Human pluripotent stem cell-derived senomorphic extracellular vesicle composition for treating cellular senescence and stem cell degeneration

Abstract

The present invention relates to a human pluripotent stem cell-derived senomorphic extracellular vesicle composition for treating cellular senescence and stem cell degeneration. Specifically, the present invention relates to a senomorphic composition comprising stem cell-derived extracellular vesicles as an active ingredient, the composition being characterized by restoring a function or morphology of senescent cells to that of normal cells, and to a pharmaceutical composition comprising the senomorphic composition as an active ingredient for preventing or treating nerve injury and ischemic diseases.

Description

Composition of human pluripotent stem cell-derived cenomomorphic extracellular vesicles for the treatment of cellular aging and stem cell degeneration

[0001] The present invention relates to a composition of human pluripotent stem cell-derived cenomorphic extracellular vesicles for the treatment of cellular aging and stem cell degeneration, and to the use thereof.

[0002] Pluripotent stem cells, including induced pluripotent stem cells and embryonic stem cells, are being actively researched as cell therapies for degenerative diseases requiring tissue transplantation due to their excellent proliferative and differentiation capabilities. However, potential carcinogenicity and the limited viability of transplanted cells are cited as limitations to their practical therapeutic efficacy.

[0003] In addition, the use of pluripotent stem cell-derived secretory bodies (secretoms) has recently been gaining attention as a potential alternative, and in particular, extracellular vesicles containing exosomes, microvesicles, microparticles, and ectosomes within the secretory bodies are considered to be key factors mediating the effects of the secretory bodies.

[0004] Extracellular vesicles derived from pluripotent stem cells are known to contain large amounts of proteins and nucleic acids associated with antioxidant and anti-inflammatory responses, cell survival, and cell growth; however, research regarding their relationship with neurological and ischemic diseases involving oxidation, inflammation, apoptosis, cellular senescence, and stem cell degeneration remains insufficient. In particular, neural stem cells endogenous in adult nervous tissue mediate the prevention, inhibition, and recovery of neurological diseases through their proliferative, differentiation, and secretory capabilities. Therefore, cellular senescence caused by oxidative stress within neural stem cells induces reduced proliferative capacity and apoptosis, while increasing the expression of aging-related inflammatory cytokines, thereby leading to stem cell degeneration and extensive damage to nervous tissue.

[0005] To overcome this, research on various drugs for neural stem cell regeneration is underway, but a drug with significantly superior efficacy has not yet been developed.

[0006] Furthermore, there is a lack of research on the anti-aging of neural stem cells and the reduction of aging-related inflammatory cytokine secretion by pluripotent stem cell-derived extracellular vesicles.

[0007] Accordingly, the inventors isolated extracellular vesicles from human pluripotent stem cells and confirmed that the isolated extracellular vesicles have an excellent effect in inhibiting cell aging and improving stem cell degeneration. Furthermore, they confirmed that extracellular vesicles obtained from pluripotent stem cells cultured under hypoxic conditions have increased expression of glutathione system-related factors, thereby more effectively inhibiting cell aging and improving stem cell degeneration. Thus, the inventors completed the present invention by identifying that a composition containing extracellular vesicles derived from pluripotent stem cells can be used for the prevention, improvement, or treatment of nerve damage and ischemic diseases.

[0008] Therefore, the object of the present invention is to provide a senomorphic composition characterized by restoring the function or morphology of senescent cells to that of normal cells, comprising stem cell-derived extracellular vesicles as an active ingredient.

[0009] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of nerve damage and ischemic diseases comprising the cenomorphic composition of the present invention as an active ingredient.

[0010] Accordingly, the present invention provides a senomorphic composition characterized by restoring the function or morphology of senescent cells to that of normal cells, comprising stem cell-derived extracellular vesicles as an active ingredient.

[0011] In one embodiment of the present invention, the stem cells may be embryonic stem cells, induced pluripotent stem cells, adult stem cells, or progenitor cells.

[0012] In one embodiment of the present invention, the senescent cells may be neural stem cells or neural progenitor cells.

[0013] In one embodiment of the present invention, the extracellular vesicles may be exosomes, microvesicles, microparticles, or ectosomes.

[0014] In one embodiment of the present invention, the extracellular vesicle may be obtained from stem cells cultured under hypoxic conditions.

[0015] In one embodiment of the present invention, the composition may have inhibition of the production of aging-related inflammatory cytokines; antioxidant activity; inhibition of the expression of aging-related markers; maintenance of the self-proliferative capacity of stem cells; and the ability to repair cell damage.

[0016] In one embodiment of the present invention, the inflammatory cytokine may be selected from the group consisting of CXCL1, MMP9, IL-6, and IL-8.

[0017] In one embodiment of the present invention, the aging-related markers may be p16 and SA-β-Gal (SA-β-galactosidase).

[0018] In one embodiment of the present invention, the extracellular vesicle may have increased expression and activity of the glutathione system.

[0019] In one embodiment of the present invention, the extracellular vesicles may have increased expression of NRF2 (Nuclear factor erythroid-2-related factor 2), GSTK1 (Glutathione S-transferase kappa 1), GSTO1 (Glutathione S-transferase omega-1) and GSTZ1 (Glutathione S-transferase Zeta 1).

[0020] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of nerve damage and ischemic diseases comprising the cenomorphic composition of the present invention as an active ingredient.

[0021] In one embodiment of the present invention, the nerve damage may be caused by apoptosis of nerve cells, morphological abnormalities of cells, neurotransmission abnormalities, oxidative stress, increased expression of aging-related markers, increased inflammatory response, or increased secretion of aging-related inflammatory cytokines and decreased secretion of neuroregenerative growth factors.

[0022] In one embodiment of the present invention, the ischemic disease may be selected from the group consisting of ischemic necrosis, ischemic cerebrovascular disease, ischemic renal disease, ischemic lung disease, ischemic disease of the limbs, ischemic heart disease, stroke, cerebral infarction, myocardial infarction, ischemic heart failure, and obstructive arteriosclerosis.

[0023] It has been confirmed through experiments that the stem cell-derived extracellular vesicles provided by the present invention inhibit the production of aging-related inflammatory cytokines, possess antioxidant activity, suppress the expression of aging-related markers, maintain the self-proliferative capacity of stem cells, and have excellent cell damage recovery capabilities. In particular, the expression of glutathione system-related factors such as NRF2, GSTK1, GSTO1, and GSTZ1 is significantly increased in stem cell-derived extracellular vesicles cultured under hypoxic conditions, and excellent anti-aging activity has been confirmed. Therefore, the stem cell-derived extracellular vesicles according to the present invention or stem cell-derived extracellular vesicles cultured under hypoxic conditions can be usefully used as cenomorphic therapeutic agents, and furthermore, they can be usefully used as pharmaceuticals for the prevention, improvement, or treatment of nerve damage and ischemic diseases.

[0024] Figure 1 shows the results of confirming the anti-aging activity of extracellular vesicle-mediated hypoxia-reperfusion injury neural stem cells derived from pluripotent stem cells.

[0025] Figure 2 shows the results confirming the cenomorphic action of pluripotent stem cell-derived extracellular vesicles on hypoxic-reperfusion injury neural stem cells.

[0026] Figure 3 shows the results of confirming the anti-aging activity of neural stem cells by glutathione S-transferase containing extracellular vesicles derived from pluripotent stem cells.

[0027] Figure 4 shows the results of confirming the anti-aging activity of neural stem cells by the enhanced glutathione system within extracellular vesicles derived from pluripotent stem cells.

[0028] As cells divide, they undergo aging, and after a certain number of divisions, they reach Hayflick's limit, a state where they can no longer divide. Cells that have reached this limit are called senescent cells.

[0029] In the past, aging research was focused on identifying the causes of aging and methods to extend lifespan at the molecular level, but recently, research on the development of methods and technologies for anti-aging in terms of prevention, recovery, and treatment has been actively underway, and in particular, research focused on delaying aging and developing therapeutic substances for aging-related diseases is increasing.

[0030] Strategies for developing drugs to combat aging can be broadly divided into the development of senolytics, which eliminate only senescent cells, and senomorphics, which improve the characteristics of senescent cells to resemble those of young cells; however, development of drugs that restore the overall characteristics of senescent cells to induce active cell division similar to that of young cells is currently lacking.

[0031] Accordingly, while conducting research to develop a cenomorphic therapeutic agent capable of improving or inhibiting cellular senescence of stem cells and improving stem cell degeneration, the inventors confirmed that stem cell-derived extracellular vesicles, particularly those cultured under hypoxic conditions, can be usefully utilized as a cenomorphic therapeutic agent by inhibiting the production of aging-related inflammatory cytokines, exhibiting excellent antioxidant activity, suppressing the expression of aging-related markers, maintaining the self-proliferative capacity of stem cells, and demonstrating the ability to repair cell damage.

[0032] Accordingly, the present invention is characterized by providing a senomorphic composition comprising a stem cell-derived extracellular vesicle as an active ingredient, which restores the function or morphology of senescent cells to that of normal cells.

[0033] The above extracellular vesicles can be obtained from stem cells, and the stem cells may be embryonic stem cells, induced pluripotent stem cells, adult stem cells, or progenitor cells, although not limited thereto; in one embodiment of the present invention, extracellular vesicles were obtained from human induced pluripotent stem cells.

[0034] In addition, the senescent cells for inducing a cenomorphic effect by treating with the composition of the present invention may be nerve cells, neural stem cells, or neural progenitor cells, and preferably may be nerve cells, neural stem cells, or neural progenitor cells that have undergone aging due to oxidative stress, inflammatory response, damage to cells or tissues, etc.

[0035] The above extracellular vesicles may be exosomes, microvesicles, microparticles, or ectosomes.

[0036] In one embodiment of the present invention, an experiment was conducted to confirm the cenomorphic effect of stem cell-derived extracellular vesicles. Extracellular vesicles were obtained from cultured human induced pluripotent stem cells and treated with neural stem cells under conditions of oxygen-glucose deprivation and reoxygenation (OGD / R), which mimic hypoxia-reperfusion injury. As a result, it was confirmed that oxidative stress was reduced by inhibiting the generation of reactive oxygen species, cellular aging could be improved by suppressing the expression of cellular aging markers, and the expression of SOX1 and PAX6, representative markers of neural stem cells, and self-proliferative activity were restored.

[0037] In addition, when stem cell-derived extracellular vesicles were treated to hypoxic-reperfusion injury neural stem cells, it was confirmed that the expression of aging-related inflammatory cytokines CXCL1, MMP9, IL-6, and IL-8, which are increased by hypoxic-reperfusion injury, was effectively suppressed. Consequently, oxidative stress within neurons was reduced and cellular aging was improved, which was shown to increase the gene expression of BDNF, GDNF, and VEGFA within neurons, which contribute significantly to the recovery of many neurological diseases.

[0038] Through these results, the inventors found that the stem cell-derived extracellular vesicles according to the present invention possess inhibition of the production of aging-related inflammatory cytokines, antioxidant activity, inhibition of the expression of aging-related markers, maintenance of stem cell self-proliferative capacity, and cell damage repair ability, and confirmed that the stem cell-derived extracellular vesicles of the present invention can be usefully utilized as cenomorphic therapeutic agents.

[0039] In addition, regarding the cenomorphic activity of stem cell-derived extracellular vesicles, the inventors analyzed the major proteins contained in the extracellular vesicles using proteomics. As a result, it was found that biological phenomena related to oxidation reactions, subsequent neuronal death, and glutathione transport were significantly related to the major proteins contained in the extracellular vesicles, and it was confirmed that the activity of glutathione S-transferase plays a very important role.

[0040] Furthermore, as a method to obtain extracellular vesicles with enhanced pharmacological efficacy as described above, the inventors cultured stem cells under hypoxic conditions, obtained extracellular vesicles from the cultured stem cells, and analyzed their activity.

[0041] As a result, extracellular vesicles obtained from stem cells cultured under hypoxic conditions showed significantly increased expression of glutathione system-related factors, such as NRF2 (Nuclear factor erythroid-2-related factor 2), GSTK1 (Glutathione S-transferase kappa 1), GSTO1 (Glutathione S-transferase omega-1), and GSTZ1 (Glutathione S-transferase Zeta 1), compared to extracellular vesicles derived from stem cells cultured under normal culture conditions.

[0042] Furthermore, extracellular vesicles obtained from stem cells cultured under hypoxic conditions were found to exhibit significantly superior antioxidant activity, reduced expression of cellular senescence-related markers, inhibition of stem cell regression, and enhanced resilience compared to extracellular vesicles derived from stem cells cultured under normal culture conditions.

[0043] Through the above results, the inventors found that a composition containing stem cell-derived extracellular vesicles or stem cell-derived extracellular vesicles cultured under hypoxic conditions can be used as a senomorphic composition, and furthermore, can be used as a therapeutic agent for nerve damage and ischemic diseases.

[0044] Therefore, the present invention may provide a pharmaceutical composition for the prevention or treatment of nerve damage and ischemic diseases, comprising as an active ingredient a cenomorphic composition containing stem cell-derived extracellular vesicles of the present invention or stem cell-derived extracellular vesicles cultured under hypoxic conditions.

[0045] The above-mentioned nerve damage may be caused by neuronal apoptosis, cell morphology abnormalities, neurotransmission abnormalities, oxidative stress, increased expression of aging-related markers, increased inflammatory response, or increased secretion of aging-related inflammatory cytokines and secretion of neuroregenerative growth factors.

[0046] In addition, the above-mentioned ischemic disease may be selected from the group consisting of ischemic necrosis, ischemic cerebrovascular disease, ischemic renal disease, ischemic lung disease, ischemic disease of the extremities, ischemic heart disease, stroke, cerebral infarction, myocardial infarction, ischemic heart failure, and obstructive arteriosclerosis.

[0047] Since the pharmaceutical composition according to the present invention contains the extracellular vesicle of the present invention as an active ingredient, it can prevent, improve, or treat nerve damage and ischemic diseases by improving oxidative stress through anti-aging of cells, suppressing the expression of aging-related markers, maintaining the characteristics and proliferative capacity of stem cells, suppressing inflammatory responses, and improving the secretion of aging-related inflammatory cytokines and neuroregenerative growth factors.

[0048] The pharmaceutical composition according to the present invention may use any one formulation selected from the group consisting of injectables, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, drops, or liquids according to conventional methods.

[0049] The above pharmaceutical composition may further include one or more additives selected from the group consisting of suitable carriers, excipients, disintegrants, sweeteners, coating agents, leavening agents, lubricants, lubricants, flavoring agents, antioxidants, buffers, bacteriostatic agents, diluents, dispersants, surfactants, binders, and lubricants commonly used in the manufacture of pharmaceutical compositions.

[0050] Specifically, the carrier, excipient, and diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. These solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition, lubricants such as magnesium stearate and talc may also be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups, and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. For non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerogelatin, etc. may be used as the base for suppositories.

[0051] According to one embodiment of the present invention, the pharmaceutical composition may be administered to a subject in a conventional manner through intravenous, intra-arterial, intra-abdominal, intramuscular, intra-arterial, intra-abdominal, intrasternal, transdermal, nasal, inhalation, local, rectal, oral, ocular, or intradermal routes.

[0052] The preferred dosage of the above active ingredient may vary depending on the subject's condition and body weight, the type and severity of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by a person skilled in the art. According to one embodiment of the present invention, although not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, and more specifically 0.1 to 100 mg / kg. The administration may be performed once a day or divided into several doses, and the scope of the present invention is not limited by this.

[0053] In the present invention, the 'object' may be a mammal including a human, but is not limited to these examples.

[0054] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art. The following experimental examples are intended to provide experimental examples that are commonly applicable to each of the embodiments according to the present invention.

[0055]

[0056] <Materials Preparation and Experimental Method>

[0057] 1) Obtaining extracellular vesicles from pluripotent stem cells

[0058] Step 0: Culture of pluripotent stem cells

[0059] The BG01 pluripotent stem cell line was feder-free in mTeSR™1 medium (Stemcell Technologies) in Matrigel (Corning) coated culture dishes at 37°C for 30 minutes. These cells were cultured under standard conditions (37°C, 5% CO2, saturated humidity), and the medium was changed daily. Subculture was performed every 5 to 6 days at a ratio of 1:10 after individualizing the cells by treating them with ACCUTASE (Stemcell Technologies) at 37°C for about 5 minutes.

[0060]

[0061] Step 1: Harvesting pluripotent stem cell-derived culture medium

[0062] To harvest extracellular vesicles from the pluripotent stem cells cultured in Step 0 above, the BG01 pluripotent stem cell line was dispensed onto a 150 pi culture dish (Corning) and cultured under the same conditions as described above. After collecting 30 ml of mTeSR™1 medium (Stemcell Technologies) cultured for 24 hours from the point when a cell density of 50% was reached, the medium was centrifuged at 300 x g for 5 minutes to remove debris such as dead cells. Subsequently, the supernatant was harvested again and centrifuged at 3000 x g for 10 minutes to remove cell debris of smaller units; the supernatant was collected and named the pluripotent stem cell-derived culture medium or pluripotent stem cell-derived secretome and used.

[0063]

[0064] Step 2: Concentration of pluripotent stem cell-derived culture medium

[0065] To purify a large amount of extracellular vesicles from the pluripotent stem cell-derived culture medium obtained in Step 1 above, the culture medium was concentrated. For concentration, 15 ml of the culture medium was placed in an Amicon Ultra-15, 100 kDa MWCO (Millipore) filter and centrifuged at a speed of 3500 x g for 15 minutes. Since the filter of this product allows only molecules weighing 100 kDa or less to pass through, extracellular vesicles much larger than that size remain in the filter without passing through; therefore, the resulting unfiltered solution was referred to as the concentrate. Concentration was performed until the total volume of the culture medium was reduced to 1 / 20 of its value before concentration.

[0066]

[0067] Step 3: Harvesting of pluripotent stem cell-derived extracellular vesicles

[0068] To harvest extracellular vesicles from pluripotent stem cell-derived culture concentrates, highly concentrated extracellular vesicles were harvested by performing two consecutive ultracentrifugations at 105,000 x g at 4°C using Beckman Coulter’s Optima™ XE-90 Ultracentrifuge and SW 41 Ti rotor. The protein concentration of the harvested extracellular vesicle solution was measured using the BCA Protein Assay Kit (Sigma-Aldrich) according to its manual.

[0069]

[0070] 2) Harvesting of extracellular vesicles from hypoxic pluripotent stem cells

[0071] After culturing the BG01 pluripotent stem cell line in the same manner as in 1) above, 30 ml of mTeSR™1 medium (Stemcell Technologies) was added when the cell density reached 50%, and the cells were placed in a Hypoxia Incubator Chamber (Stemcell Technologies). The cells were then cultured at 37°C for 24 hours under a 95% N2, 5% CO2 environment. Subsequently, extracellular vesicles were purified using the harvested 30 ml of mTeSR™1 medium in the same manner as above and used as hypoxic pluripotent stem cell-derived extracellular vesicles (HypoxEVs).

[0072]

[0073] 3) Differentiation of neural stem cells from pluripotent stem cells

[0074] All differentiation processes were carried out according to the provided protocol using the PSC Neural Induction Medium kit (Gibco). The medium for differentiation from pluripotent stem cells into neural stem cells was prepared by adding 10 ml of Neural Induction Supplement to 490 ml of Neurobasal Medium, a component of the kit.

[0075] 2.5 × 10^5-3 × 10^5 BG01 pluripotent stem cell lines were inoculated into a Matrigel-coated 6-well plate (Corning) in 2.5 ml of mTeSR™1 medium (Stemcell Technologies) containing 10 mM Y-27632 (Tocris). The next day, the medium was removed and 2.5 ml of prepared neural stem cell differentiation medium was added. The neural stem cell differentiation medium was replaced at 48-hour intervals, and 5 ml of differentiation medium was used starting from day 4 of differentiation, with differentiation completed on day 7.

[0076] On day 7 of differentiation, cells were treated with ACCUTASE (Stemcell Technologies) at 37°C for approximately 5 minutes and individualized using a cell scraper (Gibco). Neural stem cells were inoculated into Matrigel (Corning) coated culture dishes in Neural Expansion Medium containing 5 μM Y-27632 (Tocris). The medium was prepared by mixing 2 ml of Neural Induction Supplement with 49 ml of Neurobasal Medium and 49 ml of Advanced™ DMEM / F-12 (Gibco).

[0077]

[0078] 4) Immunofluorescence staining

[0079] For immunofluorescence staining, cultured neural stem cells were washed three times with PBS and fixed by treating them in a 4% paraformaldehyde (Sigma-Aldrich) solution for 20 minutes. After washing three times with PBS, blocking and perforation were performed at room temperature for 45 minutes using PBS containing 10% Donkey Serum (Abcam) and 0.1% Triton X-100 (Sigma-Aldrich). Subsequently, the neural stem cells were treated with the primary antibody in a 10% Donkey Serum (Abcam) solution and incubated overnight (12–16 hours) at 4°C. After the reaction was complete, the neural stem cells were washed three times with PBS, the secondary antibody in a 10% Donkey Serum (Abcam) solution was added, and the cells were incubated at room temperature for 1 hour and 30 minutes. Subsequently, after washing three times with PBS, the cell nuclei were stained by reacting in PBS containing 2 μg / ml DAPI (4',6-diamidino-2-phenylindole, Sigma-Aldrich) for 10 minutes. After washing three times with PBS, the finally stained neural stem cells were mounted on a slide glass, and immunofluorescence staining images were captured using a confocal microscope (Zeiss, LSM800). The information on the primary and secondary antibodies used is as listed in Tables 1 and 2 below.

[0080] Primary Antibody Primary Antibody Manufacturer Product Number CDKN2A / p16 Antibody (F-12)Santacruz Biotechnologysc-1661NESTIN Antibody Abcamab92391

[0081] Secondary Antibody Manufacturer Product Number Donkey anti-mouse IgG Alexa 647 InvitrogenA31571 Donkey anti-rabbit IgG Alexa 568 InvitrogenA10042

[0082]

[0083] 5) Perform reverse transcription and quantitative PCR (RT-qPCR).

[0084] To extract total RNA from the cells, neural stem cells were washed three times with PBS, and RNA was extracted using Trizol (Invitrogen) reagent. The concentration of the extracted RNA was measured using a Colibri Microvolume Spectrometer (Titertek-Berthold). Based on the measured concentration, reverse transcription was performed using the RevertAid Reverse Transcription (RT) Kit (Thermo Fisher Scientific) until the concentration of the generated cDNA reached 10 μl / ml. RT-qPCR analysis was performed using iQ™ SYBR Green SuperMix (Bio-Rad), and the results of amplification under conditions suitable for each primer were analyzed using the CFX-96 Real-Time PCR Detection System (Bio-Rad). The relative expression levels of each gene were measured by adjusting based on the expression of the housekeeping genes GAPDH and ACTB. Three independent experiments were conducted, and the sequences of the primers used are listed in Table 3 below.

[0085] 프라이머 서열유전자정방향 (5'->3')역방향 (5'->3')SOX1GTC ACG GGT TTC CCA ATT CGGT TCA GCG ATT GTG TTT CCPAX6ACC CGG CAG AAG ATT GTA GACTC ACA CAT CCG TTG GAC ACIL6GGA GAC TTG CCT GGT GAA AACAG GGG TGG TTA TTG CAT CTIL8GTG TGA AGG TGC AGT TTT GCAGT TTT CCT TGG GGT CCA GACXCL1CCC CAA GTT AGT TCA ATC TGG ACAT ACA TTC CCC TGC CTT CAMMP9CAG TCC ACC CTT GTG CTC TTGGG ACA GTT GCT TCT GGA GABDNFTCC CTG TAT CAA AAG GCC AACATC GCC AGC CAA TTC TCT TTGDNFTCC CAT TCA GAG AAC CTT GGTGG ATT TGG TGG AGC AAC TTVEGFATCC GGG TTT TAT CCC TCT TCCCT CTT TCT GCT GGT TTC CAGSTP1CTC TAT GGG AAG GAC CAG CAGAC AGC AGG GTC TCA AAA GGNFE2L2TGC CCT CAC CTG CTA CTT TACCA CAC TGG GAC TTG TGT TTGLSTGG TCT CCT CCT CTG GAT AAATC ACC ACC TTC TCT TCG AGGLS2GCC ACA TTC CAG TCA GAG ACAA GTG ACC TCC ACA GAA CAGCLCACC ATC ATC AAT GGG AAG GAGCG ATA AAC TCC CTC ATC CAGCLMCCA GAT TTG ACT GCA TTT GCTTC ACA ATG ACC GAA TAC CGGSSGAG GGT GGA GGT AAC AAC CTAAGC TCT GAA ATG CAC TGG ACGSTM2GTT TAT GGA CAG CCG TAT GCCTT CAG CAT TTC AGG GAG TGMGST1CCCACC TGA ATG ACC TTG ATGC AAT GGT GTG GTA GAT CCSLC7A11TGT GGG GTC CTG TCA CTA TTGAG TTC CAC CCA GAC TCG TAGGT1TGG ATG TGA CTG AGG TGG TCCGT GTA GAA CTC GGG CTT GGSRATC CCA AGC CCA CAA TAG AGGCA ATG TAA CCT GCA CCA ACGPX4CAG TGA GGC AAG ACC GAA GGAA CTG GTT ACA CGG GAA GGGAPDHTGG TAT CGT GGA AGG ACT CAGCA GGG ATG ATG TTC TGG AACTBAGA GCT ACG AGC TGC CTG ACAGC ACT GTG TTG GCG TAC AG

[0086] 6) 웨스턴 블롯팅

[0087] Extracellular vesicles were lysed in 1× RIPA buffer (Biosolution) containing a PhosSTOP™ and cOmplete™ protease inhibitor cocktail (Roche) and centrifuged at 13,000 × g for 20 minutes at 4°C. The protein concentration of the supernatant was measured using a BCA Protein Assay Kit (Sigma-Aldrich). 10–20 μg of protein was separated by SDS-PAGE on NuPAGE™ 1.0–1.5 mm, 4–12% Bis-Tris mini protein gels (Invitrogen). The proteins were transferred to an Immun-Blot PVDF membrane (polyvinylidene difluoride, Bio-Rad) and subsequently washed three times with TBST (0.1% [v / v] Tween 20 [Sigma-Aldrich] containing Tris-buffered saline [Calbiochem]). The membranes were blocked with 5% (w / v) Blotting Grade Blocker nonfat dry milk (Bio-Rad) or bovine serum albumin (BSA, Bovogen) dissolved in TBST at room temperature for 1 hour, followed by overnight (12-16 hours) with the primary antibody at 4°C, and then with the HRP-conjugated secondary antibody for 1 hour at room temperature. The membranes reacted with the antibodies were visualized using WestGlow™ ECL chemiluminescent substrate (Biomax), and images were analyzed using ImageQuant LAS4000 (GE Healthcare) and ImageQuant LAS 4000 Control Software (GE Healthcare). Information on the primary and secondary antibodies used is listed in Tables 4 and 5 below.

[0088] Primary Antibody Primary Antibody Manufacturer Product Number TSG101 Antibody Abcamab125011GSTP1 Antibody Cell Signaling Technology3369β-actin Antibody Cell Signaling Technology4970NRF2Abcamab62352MGST1Abcamab131059GSTK1Santacruz Biotechnologysc515580GSTO1Abcamab129106GSTZ1Abcamab153995

[0089] Secondary Antibody Manufacturer Product Number Goat anti-mouse IgG HRP Invitrogen 31430 Donkey anti-rabbit IgG HRPA bcamab 6802

[0090]

[0091] <Example 1>

[0092] Confirmation of anti-aging of pluripotent stem cell-derived extracellular vesicle-mediated hypoxia-reperfusion injury neural stem cells

[0093] After treating neural stem cells under oxygen-glucose deprivation and reoxygenation (OGD / R) conditions, which mimic hypoxic-reperfusion injury, with pluripotent stem cell-derived extracellular vesicles (OGD / EVs), the effects on oxidative stress, cellular senescence, stem cell characteristics, and proliferation were analyzed.

[0094]

[0095] As a result, as shown in Fig. 1, neural stem cells showed a rapid increase in reactive oxygen species (ROS) under OGD / R conditions mimicking hypoxia-reperfusion injury conditions, as confirmed by Abcam’s Cellular ROS Assay Kit [Red] (Fig. 1b), and an increase in the expression of p16, a marker of cellular senescence, was also confirmed (Fig. 1c). Meanwhile, in the group treated with 40 μg / ml of pluripotent stem cell-derived extracellular vesicles whose membrane structure was observed via transmission electron microscopy, the generation of reactive oxygen species decreased to the level of the control group, and the increase in p16 expression was also found to decrease and improve (Figs. 1a to 1c). In particular, the expression of senescence-associated β-galactosidase (SA-β-gal), a representative marker of cellular senescence, was confirmed using Sigma-Aldrich's Senescence Cells Histochemical Staining Kit, and the inhibition of SA-β-gal was confirmed (Fig. 1d).

[0096] Through these results, it was found that the pluripotent stem cell-derived extracellular vesicles of the present invention can induce cellular anti-aging of neural stem cells.

[0097]

[0098] In addition, cellular senescence caused by hypoxia-reperfusion injury was shown to inhibit the expression of SOX1 and PAX6, which mediate the stem cell characteristics of neural stem cells (Fig. 1e), and the self-proliferative capacity of neural stem cells was also shown to be suppressed (Fig. 1f). However, in the group treated with the pluripotent stem cell-derived extracellular vesicles of the present invention, it was confirmed that the expression of reactive oxygen species and p16 protein, which were increased by hypoxia-reperfusion injury, was suppressed and improved to levels similar to the control group, and it was confirmed that the expression levels of SOX1 and PAX6, which are markers of neural stem cells, and self-proliferative capacity were restored to levels similar to the control group (Figs. 1e and 1f).

[0099] Through this, the inventors found that the extracellular vesicles derived from pluripotent stem cells according to the present invention possess antioxidant and anti-aging capabilities, can inhibit the degeneration of stem cells, and can restore stem cell properties and proliferative capacity.

[0100]

[0101] <Example 2>

[0102] Confirmation of cenomorphic action of pluripotent stem cell-derived extracellular vesicles on hypoxic-reperfusion injury neural stem cells

[0103] Next, the inventors analyzed the effects of treatment with the pluripotent stem cell-derived extracellular vesicles of the present invention on the secretion of aging-related inflammatory cytokines and the regeneration of neural damage in neural stem cells with hypoxia-reperfusion injury.

[0104]

[0105] <2-1> Analysis of the Effects on the Secretion of Aging-Related Inflammatory Cytokines

[0106] As shown in Fig. 2, the expression of aging-related inflammatory cytokines IL-6, IL-8, CXCL1, and MMP9, which play a major role in the development and spread of aging and disease in various organs including the brain, increased rapidly due to hypoxia-reperfusion injury, but it was confirmed that they significantly decreased upon treatment with the pluripotent stem cell-derived extracellular vesicles of the present invention (Fig. 2a). In addition, the secretion of the inflammation-inducing factors IL-6 and IL-8 was also found to decrease in the group treated with the pluripotent stem cell-derived extracellular vesicles of the present invention (Figs. 2b, 2c). Therefore, through these results, it was found that the pluripotent stem cell-derived extracellular vesicles of the present invention can induce a cenomorphic effect by inhibiting the production of inflammatory factors involved in aging.

[0107]

[0108] <2-2> Analysis of the Effects of Neural Stem Cells on the Regenerative Capacity of Nerve Damage

[0109] Tissue-intrinsic stem cells secrete various types of molecules, such as proteins, carbohydrates, and lipids, into the tissue microenvironment to regulate tissue homeostasis and the regeneration process in the event of damage. Neural stem cells can also maintain neuronal homeostasis and prevent or repair damage through their secretory capacity and secretory factors. Since the role of aging-related inflammatory cytokines secreted by neural stem cells is significant in this process, we analyzed the effects of changes in the composition of neural stem cell-derived secretory medium (NSCM) following treatment with pluripotent stem cell-derived extracellular vesicles on the neuronal homeostasis maintenance and damage recovery capabilities of the said secretory medium.

[0110]

[0111] To this end, 2D neurons were cultured in an environment of oxygen-glucose depletion and reoxidation under identical conditions to induce hypoxia-reperfusion injury. Similar to neural stem cells, 2D neurons also showed increased intracellular production of reactive oxygen species and increased expression of the aging marker p16 in response to hypoxia-reperfusion injury (Figs. 2d, 2e). In the case of neural stem cell-derived secretory bodies (NSCM(OGD / R)) containing large amounts of aging-related inflammatory cytokines, cellular aging was accelerated by hypoxia-reperfusion injury, which was found to further exacerbate the pattern of neurological damage; however, in the case of neural stem cell-derived secretory bodies (NSCM(OGD / EVs)) in which anti-aging and the secretion of aging-related inflammatory cytokines were reduced following treatment with the pluripotent stem cell-derived extracellular vesicles of the present invention, the production of reactive oxygen species and the expression of p16 protein were found to decrease to levels similar to those of the untreated group (OGD / R) (Figs. 2d, 2e). In particular, it was confirmed that the intraneural gene expression of BDNF, GDNF, and VEGFA, which play an important role in the recovery of many neurological diseases, was significantly reduced in the NSCM (OGD / R) group but recovered to a level similar to the untreated group in the NSCM (OGD / EVs) group (Fig. 2f).

[0112] Through this, it was found that the pluripotent stem cell-derived extracellular vesicles of the present invention can reduce the anti-aging and aging-related inflammatory cytokines of neural stem cells, and can also restore the regenerative capacity of neural stem cells for nerve damage.

[0113]

[0114] <Example 3>

[0115] Confirmation of neural stem cell anti-aging induced by glutathione S-transferase contained in pluripotent stem cell-derived extracellular vesicles of the present invention

[0116] In order to identify key factors capable of inducing antioxidant and anti-aging effects in neural stem cells among the proteins contained in the extracellular vesicles derived from pluripotent stem cells of the present invention, proteomic analysis was performed on extracellular vesicles derived from pluripotent stem cells.

[0117] Among the derived proteins, only the list of the 50 with the highest content was selected. To identify the delivery proteins mediating antioxidant, anti-aging, and recovery in neural stem cells, network analysis of the Gene Ontology Biological Process of the top 50 proteins was performed using ClueGO.

[0118]

[0119] As a result, it was confirmed that biological phenomena related to glutathione transport, in addition to oxidation reactions and subsequent neuronal death, are significantly associated with pluripotent stem cell-derived extracellular vesicles (Fig. 3a). Furthermore, among the Gene Ontology Biological Processes associated with 50 proteins obtained via ShinyGO (http: / bioinformatics.sdstate.edu / go / ), GSTP1 was found to be the only factor performing a common function among the top 50 proteins involved in the inhibition of apoptosis (GO:0060548) and antioxidant roles (GO:1901701, GO:0098869) (Fig. 3b).

[0120] Accordingly, the inventors prepared pluripotent stem cell-derived extracellular vesicles with GSTP1 depleted by inhibiting GSTP1 gene expression within pluripotent stem cells with small-interfering RNA (siRNA, siGSTP1 #1 & siGSTP1 #2) (Figs. 3c, 3d). When the activity of glutathione S-transferase in the extracellular vesicles was analyzed using a Glutathione S-Transferase (GST) Assay Kit (Sigma-Aldrich), it was found that the activity of glutathione S-transferase was depleted by approximately two-thirds (Fig. 3e). When the GSTP1-depleted pluripotent stem cell-derived extracellular vesicles were treated with neural stem cells, it was confirmed that the antioxidant capacity exhibited by the pluripotent stem cell-derived extracellular vesicles had disappeared (Fig. 3f).

[0121] Through this, it was found that glutathione S-transferase contained in the extracellular vesicles derived from pluripotent stem cells of the present invention plays an important role in antioxidant capacity and anti-aging activity.

[0122]

[0123] <Example 4>

[0124] Analysis of Neural Stem Cell Anti-aging Activity of Extracellular Vesicles Obtained from Pluripotent Stem Cells Cultured Under Hypoxic Conditions

[0125] The antioxidant and anti-aging activities of neural stem cells on extracellular vesicles obtained from pluripotent stem cells cultured under hypoxic conditions were analyzed.

[0126] As confirmed in the above examples, since it was determined that treatment with extracellular vesicles obtained from pluripotent stem cells can induce antioxidant and anti-aging effects in neural stem cells, pluripotent stem cells were cultured under hypoxic stress to obtain extracellular vesicles with enhanced efficacy.

[0127]

[0128] As a result, it was found that various genes related to the production and use of glutathione, including NFE2L2, the gene for NRF2 which acts as a transcription factor of the glutathione system, were expressed in extracellular vesicles isolated from pluripotent stem cells cultured under hypoxic conditions, and among these, it was confirmed that the expression of NRF2, GSTK1, GSTO1, and GSTZ1 was significantly increased within the extracellular vesicles (Figs. 4a, 4b).

[0129] Since the roles of GSTK1, GSTO1, and GSTZ1 associated with pluripotent stem cell-derived extracellular vesicles have not been studied to date, the results of this invention imply that these genes and the proteins expressed therefrom can act on antioxidant and anti-aging effects. As such, the increase in GSTs within extracellular vesicles isolated under hypoxic conditions indicates that pluripotent stem cell-derived extracellular vesicles can suppress oxidative stress within neural stem cells and inhibit aging and stem cell degeneration (Figs. 4c, 4e). In particular, the expression of the glutathione system within neural stem cells was found to be enhanced in correlation with the increase in the transcription factor NRF2 within the extracellular vesicles (Fig. 4f).

[0130] Therefore, it was found that extracellular vesicles isolated from pluripotent stem cells cultured under hypoxic conditions have an overexpression of the glutathione system, which can contribute to the enhancement of the stem cells' antioxidant capacity, anti-aging capacity, and stem cell regenerative capacity, and furthermore, can induce an increase in the expression of the intrinsic glutathione system of target stem cells, thereby inducing a more stable therapeutic effect even after the discontinuation of drug administration.

[0131] Therefore, when pluripotent stem cells are cultured under hypoxic conditions, extracellular vesicles with overexpressed glutathione systems can be obtained, and when the obtained extracellular vesicles are treated with degenerative stem cells, the antioxidant capacity, anti-aging capacity, and stem cell characteristics of the stem cells can be effectively restored, and furthermore, the expression of the intrinsic glutathione system of the target stem cells can be increased, thereby inducing a more stable therapeutic effect on aging-related diseases even after the discontinuation of drug administration.

[0132]

[0133] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

[0134] This patent application is the result of research conducted under the following national research support program.

[0135] Project ID: 2710000024

[0136] Project Number: RS-2022-00070637

[0137] Ministry Name: Ministry of Science and ICT

[0138] Project Management (Specialized) Agency Name: Pan-Governmental Regenerative Medicine Technology Development Agency

[0139] Research Project Name: Inter-Ministerial Regenerative Medicine Technology Development Project

[0140] Research Project Title: Pathological Microenvironment Regeneration Technology for Differentiated Neuroectodermal Extracellular Vesicle / MFG-e8-Based Endogenous Stem Cells and Development of Candidate Substances for Central Nervous Tissue Regeneration Utilizing the Same

[0141] Project Executing Organization Name: Korea University Industry-Academic Cooperation Foundation

[0142] Research Period: 2024.01.01 ~ 2024.12.31.

[0143]

[0144] Project ID: 2710003452

[0145] Project No.: RS-2023-00280923

[0146] Ministry Name: Ministry of Science and ICT

[0147] Project Management (Specialized) Agency Name: National Research Foundation of Korea

[0148] Research Project Name: Individual Basic Research (Ministry of Science and ICT)

[0149] Research Project Title: Elucidation of the Regulation Mechanism of Cellular Anti-aging and Pluripotency Mediated by “Protein-Corona” on the Surface of Human Pluripotent Stem Cell-Derived Extracellular Vesicles

[0150] Project Executing Organization Name: Korea University Industry-Academic Cooperation Foundation

[0151] Research Period: 2024.03.01 ~ 2025.02.28

Claims

1. A senomorphic composition characterized by restoring the function or morphology of senescent cells to normal cells, comprising stem cell-derived extracellular vesicles as an active ingredient.

2. In Paragraph 1, A cenomorphic composition characterized in that the above-mentioned stem cells are embryonic stem cells, induced pluripotent stem cells, adult stem cells, or progenitor cells.

3. In Paragraph 1, A cenomorphic composition characterized in that the senescent cells are nerve cells, neural stem cells, or neural progenitor cells.

4. In Paragraph 1, A cenomorphic composition characterized in that the extracellular vesicles are exosomes, microvesicles, microparticles, or ectosomes.

5. In Paragraph 1, A cenomorphic composition characterized in that the above extracellular vesicles are obtained from stem cells cultured under hypoxic conditions.

6. In Paragraph 1, The above composition is, Inhibition of the production of aging-related inflammatory cytokines; Antioxidant activity; Inhibition of expression of aging-related markers; Maintenance of stem cell self-proliferative capacity; and A cenomorphic composition characterized by having the ability to repair cell damage.

7. In Paragraph 6, A cenomorphic composition characterized in that the above-mentioned inflammatory cytokine is selected from the group consisting of CXCL1, MMP9, IL-6, and IL-8.

8. In Paragraph 6, A cenomorphic composition characterized in that the aging-related markers are p16 and SA-β-Gal (SA-β-galactosidase).

9. In Paragraph 1, A cenomorphic composition characterized in that the extracellular vesicles have increased expression and activity of the glutathione system.

10. In Paragraph 9, A cenomorphic composition characterized by the extracellular vesicles having increased expression of NRF2 (Nuclear factor erythroid-2-related factor 2), GSTK1 (Glutathione S-transferase kappa 1), GSTO1 (Glutathione S-transferase omega-1), and GSTZ1 (Glutathione S-transferase Zeta 1).

11. A pharmaceutical composition for the prevention or treatment of nerve damage and ischemic disease comprising the composition of claim 1 as an active ingredient.

12. In Paragraph 11, A pharmaceutical composition for the prevention or treatment of nerve damage and ischemic disease, characterized in that the nerve damage is caused by apoptosis of nerve cells, morphological abnormalities of cells, neurotransmission abnormalities, oxidative stress, increased expression of aging-related markers, increased inflammatory response, or increased secretion of aging-related inflammatory cytokines and secretion of neuroregenerative growth factors.

13. In Paragraph 11, A pharmaceutical composition for the prevention or treatment of nerve damage and ischemic disease, characterized in that the above-mentioned ischemic disease is selected from the group consisting of ischemic necrosis, ischemic cerebrovascular disease, ischemic renal disease, ischemic lung disease, ischemic disease of the limbs, ischemic heart disease, stroke, cerebral infarction, myocardial infarction, ischemic heart failure, and obstructive arteriosclerosis.

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