Composition comprising component derived from induced pluripotent stem cell culture medium and uses thereof

Abstract

The present invention relates to a composition for the prevention, improvement, or treatment of neuronal damage diseases comprising a component derived from the supernatant of an induced pluripotent stem cell (iPSC) culture medium. The exosomes of the present invention can be isolated from a culture medium for induced pluripotent stem cells to improve nerve cell damage or regenerate skin, and thus can be effectively used as a composition for the prevention, improvement, or treatment of nerve cell damage-related diseases, particularly abnormalities in taste or smell caused by coronavirus infection or Alzheimer's disease, or as a composition for skin regeneration.

Description

Technology Field

[0001] The present invention relates to a composition for the prevention, improvement, or treatment of neuronal damage diseases comprising a component derived from the supernatant of an induced pluripotent stem cell (iPSC) culture medium. Background Technology

[0003] Extracellular vesicles (EVs) are nano-sized vesicles surrounded by a lipid bilayer, secreted by various cells such as prokaryotes, higher eukaryotes, or plants. Extracellular vesicles are responsible for interactions between cells through the transport of DNA, RNA, proteins, or lipids within the cell.

[0004] Recently, research is being conducted to clinically apply extracellular vesicles for drug development. In particular, extracellular vesicles obtained from human cell culture are widely used for the treatment of diseases. However, a large quantity of extracellular vesicles is required to demonstrate therapeutic activity in vivo. Therefore, there is a need for new sources or isolation methods for extracellular vesicles.

[0005] Exosomes are small, membrane-bound vesicles secreted by various types of cells. The diameter of exosomes is reported to be approximately 30–100 nm. Electron microscopy studies have observed that exosomes do not detach directly from the plasma membrane but rather originate from specific intracellular compartments called multivesicular bodies, from which they are released and secreted outside the cell. In other words, when the fusion of the multivesicular bodies and the plasma membrane occurs, the vesicles are released into the extracellular environment, which are referred to as exosomes. Although the specific molecular mechanisms by which these exosomes are produced have not been definitively elucidated, it is known that not only red blood cells but also various types of immune cells—including B lymphocytes, T lymphocytes, dendritic cells, platelets, and macrophages—as well as tumor cells and stem cells, produce and secrete exosomes while alive. In particular, exosomes derived from stem cells are known to play a role in intercellular communication, as they contain nuclear components in addition to receptors and proteins. In addition, exosomes derived from the above stem cells contain relatively less animal serum compared to stem cells, so the risk of zoonosis caused by animal serum infection can also be excluded. Prior art literature

[0007] Republic of Korea Patent Application No. 10-2017-0163897 The problem to be solved

[0008] Accordingly, the inventors of the present invention made diligent efforts to provide exosomes isolated from a new source or the therapeutic effects thereof, and confirmed that exosomes isolated from the supernatant of an induced pluripotent stem cell (iPSC) culture medium have the effect of improving nerve cell damage or regenerating skin, thereby completing the present invention.

[0009] Accordingly, the objective of the present invention is to provide a composition for the prevention, improvement, or treatment of neuronal damage diseases comprising a component derived from the supernatant of an induced pluripotent stem cell (iPSC) culture medium.

[0010] Another objective of the present invention is to provide a composition for skin regeneration comprising a component derived from the supernatant of an induced pluripotent stem cell (iPSC) culture medium. means of solving the problem

[0012] The present invention provides a pharmaceutical composition for the prevention or treatment of neuronal damage diseases comprising a component derived from the supernatant of an induced pluripotent stem cell (iPSC) culture medium.

[0013] According to a preferred embodiment of the present invention, the induced pluripotent stem cells are heterozygous or homozygous.

[0014] According to a preferred embodiment of the present invention, the induced pluripotent stem cells are induced pluripotent stem cells before differentiation or cells differentiated from induced pluripotent stem cells.

[0015] According to a preferred embodiment of the present invention, the cells differentiated from the induced pluripotent stem cells are cortical neural stem cells.

[0016] According to a preferred embodiment of the present invention, the component derived from the supernatant is a secretome secreted from induced pluripotent stem cells.

[0017] According to a preferred embodiment of the present invention, the secreted protein is an exosome.

[0018] According to a preferred embodiment of the present invention, the neuronal damage disease is one or more selected from the group consisting of taste disorders, olfactory disorders, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis, ischemic stroke, demyelinating disease, multiple sclerosis, epilepsy, degenerative neurological disease, and spinal cord injury.

[0019] According to a preferred embodiment of the present invention, the taste abnormality or olfactory abnormality is a taste abnormality or olfactory abnormality caused by coronavirus infection.

[0020] According to a preferred embodiment of the present invention, the composition is in the form of a spray, gargle, or injectable formulation.

[0021] In addition, the present invention provides a composition for improving neuronal damage comprising a component derived from the supernatant of an induced pluripotent stem cell (iPSC) culture medium.

[0022] According to a preferred embodiment of the present invention, the composition is in the form of a spray, gargle, or injectable formulation.

[0023] In addition, the present invention provides a skin regeneration composition comprising a component derived from the supernatant of an induced pluripotent stem cell (iPSC) culture medium.

[0024] According to a preferred embodiment of the present invention, the composition is in the form of a spray.

[0025] In addition, the present invention comprises: i) a step of subculture induced pluripotent stem cells (iPSCs);

[0026] ii) a step of centrifuging the above-mentioned subcultured iPSC culture medium to obtain the supernatant;

[0027] iii) A step of filtering the obtained supernatant using a syringe filter;

[0028] iv) a step of concentrating the filtered supernatant by freeze-drying or ultrafiltration; and

[0029] v) a step of separating secreted proteins from the concentrated sample using size exclusion chromatography or ultracentrifugation;

[0030] A method for isolating secreted proteins derived from induced pluripotent stem cells, including Effects of the invention

[0032] The components isolated from the culture medium of induced pluripotent stem cells of the present invention can improve neuronal damage or regenerate skin, and thus can be effectively used as a composition for the prevention, improvement, or treatment of neuronal damage-related diseases, particularly abnormalities in taste or smell caused by coronavirus infection or Alzheimer's disease, or as a composition for skin regeneration. Brief explanation of the drawing

[0034] Figure 1 shows the therapeutic effects of exosomes isolated from the culture medium of induced pluripotent stem cells (iPSCs) or iPSC-derived neural stem cells. Figure 2 shows the results of subculturing induced pluripotent stem cells (iPSCs) for 2 or 4 days. Figure 3 shows the process of isolating exosomes by expanding and culturing induced pluripotent stem cells (iPSCs). Figure 4 illustrates the process of isolating exosomes from induced pluripotent stem cells (iPSCs). Exosomes can be isolated by lyophilizing or ultrafiltration of components derived from the iPSC culture medium. Figure 5 shows the results of centrifuging the supernatant of the induced pluripotent stem cell (iPSC) culture medium. The centrifuged supernatant of the induced pluripotent stem cell (iPSC) culture medium was eluted through a column into a total of 33 fractions. Figure 6 shows the results of measuring exosome particles using Nanoparticle Tracking Analysis (NTA) by collecting fractions containing extracellular vesicles (EVs). Figure 7 shows 9.4 x 10 isolated exosomes 7 It indicates that it has been separated by particles / ml concentration. Figure 8 illustrates the process of induced pluripotent stem cells (iPSCs) differentiating into neurons. After 24 days of differentiation, iPSCs can differentiate into cortical neural stem cells with a Tbr2+ marker, and cortical neural stem cells can subsequently differentiate into mature cortical neurons. Figure 9 illustrates the differentiation of cortical neural stem cells and the isolation of exosomes using induced pluripotent stem cells (iPSCs). Secreted proteins can be obtained by freeze-drying the culture medium. Figure 10 illustrates the concept of an Alzheimer's treatment using the secreted protein of the present invention. Figure 11 shows the steps for manufacturing a mouse model and evaluating cognitive ability to evaluate the in vivo efficacy of the induced pluripotent stem cell-derived secretome (iPSC-Sec) and extracellular vesicles (EVs) of the present invention. Figure 12 shows the results of Barnes Maze experiments in mouse models administered the induced pluripotent stem cell-derived secretome (iPSC-Sec) and extracellular vesicles (EVs) of the present invention. Figure 13 shows the change in body weight of mouse models administered the induced pluripotent stem cell-derived secretome (iPSC-Sec) and extracellular vesicles (EVs) of the present invention. Figure 14 shows the change in the concentration of amyloid beta 42 in mouse models administered the induced pluripotent stem cell-derived secretome (iPSC-Sec) and extracellular vesicles (EVs) of the present invention. Figure 15 shows the NF-L concentrations of mouse models administered the induced pluripotent stem cell-derived secretome (iPSC-Sec) and extracellular vesicles (EVs) of the present invention. FIG. 16 shows the results of staining brain tissues with Thioflavin S of mouse models administered the induced pluripotent stem cell-derived secretome (iPSC-Sec) and extracellular vesicles (EVs) of the present invention (Group 1: G1, Group 2: G2, Group 3: G3, Group 4: G4, Group 5: G5, Group 6: G6, Group 7: G7, Group 8: G8, Group 9: G9, Group 10: G10). Specific details for implementing the invention

[0035] The present invention will be described in more detail below.

[0037] The term "prevention" in the present invention refers to any act that causes neuronal damage-related diseases to be suppressed or their onset to be delayed due to components derived from the supernatant of the iPSC culture medium of the present invention.

[0038] The term "improvement" or "treatment" of the present invention refers to any act that causes parameters related to neuronal damage-related diseases, such as the severity of symptoms, to be improved or beneficial due to components derived from the supernatant of the iPSC culture medium of the present invention.

[0040] The exosome of the present invention can provide cell proliferation, wound healing, anti-apoptotic or anti-inflammatory effects, and can be utilized as a drug delivery means or a novel iPSC induction means.

[0041] The advantages and disadvantages of lyophilization or ultrafiltration when isolating exosomes according to the present invention are as shown in [Table 1] below.

[0043] Lyophilization ultrafiltration merit Bulk production possible, low cost, long-term storage possible (powder) Fast, easy to use, and capable of immediate processing. disadvantage Exosomes may be damaged during freeze-drying; the pretreatment process takes a long time. High cost for large-scale processing; loss of filter membranes; potential for exosome damage during concentration.

[0045] The spray formulation containing the above exosomes is sprayed into the nose to allow the drug to pass through the olfactory epithelium toward the brain.

[0046] The olfactory epithelium contains olfactory nerve cells, which allow it to cross the blood-brain barrier (BBB) ​​and serve as a pathway for directly delivering drugs to the brain or cerebrospinal fluid (CSF).

[0047] The secretome or exosomes of cortical neural stem cells are predicted to contain large amounts of neuronal-related proteins or miRNAs, and by delivering them directly from the nose to the brain, the disadvantages of existing drug delivery methods, in which drugs cannot cross the BBB, can be compensated for.

[0049] Accordingly, the present invention can provide a pharmaceutical composition for the prevention or treatment of neuronal damage diseases comprising a component derived from the supernatant of an induced pluripotent stem cell (iPSC) culture medium.

[0050] According to a preferred embodiment of the present invention, the induced pluripotent stem cells may be heterozygous or homozygous.

[0051] According to a preferred embodiment of the present invention, the induced pluripotent stem cells may be induced pluripotent stem cells before differentiation or cells differentiated from induced pluripotent stem cells.

[0052] According to a preferred embodiment of the present invention, the cells differentiated from the induced pluripotent stem cells may be cortical neural stem cells.

[0053] According to a preferred embodiment of the present invention, the supernatant-derived component may be a concept that includes all components contained in the supernatant of the culture medium in which induced pluripotent stem cells are cultured, including secretomes secreted from induced pluripotent stem cells.

[0054] According to a preferred embodiment of the present invention, the secreted protein may be an exosome.

[0055] According to a preferred embodiment of the present invention, the neuronal damage disease may be one or more selected from the group consisting of taste disorders, olfactory disorders, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis, ischemic stroke, demyelinating disease, multiple sclerosis, epilepsy, degenerative neurological disease, and spinal cord injury.

[0056] According to a preferred embodiment of the present invention, the taste abnormality or olfactory abnormality may be a taste abnormality or olfactory abnormality caused by coronavirus infection.

[0057] According to a preferred embodiment of the present invention, the composition may be in the form of a spray, gargle, or injectable formulation.

[0058] The pharmaceutical composition of the present invention may be in various parenteral formulations. When formulating the composition, it may be prepared using one or more buffers (e.g., saline solution or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.

[0059] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, or suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrogol, Tween 61, cocoa paste, laurin paste, glycerol, gelatin, etc. may be used as bases for suppositories.

[0060] The composition of the present invention may be administered parenterally and, when administered parenterally, may be formulated according to methods known in the art in the form of a topical application; an injectable for intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, or intracerebrovascular injection; a transdermal administration; or a nasal inhalant.

[0061] The above-mentioned injectable must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Suitable carriers for the injectable may include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils, as solvents or dispersion media. More preferably, suitable carriers may include Hanks' solution, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol, and 5% dextrose. To protect the injectable from microbial contamination, various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal may be additionally included. Additionally, the injectable may, in most cases, further include isotonic agents such as sugars or sodium chloride.

[0062] Transdermal administration methods include forms such as ointments, creams, lotions, gels, topical solutions, pastes, liniments, and aerosols. In the above, transdermal administration means administering a pharmaceutical composition topically to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin.

[0063] In the case of an inhalation dose, the compound used according to the present invention can be conveniently delivered in the form of an aerosol spray from a pressurized pack or atomizer using a suitable propellant, for example, dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges used in an inhaler or blower can be formulated to contain the compound and a powder mixture of a suitable powder base, such as lactose or starch.

[0065] In addition, the present invention can provide a composition for improving neuronal damage comprising a component derived from the supernatant of an induced pluripotent stem cell (iPSC) culture medium.

[0066] According to a preferred embodiment of the present invention, the composition may be in the form of a spray, gargle, or injectable formulation.

[0067] The above spray formulation can be conveniently delivered in the form of an aerosol spray from a pressurized pack or atomizer using a suitable propellant, e.g., dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges used in an inhaler or blower can be formulated to contain the compound and a powder mixture of a suitable powder base, such as lactose or starch.

[0068] The above gargle formulation may include all gargle formulations commonly used in the relevant technical field as gargling products, such as toothpaste, oral spray, oral ointment, mouthwash, or mouthwash.

[0069] Since the above-mentioned injectable formulation is identical in concept to that used in the formulation of the above-mentioned pharmaceutical composition, the description is replaced by the description thereof.

[0071] In addition, the present invention can provide a composition for skin regeneration comprising a component derived from the supernatant of an induced pluripotent stem cell (iPSC) culture medium.

[0072] According to a preferred embodiment of the present invention, the composition may be in the form of a spray.

[0073] The above spray formulation can be conveniently delivered in the form of an aerosol spray from a pressurized pack or atomizer using a suitable propellant, e.g., dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges used in an inhaler or blower can be formulated to contain the compound and a powder mixture of a suitable powder base, such as lactose or starch.

[0075] In addition, the present invention comprises: i) a step of subculture induced pluripotent stem cells (iPSCs);

[0076] ii) a step of centrifuging the above-mentioned subcultured iPSC culture medium to obtain the supernatant;

[0077] iii) A step of filtering the obtained supernatant using a syringe filter;

[0078] iv) a step of concentrating the filtered supernatant by freeze-drying or ultrafiltration; and

[0079] v) a step of separating secreted proteins from the concentrated sample using size exclusion chromatography or ultracentrifugation;

[0080] A method for isolating secreted proteins derived from induced pluripotent stem cells, including [the above], can be provided.

[0081] The separation in step v) above may include all separations using methods used by a person skilled in the art to separate secreted proteins from a concentrated sample, but preferably refers to separation using size exclusion chromatography or ultracentrifugation.

[0083] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0085] Induced pluripotent stem cell passage and exosome isolation

[0086] Induced pluripotent stem cells (iPSCs) were cultured in vitronectin-coated dishes and incubated in a 37°C incubator under 10% CO2 conditions. The medium was changed daily with 6 ml of Fresh Essential 8 (E8) medium, and subculture was performed when the iPSCs reached 70% capacity. Trypsin-EDTA (TE) was used during subculture, and 10 μM rho-associated kinase (ROCK) inhibitor was added to the medium. On the fourth day after seeding, all culture medium was collected, and centrifugation was performed at 3,000 xg for 15 minutes at 4°C to remove cell debris. After centrifugation, the supernatant was transferred to a new conical tube, and the culture medium was filtered using a 0.2 mm syringe filter. The culture medium was concentrated by freeze-drying or ultrafiltration (4,500 xg, 30 min, 4 ℃), and then exosomes were isolated using a column.

[0087] For exosome isolation, the size exclusion chromatography (SEC) was set at room temperature and equilibrated with 10 mL of DPBS, after which the concentrated sample (0.5–1 mL) was added to the SEC. Once all the concentrated sample had entered the column, DPBS was added for elution. A total of 33 fractions were obtained, with 0.5 mL of eluted sample per fraction. After obtaining all fractions, cleaning in place (CIP) was performed using 2 mL of 0.5 M NaOH, and the column was then cleaned again using DPBS to remove any remaining NaOH. The particle count of the isolated and purified exosomes was measured using an NTA instrument, and their concentration was determined via ELISA.

[0089] Differentiation from induced pluripotent stem cells into cortical neurons

[0090] Frozen iPSCs were thawed and expanded. When the iPSCs reached 70% capacity, the cells were dissociated using gentle cell dissociation. After seeding in laminin-coated dishes, the culture medium containing Compound C and SB431542 was changed daily for 10 days. The culture medium was prepared by mixing N2 and B27 culture media in a 1:1 ratio. As a result, differentiation into Neuron progenitor cells (NPCs) was possible. Split NPCs were seeded in laminin-coated dishes, and the culture medium was changed every two days for 8 days. FGF2 was added on days 16 and 17 of differentiation. Consequently, differentiation into Neural stem cells (NSCs) was possible. Split NSCs were seeded in laminin-coated dishes, and the culture medium was changed every two days for 6 days. As a result, differentiation into Cortical neural stem cells was possible. After differentiation, the culture medium can be collected and freeze-dried to obtain a secretome or isolate exosomes.

[0092] In vivo efficacy evaluation

[0093] We intended to evaluate the in vivo efficacy of the induced pluripotent stem cell-derived secretome (iPSC-Sec) and extracellular vesicles (EVs) isolated in <Example 1> above.

[0094] As an animal model of Alzheimer's disease, induced pluripotent stem cell-derived secretomes or EVs were administered intranasally for 4 weeks to 13-week-old 5xFAD mice expressing human APP and PSEN1 transgenes (Fig. 11). The specific conditions are as shown in [Table 2] below.

[0096] experimental group explanation method Group 1 Alzheimer's (AD) DW 25 µl Group 2 iPSC-Sec 5㎍ / g Dissolve lyophilized iPSC-Sec in distilled water to the appropriate concentration within 25 µl and use, administer once a week for 4 weeks. Group 3 iPSC-Sec 10㎍ / g Dissolve lyophilized iPSC-Sec in distilled water to the appropriate concentration within 25 µl and use, administer once a week for 4 weeks. Group 4 iPSC-Sec 50㎍ / g Dissolve lyophilized iPSC-Sec in distilled water to the appropriate concentration within 25 µl and use, administer once a week for 4 weeks. Group 5 EVs 5.00E+05 EV 5x10 5 particles / germ in 25 µl, administered once a week for 4 weeks Group 6 EVs 5.00E+07 EV 5x10 7 particles / germ in 25 µl, administered once a week for 4 weeks Group 7 Media Only 50㎍ / g Dissolve the powder, which is obtained by freezing only the media corresponding to the maximum concentration of iPSC-Sec (50 µg / g), in distilled water and use it. Group 8 F. PBS Filter PBS 25 µl serving as the control group for EV treatment Group 9 Donepezil Donepezil, a positive drug for Alzheimer's disease, was administered intraperitoneally (IP) three times a week. Group 10 Wilde type healthy control for disease models

[0098] <3-1> Cognitive Ability Assessment

[0099] When the mice reached 17 weeks of age, the Barnes Maze was administered to measure cognitive improvement ability. For the Barnes Maze, a pre-maze was performed once a day for two days, and the main maze was performed on the third day to measure the mice's movement patterns and time. The time taken to first find and enter the target containing food was measured as latency (Fig. 11).

[0100] It was confirmed that the media-only group (Group 7) showed an increasing trend compared to the AD group, while the iPSC-Sec group containing secretome showed a slight decrease in latency. It was also confirmed that the EV-treated group showed a slight decrease in latency compared to the disease model F. PBS group (Group 8). The same pattern was observed for the target distance where food was located. When looking at the speed within the target zone, it was confirmed that the speed of the EV-treated group increased slightly compared to the group treated with F. PBS (Group 8) in the Alzheimer's model (Fig. 12).

[0102] <3-2> Measuring Mouse Body Weight

[0103] When the body weight of the mouse group was measured weekly, no significant difference due to the drug was confirmed up to 16 weeks (Fig. 13).

[0105] <3-3> Measurement of Amyloid Beta and NFL Concentrations

[0106] Brains of three animals from each group were homogenized, and ELISA for amyloid beta 42 and NF-L (neurofilament light chain) was performed. Amyloid beta 42 is known to accumulate significantly in the brain in Alzheimer's disease. NF-L is known to increase in the CSF of Alzheimer's patients.

[0107] The Amyloid beta 42 Mouse ELISA Kit (Invitrogen, # KMB3441) was used, and the procedure was performed according to the manufacturer's protocol. The amount of amyloid beta 42 measured via the BCA assay, which can measure total protein levels, was normalized. As an early Alzheimer's model, the data was obtained by injecting 5xFAD mice starting at 13 weeks of age, sacrificing them at 17 weeks of age, and extracting their brains.

[0108] It was confirmed that compared to the wild type (group 10), amyloid beta 42 increased in the Alzheimer's disease patients (AD group) and F. PBS group (group 8), and decreased slightly in the secretome-treated group iPSC-Sec 10 μg / g (Fig. 14).

[0109] When measuring NF-L (neurofilament light chain) in the brains of mice, compared to the wild type, the levels increased in the AD group and EV group, and showed a decreasing trend in the AD control group F. PBS, iPSC-Sec 50 μg / g (Fig. 15).

[0111] <3-4> Thioflavin staining

[0112] The brains of 5xFAD mice from each experimental group were extracted and frozen sections were performed.

[0113] Brain tissue was sectioned into 30 µm pieces and stained with Thioflavin S, which can stain amyloid beta plaques. Staining was performed by incubating in Thioflavin S solution for 8 minutes, and after washing with ethanol and mounting, the plaques were examined under a fluorescence microscope.

[0114] Amyloid beta plaques were expressed in Alzheimer's patients G1 (Group 1) and G8 (Group 8), but were not significantly expressed in the hippocampus, which is associated with cognitive function. Plaques were expressed in the cerebral cortex, but no significant difference in amyloid beta plaques was observed in the staining between the secretome-treated groups G2, G3, and G4 and the EV-treated groups G5 and G6 (Fig. 16).

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of neuronal damage, comprising a component derived from the supernatant of a culture medium for cortical neural stem cells differentiated from induced pluripotent stem cells (iPSCs), wherein the composition is characterized in that it is a spray formulation for nasal administration. Claim 2 A composition according to claim 1, characterized in that the induced pluripotent stem cells are heterozygous or homozygous. Claim 3 delete Claim 4 delete Claim 5 A composition according to claim 1, characterized in that the component derived from the supernatant is a secretome secreted from induced pluripotent stem cells. Claim 6 A composition according to claim 5, characterized in that the secreted protein is an exosome. Claim 7 A composition according to claim 1, characterized in that the neuronal damage disease is one or more selected from the group consisting of Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotriophic lateral sclerosis, ischemic stroke, demyelinating disease, multiple sclerosis, epilepsy, neurodegenerative disease, and spinal cord injury. Claim 8 delete Claim 9 delete Claim 10 A composition for improving neuronal damage comprising a component derived from the supernatant of a culture medium for cortical neural stem cells differentiated from induced pluripotent stem cells (iPSCs), wherein the composition is characterized in that it is a spray formulation for nasal administration. Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 i) a step of subculture induced pluripotent stem cells (iPSCs); ii) a step of differentiating the subcultured iPSCs into cortical neural stem cells; iii) a step of centrifuging the culture medium of the differentiated cortical neural stem cells to obtain a supernatant; iv) a step of filtering the obtained supernatant with a syringe filter; v) a step of concentrating the filtered supernatant by freeze-drying or ultrafiltration; and vi) a step of separating the secreted proteins from the concentrated sample using size exclusion chromatography or ultracentrifugation; comprising a method for separating secreted proteins derived from induced pluripotent stem cells for nasal administration.

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