease, stroke, migraine, anemia, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), mitochondrial encephalomyopathy, liver cirrhosis, Fanconi syndrome, atypical phenylketonuria, asthma, diabetes, arteriosclerosis, hypertension, hypercholesterolemia, optic nerve disease, obesity, depression, and cancer.

12. A health functional food for the prevention or improvement of diseases caused by mitochondrial dysfunction, containing exosomes derived from placental mesenchymal stem cells as an active ingredient.

13. Use of a composition comprising placental mesenchymal stem cell-derived exosomes as an active ingredient for preventing or treating optic nerve disease.

14. A method for preventing or treating optic nerve disease, comprising administering to a subject in need thereof a composition comprising an effective amount of placental mesenchymal stem cell-derived exosomes as an active ingredient.

15. Use of a composition comprising placental mesenchymal stem cell-derived exosomes as an active ingredient for use in the manufacture of a medicine for preventing or treating optic nerve disease.

16. Use of a composition comprising placental mesenchymal stem cell-derived exosomes as an active ingredient for preventing or treating diseases caused by mitochondrial dysfunction.

17. A method for preventing or treating a disease caused by mitochondrial dysfunction, comprising administering to a subject in need thereof a composition containing an effective amount of placental mesenchymal stem cell-derived exosomes as an active ingredient.

18. Use of a composition comprising placental mesenchymal stem cell-derived exosomes as an active ingredient for use in the manufacture of a medicine for preventing or treating a disease caused by mitochondrial dysfunction.

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