Method for preparing extracellular vesicles derived from glia-like cells differentiated from stem cells, and composition for alleviating or treating nerve damage, comprising same as active ingredient

Abstract

The present invention relates to a method for preparing extracellular vesicles derived from glia-like cells differentiated from stem cells, and a composition for preventing or treating nervous system diseases caused by nerve damage, comprising same as an active ingredient. The extracellular vesicles, of the present invention, which are separated after pretreating, with a specific peptide, the glia-like cells differentiated from stem cells, have neuroprotective and regenerative effects, and thus can be effectively used for preventing or treating nervous system diseases caused by nerve damage. In addition, the method for preparing extracellular vesicles, of the present invention, can produce, in a high yield, stem cell-derived extracellular vesicles having a nerve regeneration effect, and thus is useful.

Description

Method for producing extracellular vesicles derived from glioma cells differentiated from stem cells and composition for alleviating or treating nerve damage comprising the same as an effective ingredient

[0001] The present invention relates to a method for producing extracellular vesicles derived from glioma cells differentiated from stem cells and a composition for preventing or treating nervous system diseases caused by nerve damage, comprising the same as an active ingredient.

[0002] Neuropathy, caused by nerve damage from diabetes, inflammation, chemotherapy, trauma, and other factors, causes severe chronic pain and motor impairment.

[0003] Research has been ongoing to suppress or alleviate pain or permanent functional paralysis caused by long-term nerve damage.

[0004] To date, various potential pharmacological treatments have been attempted, including steroids, antioxidants, glutamate receptor antagonists, ion channel inhibitors, gangliosides, antibodies to axon regeneration inhibitors, anti-inflammatory agents, and neurotrophic factors. These drugs have numerous drawbacks, including unclear therapeutic efficacy and adverse effects due to overdose.

[0005] Additionally, medications known to suppress pain, such as tricyclic antidepressants, anticonvulsants, serotonin and noradrenaline reuptake inhibitors, and opioids, are used to reduce pain caused by peripheral neuropathy. However, these medications cause various side effects and only serve to suppress symptoms. Currently, there is no known way to treat the underlying cause, the damaged nerves.

[0006] Thus, treatment of nerve damage using drugs or physiotherapy has reached its limits, and many experimental studies have been conducted focusing on treatment using stem cells or their secretions.

[0007] Recently, studies have reported that cell secretomes contain various bioactive factors that regulate cell behavior. In particular, cell secretomes contain 'exosomes' or 'extracellular vesicles' that have intercellular signaling functions, and research on their components and functions is actively underway.

[0008] Additionally, extracellular vesicles reflect the state of the secreting source cell (donor cell), exhibit various biological activities depending on the cell from which they are secreted, and play an important role in cell-to-cell interactions by transferring genetic material and proteins between cells.

[0009] Furthermore, extracellular vesicles are safer than stem cells, with less immune rejection and a lower risk of tumor formation. Furthermore, extracellular vesicles are much easier to store and transport than stem cells. They can cross the blood-brain barrier, which cells cannot, making them highly effective in treating nerve damage and offering the advantage of fewer systemic side effects.

[0010] In addition, treatments using extracellular vesicles are attracting attention as a new approach that can overcome the shortcomings of existing cell therapy methods, such as problems in securing and maintaining therapeutically effective amounts of cells because they do not use cells.

[0011] Accordingly, the present inventors have studied stem cell-derived extracellular vesicles that contain various beneficial ingredients and are composed of a lipid bilayer, thereby functioning as a stable drug delivery system in their own right. Specifically, we aimed to develop extracellular vesicles that retain the unique effects of mesenchymal stem cells (hereinafter referred to as "glial-like cells"), which have enhanced neuroprotective and regenerative effects by secreting a large amount of glial growth factors. In addition, since the number of extracellular vesicles secreted by nucleated cells is generally only about 500 to 1,000 per cell, we have also studied to develop an efficient method for obtaining the extracellular vesicles.

[0012] As a result, the inventors of the present invention have completed the present invention by confirming that when the above-mentioned pseudoglial cells are pretreated with a human protein-derived peptide, extracellular vesicles having the inherent efficacy of the above-mentioned pseudoglial cells, i.e., mesenchymal stem cells with enhanced neuroprotective and regenerative efficacy, can be obtained with high purity and high yield.

[0013] [Prior Art Literature]

[0014] [Patent Document]

[0015] (Patent Document 001) KR 10-2024-0032613 A

[0016] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating a nervous system disease caused by nerve damage.

[0017] Another object of the present invention is to provide a method for producing stem cell-derived extracellular vesicles with improved nerve regeneration efficacy and yield.

[0018] In order to achieve the above-mentioned purpose, the present invention provides a pharmaceutical composition for preventing or treating a nervous system disease caused by nerve damage, which comprises as an active ingredient an extracellular vesicle derived from glial-like cells (ghMSCs) differentiated from human mesenchymal stem cells (hMSCs), wherein the glial-like cells are characterized in that they are pretreated with a peptide containing the amino acid sequence of SEQ ID NO: 1.

[0019] According to a preferred embodiment of the present invention, the mesenchymal stem cells may be bone marrow-derived mesenchymal stem cells.

[0020] According to a preferred embodiment of the present invention, the extracellular vesicles may be obtained by a method comprising the following steps:

[0021] a) A step of differentiating human bone marrow-derived mesenchymal stem cells to obtain glioma cells;

[0022] b) culturing the above-described cells in a medium composition containing a peptide having an amino acid sequence of sequence number 1; and

[0023] c) A step of collecting the culture medium of step b) containing the extracellular vesicles and removing the stem cells through centrifugation.

[0024] According to a preferred embodiment of the present invention, the extracellular vesicles may have an average diameter of 50 to 300 nm.

[0025] According to a preferred embodiment of the present invention, the composition comprises 10 6 10 inland 14 It may be included in a concentration of 100 mg / mL.

[0026] According to a preferred embodiment of the present invention, the nervous system disease caused by nerve damage may be a nervous system disease caused by nerve damage in the central nervous system or nerve damage in the peripheral nervous system.

[0027] According to a preferred embodiment of the present invention, the nervous system disease caused by nerve damage of the central nervous system may be at least one selected from organic diseases and dysfunctions of the central nervous system, epilepsy, multiple sclerosis, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Alzheimer's, dementia with Lewy bodies, Huntington's disease, Parkinson's disease, schizophrenia, traumatic brain injury, stroke, Pick's disease, Creutzfeldt-Jakob disease, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, spinocerebellar degeneration, cerebellar atrophy, post-traumatic stress disorder, amnesia, vascular dementia, and cerebral infarction.

[0028] According to a preferred embodiment of the present invention, the nervous system disease caused by nerve damage of the peripheral nervous system is selected from peripheral neuropathy, diabetic neuropathy, peripheral neuropathic pain, peripheral neuropathy due to chemotherapy, complex regional pain syndrome, optic neuropathy, mononeuropathy, multiple mononeuropathy (mononeuritis multiplex), polyneuropathy, Guillain-Barré syndrome (acute inflammatory demyelinating polyneuropathy), chronic inflammatory demyelinating polyneuropathy, hereditary neuropathy, plexus disorder, glaucoma, macular degeneration, amyotrophic lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, polio, post-polio syndrome, spastic-person syndrome, Isaacs syndrome, myasthenia gravis, neonatal myasthenia, botulism, Eaton-Lambert syndrome, thoracic outlet syndrome, Charcot-Marie-Tooth disease, and spinal muscular atrophy. There may be more than one type.

[0029] In addition, in order to achieve another object of the present invention, the present invention provides a method for producing stem cell-derived extracellular vesicles with improved nerve regeneration efficacy and yield, comprising the following steps:

[0030] a) A step of differentiating human bone marrow-derived mesenchymal stem cells to obtain glia-like cells (ghMSCs);

[0031] b) culturing the above-described cells in a medium composition containing a peptide having an amino acid sequence of sequence number 1; and

[0032] c) A step of collecting the medium composition of step b) containing the extracellular vesicles and removing the pseudoglia through centrifugation.

[0033] Extracellular vesicles isolated from the stem cells of the present invention, which are differentiated from the glioma cells by pretreatment with a specific peptide, have neuroprotective and regenerative properties, and thus can be usefully used in the prevention or treatment of neurological diseases caused by nerve damage.

[0034] In addition, the method for producing extracellular vesicles of the present invention is useful because it can produce stem cell-derived extracellular vesicles with nerve regeneration efficacy at a high yield.

[0035] Figure 1 shows the results of analyzing the characteristics of exosomes (Example 1; Pep-EV(N)) derived from glioma cells (hereinafter referred to as 'ghMSCs') pretreated with a peptide of SEQ ID NO: 1 according to an embodiment of the present invention. Figures 1a, 1b, 1c, 1d and 1e show the results of comparing the exosome production per cell between exosomes (Example 1; Pep-EV(N)(F)) derived from glioma cells (hereinafter referred to as 'ghMSCs') pretreated with a peptide of SEQ ID NO: 1 according to an embodiment of the present invention and exosomes (Comparative Example 1; Natural-EV(N)(F)) derived from ghMSCs not pretreated with the peptide. In the above Figures 1a, 1b, 1c, 1d and 1e, (N) refers to exosomes obtained from cells cultured in Neurobasal-A medium, and (F) refers to exosomes obtained from cells cultured in α-MEM medium containing 10% exosome-depleted FBS. Figure 1f is the result of Western blot analysis to confirm the expression of exosome-positive markers CD9 and CD63 in Pep-EV(N) of Example 1. Figure 1g is a photograph taken by observing the morphology of Pep-EV(N) of Example 1 and Natural-EV(N) of Comparative Example 1 using a transmission electron microscope (TEM).

[0036] Figure 2 shows the apoptosis inhibitory effect of ghMSC-derived exosomes pretreated with a peptide of sequence number 1 according to one embodiment of the present invention in a nerve injury model. Figure 2A is an image of apoptosis observed using a fluorescence microscope 7 days after treating a nerve injury model with exosomes isolated from ghMSCs, Figure 2B is a graph showing a quantitative analysis of the apoptosis image of Figure 2A, and Figure 2C is a table showing the graph of Figure 2B in numerical form. In the above Figure 2, 'Normal' means 'normal group or control group', 'LPC+Vehicle' means 'nerve damage-induced group or negative control group', 'LPC+hMSC' means 'hMSC (human mesenchymal stem cell) administration group', 'LPC+ghMSC' means 'ghMSC administration group or positive control group', 'LPC+Natural-EV(N)' means 'ghMSC-derived exosome administration group not pretreated with peptide' of Comparative Example 1, and 'LPC+Pep+EV(N)' means 'ghMSC-derived exosome administration group pretreated with peptide' of Example 1.

[0037] Figure 3 shows the nerve regeneration effect of ghMSC-derived exosomes pretreated with a peptide of sequence number 1 according to one embodiment of the present invention in a nerve injury model. Figure 3A is an image of the degree of NF-M staining observed under a fluorescence microscope 7 days after treating a nerve injury model with exosomes isolated from ghMSCs, Figure 3B is a graph showing the quantitative analysis of the NF-M fluorescence intensity of Figure 3A, and Figure 3C is a table showing the graph of Figure 3B in numerical form. In the above Figure 3, 'Normal' means 'normal group or control group', 'LPC+Vehicle' means 'nerve damage-induced group or negative control group', 'LPC+hMSC' means 'hMSC (human mesenchymal stem cell) administration group', 'LPC+ghMSC' means 'ghMSC administration group or positive control group', 'LPC+Natural-EV(N)' means 'ghMSC-derived exosome administration group not pretreated with peptide' of Comparative Example 1, and 'LPC+Pep+EV(N)' means 'ghMSC-derived exosome administration group pretreated with peptide' of Example 1.

[0038] FIG. 4 is a graph showing the neuroinflammation inhibitory effect of ghMSC-derived exosomes pretreated with a peptide of sequence number 1 according to one embodiment of the present invention in a neuroinflammation model, comparing the NO production, TNF-α expression, and IL-6 expression of the Example 1 treatment group with the control group, negative control group (LPS), and comparative example 1 treatment group.

[0039] In a previous study, the present inventors obtained ghMSCs with increased paracrine activity by inducing differentiation of late-stage human mesenchymal stem cells (hMSCs) with low neural function recovery effects into glia-like cells.

[0040] Meanwhile, the ghMSCs had limitations in that they could not overcome the shortcomings of existing cell therapy, such as problems in securing and maintaining therapeutically effective amounts of cells, and the risk of immune rejection and tumor formation.

[0041] Accordingly, an attempt was made to develop extracellular vesicles with the unique efficacy of the ghMSCs, but the extracellular vesicles isolated from the ghMSCs using the existing method had a problem in that the neuroprotective and regenerative effects were significantly lower than those of the ghMSCs.

[0042] As a result of extensive research efforts to solve the above problems, the present inventors have specifically confirmed that extracellular vesicles isolated from ghMSCs pretreated with human protein-derived peptides have neuroprotective and regenerative effects similar to or greater than those of ghMSCs.

[0043]

[0044] Hereinafter, the present invention will be described in detail.

[0045] One aspect of the present invention relates to a pharmaceutical composition for preventing or treating a nervous system disease caused by nerve damage, comprising as an active ingredient an extracellular vesicle derived from glial-like cells ('ghMSC') differentiated from human mesenchymal stem cells (hMSC), characterized in that the glial-like cells are pretreated with a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0046] In relation to the above aspect, the present invention also relates to a method for preventing, improving or treating a nervous system disease caused by nerve damage, comprising administering to a subject in need thereof an effective amount of a composition comprising, as an active ingredient, extracellular vesicles derived from glial-like cells (ghMSCs) differentiated from human mesenchymal stem cells (hMSCs), wherein the glial-like cells are characterized in that they are pretreated with a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0047] In relation to the above aspect, the present invention also relates to the use of a composition comprising, as an active ingredient, extracellular vesicles derived from glia-like cells ('ghMSCs') differentiated from human mesenchymal stem cells (hMSCs) for the manufacture of a drug for preventing, improving or treating a nervous system disease caused by nerve damage, wherein the glia-like cells are characterized in that they are pretreated with a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0048] In relation to the above aspect, the present invention also relates to a composition comprising, as an active ingredient, extracellular vesicles derived from glial-like cells ('ghMSCs') differentiated from human mesenchymal stem cells (hMSCs) for use in the prevention, improvement or treatment of nervous system diseases caused by nerve damage, characterized in that the glial-like cells are pretreated with a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0049] The term "stem cell" in the present invention refers to undifferentiated cells prior to differentiation into each cell constituting a tissue, and collectively refers to cells that have the ability to differentiate into specific cells under a specific differentiation stimulus (environment). Unlike differentiated cells that have stopped dividing, stem cells can produce cells identical to themselves through cell division (self-renewal), and when a differentiation stimulus is applied, they have the flexibility of differentiation (plasticity) to differentiate into various cells depending on the nature of the stimulus.

[0050] The term "mesenchymal stem cell" in the present invention refers to a stem cell with multipotency capable of differentiating into adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, and cardiomyocytes. The mesenchymal stem cell used in the present invention is not particularly limited, but is more preferably a bone marrow-derived mesenchymal stem cell.

[0051] Specifically, the bone marrow-derived mesenchymal stem cells of the present invention can be identified through their swirl-shaped morphology and the degree of expression of basic cell surface markers CD73(+), CD90(+), CD105(+), CD34(-), and CD45(-), and have the function of regulating immune responses along with multipotency.

[0052] In one embodiment, the peptide comprising the amino acid sequence of SEQ ID NO: 1 may be a peptide derived from the Noxa protein.

[0053] The above-mentioned green protein may bind to and inhibit Mcl1 and Bcl2A1 using the BH3 (Bcl-2 homology 3) domain, thereby activating BAX and BAK proteins, causing cytochrome-c to leak into the cytoplasm, and activating the Caspase system to cause apoptosis.

[0054] In the present invention, the peptide may be manufactured using a method of direct chemical synthesis using solid phase peptide synthesis, a method of synthesis using an automatic synthesizer, or a method of manufacturing by inserting a base sequence encoding the peptide into a vector and expressing the peptide, but is not limited thereto.

[0055] In one embodiment, the peptide may be a peptide having a homology of at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% to a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0056] The term “glia-like cell ('ghMSC'; glia-like human mesenchymal stem cell)” in the present invention refers to a late-passage mesenchymal stem cell differentiated from a human mesenchymal stem cell, particularly a bone marrow-derived mesenchymal stem cell, which has a large amount of glial cell growth factor secretion function and thus has enhanced neuroprotective and regenerative efficacy.

[0057] The above-described glioma cells can be obtained by differentiating them by a method including, but not limited to, a step of first culturing human mesenchymal stem cells in a medium containing β-mercaptoethanol; a step of second culturing the first culture in a medium containing all-trans-retinoic acid; and a step of third culturing the second culture in a medium containing human basic fibroblast growth factor (hbFGF), human platelet derived growth factor AA (hPDGF-AA), forskolin, and heregulin-β1 (HRG-β1).

[0058] In the above primary culture step, the β-mercaptoethanol may be included in an amount of 0.05 mM to 20 mM, 0.1 mM to 10 mM, 0.2 mM to 5 mM, or 0.5 mM to 2 mM relative to the entire primary culture medium. In the above secondary culture step, the tretinoin may be included in an amount of 0.01 μg / mL to 2 μg / mL, 0.05 μg / mL to 1 μg / mL, or 0.1 μg / mL to 0.5 μg / mL relative to the entire secondary culture medium. In addition, in the 3rd culture step, human fibroblast growth factor may be included in the entire 3rd culture medium at 0.01 ng / mL to 1 ng / mL, 0.1 ng / mL to 100 ng / mL, 1 ng / mL to 50 ng / mL, or 5 ng / mL to 20 ng / mL, human platelet-derived growth factor may be included at 0.1 ng / mL to 100 ng / mL, 0.5 ng / mL to 20 ng / mL, or 1 ng / mL to 10 ng / mL, forskolin may be included at 0.5 μM to 100 μM, 1 μM to 50 μM, or 5 μM to 20 μM, and heregulin beta-1 may be included at 1 ng / mL to 1 μg / mL, 10 ng / mL to 500 ng / mL, It can be included to be 50 ng / mL to 300 ng / mL, but is not limited thereto. In addition, the medium composition may further include, but is not limited to, compositions such as DMEM or FBS for providing nutrients commonly used in cell culture, and antibacterial / antiviral substances such as a penicillin / streptomycin mixture.

[0059] The term “extracellular vesicle” in the present invention refers to a vesicle with a lipid bilayer structure and a diameter ranging from 30 to 1,000 nm that is secreted into the extracellular environment through the fusion of the multivesicular body and the plasma membrane in various cells.

[0060] In one embodiment, the extracellular vesicles of the present invention may be obtained by a method comprising the steps of: a) differentiating human bone marrow-derived mesenchymal stem cells to obtain glioma cells; b) culturing the glioma cells in a medium composition comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1; and c) collecting the medium composition of step b) containing the extracellular vesicles and removing the stem cells through centrifugation.

[0061] In one embodiment, the concentration of the peptide included in the medium composition may be 0.1 to 5.0 μM, 0.1 to 4.5 μM, 0.1 to 4.0 μM, 0.1 to 3.5 μM, 0.1 to 3.0 μM, 0.1 to 2.5 μM, 0.1 to 2.0 μM, 0.1 to 1.5 μM, 0.5 to 5.0 μM, 0.5 to 4.5 μM, 0.5 to 4.0 μM, 0.5 to 3.5 μM, 0.5 to 3.0 μM, 0.5 to 2.5 μM, 0.5 to 2.0 μM or 0.5 to 1.5 μM, for example, but not limited to, 0.5 to 1.5 μM.

[0062] In one embodiment, the medium composition of step b) may further include a mixture for cell growth and proliferation in vitro, which is a cell medium composition for culturing stem cells and includes elements essential for cell growth and proliferation, such as sugars, amino acids, various nutrients, and minerals.

[0063] Ingredients that may be additionally included in the above medium composition include, for example, glycerin, L-alanine, L-arginine hydrochloride, L-cysteine ​​hydrochloride-monohydrate, L-glutamine, L-histidine hydrochloride-monohydrate, L-lysine hydrochloride, L-methionine, L-proline, L-serine, L-threonine, L-valine, L-asparagine-monohydrate, L-aspartic acid, L-cystine 2HCl, L-glutamic acid, L-isoleucine, L-leucine, L-phenylalanine, L-tryptophan, L-tyrosine disodium salt dihydrate, i-inositol, thiamine hydrochloride, niacinamide, pyridoxine hydrochloride, biotin, D-calcium pantothenate, folic acid, riboflavin, vitamin B. 12 , sodium chloride (NaCl), sodium bicarbonate (NaHCO3), potassium chloride (KCl), calcium chloride (CaCl2), sodium bicarbonate monohydrate (NaH2PO4-H2O), copper sulfate pentahydrate (CuSO4-5H2O), ferric sulfate heptahydrate (FeSO4-7H2O), magnesium chloride (anhydrous), magnesium sulfate (MgSO4), disodium biphosphate (Na2HPO4), zinc sulfate heptahydrate (ZnSO4-7H2O), D-glucose (dextrose), sodium pyruvate, hypoxanthine Na, linolenic acid, lipoic acid, putrescine 2HCl, and thymidine.

[0064] The culture medium composition according to the present invention can be manufactured artificially and used, or can be purchased and used as a commercially available medium. Examples of commercially available culture media include, but are not limited to, IMDM (Iscove's Modified Dulbecco's Medium), α-MEM (Alpha Modification of Eagle's Medium), F12 (Nutrient Mixture F-12), and DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12).

[0065] In one embodiment, the “culturing” may be a rotational shaking culture in which the ghMSCs are suspended as single cells in a medium composition containing the peptide.

[0066] In the present invention, the term “orbital shaking culture” may mean, but is not limited to, culturing cells at a constant rotation speed by placing a flask on a substrate that rotates horizontally in a circle of a constant radius.

[0067] In one embodiment, the floating culture may be performed using an orbital shaker.

[0068] In the above step c), the stem cells are removed from the medium composition collected in the above step b) through multiple centrifugations.

[0069] In one embodiment of the present invention, it was specifically confirmed that ghMSC-derived extracellular vesicles pretreated with a peptide comprising the amino acid sequence of SEQ ID NO: 1 increased the exosome production per cell by at least 4 times, preferably by at least 10 times, compared to ghMSC-derived extracellular vesicles not pretreated with the peptide.

[0070] In one embodiment, the extracellular vesicles of the present invention have an average diameter of 50 to 300 nm, more specifically 100 to 250 nm, more specifically 150 to 220 nm, more specifically 180 to 200 nm, and more specifically 185 to 195 nm. Extracellular vesicles having a fine diameter in this range are called exosomes.

[0071] In one embodiment, the composition of the present invention comprises 10 6 10 inland14 It may be included in a concentration of 10 / mL, preferably 10 8 10 inland 14 Concentration of 10 / mL, more preferably 10 8 10 inland 12 Concentration of 10 / mL, more preferably 10 10 10 inland 12 Concentration of 10 / mL, more preferably 10 11 10 inland 12 May be included in concentrations of 100 mg / ml.

[0072] The composition of the present invention exhibits neuroprotective activity and nerve regenerative activity, and thus can be used for the prevention or treatment of neurological diseases caused by various nerve damages.

[0073] In one embodiment, “nerve injury” may mean damage to nerve tissue or nerve cells, including axonal degeneration or Wallerian degeneration. In addition, the nerve injury may include (in the periphery) nerve compression without Wallerian degeneration, axonal damage with Wallerian degeneration, or nerve transection in which the continuity of axons is interrupted, depending on the degree of injury.

[0074] In one embodiment, the “neurological disease caused by nerve damage” may be a nervous system disease caused by nerve damage in the central nervous system or nerve damage in the peripheral nervous system, and preferably may be a nervous system disease caused by nerve damage in the peripheral nervous system, and depending on the site and degree of damage, etc., may be accompanied or not by symptoms such as paralysis, convulsions, seizures, cognitive impairment, speech impairment, memory impairment, abnormal behavior, emotional control disorder, dizziness, vomiting, gait disturbance, hormonal imbalance, balance disorder, pain, sensory abnormality, decreased motor function, numbness, tingling, burning sensation, etc.

[0075] For example, in the case of a nervous system disease caused by nerve damage in the central nervous system, the main symptoms may include paralysis, convulsions, seizures, cognitive impairment, speech impairment, memory impairment, abnormal behavior, emotional control disorder, dizziness, vomiting, gait disturbance, hormonal imbalance, and balance disturbance. In the case of a nervous system disease caused by nerve damage in the peripheral nervous system, the main symptoms may include pain, sensory abnormalities, decreased motor function, numbness, tingling, and burning sensations. The central nervous system is the nervous system that includes the brain and spinal cord. If the spinal cord is damaged due to trauma or disease, nerve transmission between the brain and the body may not occur normally, which may result in paralysis of movement or sensation. The peripheral nervous system is the nervous system that includes 12 pairs of cranial nerves, 31 pairs of spinal nerves, and the autonomic nerves. Depending on the site of damage, it may cause symptoms such as facial paralysis due to facial nerve damage, blindness due to optic nerve damage, eyelid paralysis due to oculomotor nerve damage, and hearing loss or dizziness due to auditory nerve damage.

[0076] In one embodiment, the nervous system disease caused by nerve damage of the central nervous system may be any one selected from the group consisting of organic diseases and dysfunctions of the central nervous system, epilepsy, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, dementia with Lewy bodies, Huntington's disease, Parkinson's disease, schizophrenia, traumatic brain injury, stroke, Pick's disease, Creutzfeldt-Jakob disease, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, spinocerebellar degeneration, cerebellar atrophy, post-traumatic stress disorder, amnesia, vascular dementia, and cerebral infarction. In the present specification, "organic diseases and dysfunctions of the central nervous system" may mean diseases that exhibit symptoms such as paralysis, convulsions, seizures, cognitive impairment, speech impairment, memory impairment, abnormal behavior, emotional control disorder, dizziness, vomiting, gait disturbance, hormonal abnormality, balance disorder, and pain due to nerve damage of the central nervous system. In certain embodiments, the neurological disease caused by nerve damage in the central nervous system may be associated with central nervous system excitotoxicity. Examples thereof include epilepsy, multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease, dementia with Lewy bodies, Huntington's disease, Parkinson's disease, schizophrenia, traumatic brain injury, stroke, and other degenerative brain diseases associated with central nervous system excitotoxicity.

[0077] In one embodiment, the nervous system disease caused by nerve damage of the peripheral nervous system is peripheral neuropathy, diabetic neuropathy, peripheral neuropathic pain, peripheral neuropathy due to chemotherapy, complex regional pain syndrome, optic neuropathy, mononeuropathy, mononeuropathy multiplex (mononeuritis multiplex), polyneuropathy, Guillain-Barré syndrome (acute inflammatory demyelinating polyneuropathy), chronic inflammatory demyelinating polyneuropathy, hereditary neuropathy, plexus disorder, glaucoma, macular degeneration, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), progressive muscular atrophy, progressive bulbar palsy, polio, post-polio syndrome, spastic-person syndrome, Isaacs syndrome, myasthenia gravis, neonatal myasthenia, botulism, Eaton-Lambert syndrome, thoracic outlet syndrome, It may be any one selected from the group consisting of Charcot-Marie-Tooth disease, and spinal muscular atrophy.

[0078] In one embodiment, the diabetic neuropathy may be polyneuropathy or focal neuropathy. In one embodiment, the polyneuropathy may be any one selected from the group consisting of hyperglycemic neuropathy, distal symmetric polyneuropathy, autonomic neuropathy, acute sensory neuropathy, acute painful sensory neuropathy, and chronic sensorimotor neuropathy. In one embodiment, the local peripheral neuropathy may be one selected from the group consisting of cranial neuropathy, truncal neuropathy, limb neuropathy, thoracolumbar radiculoneuropathy, and lumbosacral radiculoplexus neuropathy.

[0079] In one embodiment, the peripheral neuropathic pain may include pain caused by peripheral nervous system abnormality or damage, trigeminal neuralgia, diabetes neuropathy pain, phantom limb pain, pain caused by viral infection, trauma, cancer, or alcoholism, pain following chemotherapy, atypical facial pain, post-herpetic neuralgia, and neuropathic pain resulting from neurological disorders.

[0080] The term "treatment" in the present invention, unless otherwise stated, means reversing, alleviating, inhibiting the progression of, or preventing symptoms caused by the nerve damage.

[0081] In one embodiment of the present invention, when the extracellular vesicles derived from glial cells of the present invention were administered to an ex vivo nerve injury model, it was specifically confirmed that the damaged nerve tissue was significantly regenerated or recovered by LPC (lysophosphatidylcholine).

[0082] In one embodiment, the pharmaceutical composition of the present invention may comprise the extracellular vesicles derived from the apoptotic cells alone, or may further comprise one or more pharmaceutically acceptable carriers, excipients or diluents.

[0083] Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, carriers for parenteral administration may include water, suitable oils, saline solution, aqueous glucose, glycols, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers include those described in the following reference (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0084] The composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intrathecal administration, suprachoroidal injection, transdermal administration, subcutaneous administration, intraperitoneal administration, intranasal administration, enteral administration, topical administration, sublingual administration, or rectal administration, and intravenous administration is preferred.

[0085] The pharmaceutical composition of the present invention can be formulated as a preparation for oral administration or parenteral administration according to the administration route described above.

[0086] In the case of preparations for oral administration, the composition of the present invention can be formulated into powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. using methods known in the art. For example, oral preparations can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture to obtain a tablet or dragee. Examples of suitable excipients may include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches including corn starch, wheat starch, rice starch, and potato starch; cellulosics including cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; fillers such as gelatin and polyvinylpyrrolidone. Additionally, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrating agent, depending on the case. Furthermore, the pharmaceutical composition of the present invention may additionally include an anticoagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, and the like.

[0087] For parenteral administration, preparations can be formulated into injections, ointments, creams, lotions, oils, gels, aerosols, and nasal inhalants using methods known in the art. These formulations are described in the literature (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour), a commonly known prescription manual for all pharmaceutical chemistry.

[0088] Preferably, the pharmaceutical composition of the present invention may be prepared in any one form selected from the group consisting of oral preparations, injections, and ointments, and more preferably, it may be an injection.

[0089] The pharmaceutical composition of the present invention can provide desirable neuroprotective and regenerative effects when it contains an effective amount of the stem cell-derived extracellular vesicles. As used herein, the term "effective amount" refers to an amount that exhibits a greater response than a negative control, and preferably refers to an amount sufficient to alleviate or treat nerve damage. The apoptotic cell-derived extracellular vesicles are present in an amount of 10% relative to the total content of the pharmaceutical composition. 6 10 inland 14 It may be included in a concentration of 10 / mL, preferably 10 8 10 inland 14 Concentration of 10 / mL, more preferably 10 8 10 inland 12 Concentration of 10 / mL, more preferably 10 10 10 inland 12 Concentration of 10 / mL, more preferably 10 11 10 inland 12can be included at a concentration of 100 mg / ml. At this time, if the content of the glioma-derived extracellular vesicles is below the lower limit, the cell viability is excellent, but the therapeutic effect on nerve damage may not appear to the desired degree. On the other hand, if it exceeds the upper limit, the therapeutic effect on nerve damage may not increase as the concentration increases, or toxicity may occur. Meanwhile, as a result of an ex vivo experiment, when the concentration of the glioma-derived extracellular vesicles of the present invention is within the above range, a significant effect on the treatment of nerve damage was observed, but no side effects such as cytotoxicity were observed. The effective amount of the glioma-derived extracellular vesicles included in the pharmaceutical composition of the present invention will vary depending on the form in which the composition is formulated, etc.

[0090] The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient as a single dose, or may be administered as multiple doses over a long period of time using a fractionated treatment protocol. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease.

[0091] The appropriate dosage of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the pharmaceutical composition of the present invention is within the range of 0.001-100 mg / kg for adults.

[0092]

[0093] In addition, another aspect of the present invention relates to the use of extracellular vesicles isolated from pseudoglial cells pretreated with a peptide comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of a medicament for the prevention or treatment of a neurological disease caused by nerve damage. As described above, extracellular vesicles isolated from pseudoglial cells pretreated with a peptide comprising the amino acid sequence of SEQ ID NO: 1 can be used for the prevention or treatment of a neurological disease caused by nerve damage.

[0094] In addition, another aspect of the present invention provides a method for preventing or treating a neurological disease caused by nerve damage, comprising administering to a mammal an extracellular vesicle isolated from a glial cell pretreated with an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0095] The term “mammal” as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

[0096] The term “effective amount” as used herein means the amount of an active ingredient or pharmaceutical composition that is believed by a researcher, veterinarian, physician, or other clinician to induce a biological or medical response in a tissue, animal, or human, including an amount that induces alleviation of the symptoms of the disease or disorder. The effective amount and frequency of administration of the active ingredient of the present invention may vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art and may be adjusted according to various factors including the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient’s age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the treatment period, and concurrently used drugs. In the method for alleviating or treating the present invention, in the case of adults, it is preferable to administer a composition containing extracellular vesicles isolated from pseudoglia pretreated with a peptide containing the amino acid sequence of SEQ ID NO: 1 at a dose of 0.001 g / kg to 10 g / kg once to several times a day.

[0097] In the treatment method of the present invention, a composition comprising extracellular vesicles isolated from pseudoglia pretreated with a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient can be administered in a conventional manner via oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes.

[0098]

[0099] In addition, another aspect of the present invention relates to a method for producing stem cell-derived extracellular vesicles with improved nerve regeneration efficacy and yield, comprising the following steps:

[0100] a) A step of differentiating human bone marrow-derived mesenchymal stem cells to obtain glia-like cells (ghMSCs);

[0101] b) culturing the above-described cells in a medium composition containing a peptide having an amino acid sequence of sequence number 1; and

[0102] c) A step of collecting the medium composition of step b) containing the extracellular vesicles and removing the pseudoglia through centrifugation.

[0103] The step a) above may be a method for obtaining glioma cells by differentiating them by a method including the steps of: first culturing human bone marrow-derived mesenchymal stem cells in a medium containing β-mercaptoethanol; second culturing the first culture in a medium containing all-trans-retinoic acid; and third culturing the second culture in a medium containing human basic fibroblast growth factor (hbFGF), human platelet-derived growth factor AA (hPDGF-AA), forskolin, and heregulin-β1 (HRG-β1).

[0104] According to the present invention, extracellular vesicles having nerve regeneration efficacy can be produced with high yield and high purity.

[0105]

[0106] Hereinafter, the present invention will be described in detail by examples, but the present invention is not limited to the following examples.

[0107]

[0108] <Example>

[0109] Experimental method

[0110] Culture of human mesenchymal stem cells (hMSCs)

[0111] Adult human mesenchymal stem cells (hMSCs) extracted from normal human bone marrow (STEMCELL Technologies Inc., Vancouver, Canada) were cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM, low glucose) supplemented with 10% FBS (Gibco, Waltham, MA, USA). Cells (passages 6–12) were subcultured at 37°C in a 5% CO2 environment to obtain late-passage hMSCs.

[0112]

[0113] Induction of glial-like cells induced derived from human mesenchymal stem cells (ghMSC)

[0114] The obtained late-stage human mesenchymal stem cells (hMSCs) were cultured for 24 hours in primary differentiation medium [DMEM-low glucose medium, 10% FBS, 1% penicillin-streptomycin mixture, 1 mM β-mercaptoethanol (M3148, Sigma-Aldrich)]. Thereafter, the human mesenchymal stem cells were washed with phosphate buffered saline (PBS) and cultured in secondary differentiation medium [DMEM-low glucose medium, 10% FBS, 1% penicillin-streptomycin mixture, 0.28 μg / mL tretinoin (all-trans-retinoic acid; R2625, Sigma-Aldrich)]. After 3 days, the mesenchymal stem cells were washed with PBS and replaced with 3rd differentiation medium [DMEM-low glucose medium, 10% FBS, 1% penicillin-streptomycin mixture, 10 μM forskolin (F6886, Sigma-Aldrich), 10 ng / mL human basic-fibroblast growth factor (hbFGF; 100-18B, PeproTech), 5 ng / mL human platelet derived growth factor-AA (hPDGF-AA; 100-13A, PeproTech), 200 ng / mL heregulin-beta-1 (HRG-β1; 396-HB, R&D SYSTEMS)] and cultured for 8 days to obtain differentiated glioma cells (ghMSCs). At this time, the 3rd differentiation medium was replaced once every two days.

[0115]

[0116] Isolation of extracellular vesicles

[0117] Example 1: Pep-EV (N)

[0118] The ghMSCs were seeded on a 100 mm dish (172931, Thermofisher scientific) and cultured. When the cells reached 80-90% confluence, the medium was replaced with Neurobasal-A (NB) (21103049, Gibco). 18 hours later, the dish was replaced with the NB medium, and cells were suspended using TrypLE™ Select (12563029, Gibco). The suspended cells were centrifuged to obtain a pellet. The pellet was placed in a 50 ml conical tube (50050, SPL), and a composition containing sucrose buffer and the peptide represented by SEQ ID NO: 1 (10 μM) was added. The mixture was then incubated with rotational shaking on an orbital shaker (60 rpm) for 30 minutes. Afterwards, the above culture medium was collected, centrifuged at 300 g for 3 minutes to remove stem cell debris, centrifuged at 2,000 g for 10 minutes, the supernatant was transferred to a new tube, centrifuged again at 10,000 g for 30 minutes, and the supernatant was centrifuged again at 178,000 g for 2 hours to obtain a pellet (extracellular vesicles, Pep-EV(N)).

[0119] Example 2: Pep-EV (F)

[0120] The Pep-EV (N) was prepared in the same manner as above, but α-MEM (12561072, Gibco) medium containing 10% exosome-depleted FBS was used instead of NB medium, and extracellular vesicles (Pep-EV (F)) were obtained by culturing for 48 hours instead of 18 hours.

[0121] Comparative Example 1: Natural-EV (N)

[0122] The ghMSCs were seeded on a 100 mm dish (172931, Thermofisher scientific) and cultured. When the cells reached 80-90% confluence, the medium was replaced with Neurobasal-A (NB) (21103049, Gibco). The culture medium from the dish replaced with the NB medium was collected 18 hours later, centrifuged at 300 g for 3 minutes to remove stem cell debris, centrifuged at 2,000 g for 10 minutes, and the supernatant was transferred to a new tube, centrifuged again at 10,000 g for 30 minutes, and the supernatant was centrifuged again at 178,000 g for 2 hours to obtain a pellet (extracellular vesicles, Natural-EV(N)).

[0123] Comparative Example 2: Natural-EV (F)

[0124] It was prepared in the same manner as the above Natural-EV (N), but instead of the NB medium, α-MEM (12561072, Gibco) medium containing 10% exosome-depleted FBS was used, and extracellular vesicles (Natural-EV (F)) were obtained by culturing for 48 hours instead of 18 hours.

[0125]

[0126] Analysis of extracellular vesicle production

[0127] The size, number, and concentration of EVs were measured by dynamic light scattering (DLS) using a ZetaView nanoparticle tracking analyzer (Particle Metrix, Germany, TWIN PMX-220). ZetaView measurements were performed with the following settings: focus, automatic; number of particles per frame, 150–200; frames per second, 30; camera sensitivity, 80; shutter speed, 100; scattering intensity, automatic; and temperature, 25°C.

[0128]

[0129] Confirmation of exosome-related marker expression - Western blot analysis

[0130] Western blot analysis was performed to confirm the protein expression pattern of extracellular vesicles.

[0131] Specifically, cells were lysed using RIPA buffer (CBR002, LPS solution) containing protease inhibitor cocktail (87786, Invitrogen), and WCL (Whole cell lysate) was isolated. The WCL and EV (exosomes) were electrophoresed on 4-12% Bis-Tris Plus Gels (NW04125BOX, Invitrogen) and then transferred to a NC (nitrocellulose) membrane (IB23001, Invitrogen). The primary antibody (1:1,000) was incubated overnight at 4 °C and washed three times with 1x TBST (TLP-118.1, TrnasLab). Subsequently, the secondary antibody was reacted for 2 hours at room temperature and washed with 1x TBST. All antibodies were diluted in 1x blocking buffer (TLP-115.1G, Translab) and visualized using Invitrogen™ iBright™ Imagers (CL-1000).

[0132] At this time, the primary antibodies that can target markers of extracellular vesicles used were positive markers CD9 (ab263023, Abcam), CD63 (ab59479, Abcam), and CD81 (ab109201, Abcam). In addition, each primary antibody was detected using a species-appropriate secondary antibody according to the information provided by the manufacturer.

[0133]

[0134] Confirmation of exosome morphology - transmission electron microscopy analysis

[0135] Exosomes were attached onto 300-mesh formvar / carbon-coated copper grids (FCF300-CU, Electron Microscopy Sciences), negatively stained with 1% phosphotungstic acid hydrate (sigma, P4006), and the exosome morphology was confirmed using a Transmission Electron Microscope (TEM; Hitachi, HT7800).

[0136]

[0137] Organotypic spinal cord slice culture

[0138] Creation of a nerve injury model

[0139] The spinal cords of postnatal day 16 rats were removed and sliced ​​into 350 μm thick slices using a chopper. The spinal cord slices were cultured in a medium containing a mixture of 50% MEM + HEPES, 25% horse serum, 25% HBSS (Hank's balanced salt solution), 6.5 mg / mL glucose, and 1% penicillin / streptomycin. After culturing for 7 days, the spinal cord slices were treated with a culture medium containing 0.5 mg / mL LPC (lysophosphatidylcholine) for 17 hours to induce demyelination and nerve damage, thereby creating a nerve injury model.

[0140]

[0141] PI (Propidium iodide) Uptake Analysis and Image Analysis

[0142] The spinal cord slice tissue was treated with PI Solution (5 μg / mL) before exposure to LPC, and the basal fluorescence intensity (F0) was recorded through a rhodamine filter of a fluorescence microscope. After exposure to LPC for 17 hours, PI Solution (5 μg / mL) was treated to measure PI uptake intensity (F t) was measured. Some spinal cord slice tissues were cultured at 4°C for 24 hours to induce apoptosis after replacing the medium with PBS for evaluation of apoptosis, and then treated with PI Solution (5 μg / mL) to measure the final PI uptake intensity (F fin ) was measured. Image evaluation was performed using Image J, which was set to 8-bit, and the Gray-value mean intensity value was measured, and the cell death rate was evaluated using the formula below.

[0143] [Mathematical Formula 1]

[0144] Apoptosis rate (%) = (F t - F0) / (F fin - F0) × 100

[0145] (F0: Basal fluorescence intensity, F t : Fluorescence intensity after exposure to LPC, F fin : Final fluorescence intensity when all cells are dead)

[0146]

[0147] Evaluation of the neuroprotective effect of ghMSC-derived exosomes in a nerve injury model

[0148] The above nerve injury model was treated with hMSC (human bone marrow-derived mesenchymal stem cells), ghMSC (glial cells, positive control), Natural-EV(N) (Comparative Example 1), and Pep-EV(N) (Example 1), respectively, and cultured for 7 days. At this time, the cells (hMSC, ghMSC) were 3×10 4 At a concentration of 1.5 μl / cell / slice, exosomes (Natural-EV(N), Pep-EV(N)) were 6×10 7 The cells were treated at a concentration of 6 μl / slice of particles. After the above culture, PI Solution (5 μg / mL) was treated and observed under a fluorescence microscope to compare the fluorescence intensity of cell death in the spinal cord tissue.

[0149]

[0150] Evaluation of the nerve regeneration effect after ghMSC-derived exosome treatment in a nerve injury model

[0151] The above nerve injury model was treated with hMSC (human bone marrow-derived mesenchymal stem cells), ghMSC (glial cells), Natural-EV(N) (Comparative Example 1), and Pep-EV(N) (Example 1), respectively, and cultured for 7 days. At this time, the cells (hMSC, ghMSC) were 3×10 4 At a concentration of 1.5 μl / cell / slice, exosomes (Natural-EV(N), Pep-EV(N)) were 6×10 7 The cultured spinal cord tissue was treated at a concentration of 100 μl / slice. The cultured spinal cord tissue was fixed with 4% paraformaldehyde and stained with NF-M (neurofilament medium polypeptide) and huNu (human nuclei). The degree of NF-M staining was observed under a fluorescence microscope, and the degree of nerve regeneration in the spinal cord tissue was compared through fluorescence intensity.

[0152]

[0153] Neuroinflammation treatment efficacy trial using BV2 microglia

[0154] BV2 microglia cells were seeded at 2 × 10 in 24-well culture dishes. 5 cells / well and cultured for 24 hours. Afterwards, Natural-EV(N) (Comparative Example 1) and Pep-EV(N) (Example 1) were mixed with DMEM-low glucose, respectively, and 1×10 8 The cells were pretreated with 100 ng / ml of LPS for 6 hours. The pretreated cells were treated with 100 ng / ml of LPS for 24 hours. After 24 hours, the cell supernatants from each group were collected and centrifuged at 3,000 rpm for 1 minute to remove cell debris and separate the supernatants.

[0155] To measure nitric oxide production in BV2 cells induced by LPS, Griess reagent was prepared by mixing 5% phosphoric acid / 1% sulfanilamide and 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride in a 1:1 ratio (w / w). 100 μl of the Griess reagent and 100 μl of the cell supernatant were added to a 96-well culture dish and incubated at room temperature for 10 minutes. Nitric oxide concentration was measured using a Bio-RAD x-Mark TM The absorbance was measured at 540 nm using a spectrophotometer and quantified.

[0156] To confirm the expression levels of TNF-α and IL-6 in BV2 cells induced with LPS, RT-qPCR was performed using mTNF-α and mIL-6 primers to quantify gene expression levels.

[0157]

[0158] Experimental results

[0159] Experimental Example 1: Characterization of Exosomes

[0160] 1-1: Comparison of exosome production per cell

[0161] The production amount of exosomes according to the above examples and comparative examples was converted numerically and shown in Table 1 below.

[0162] Cell line Harvest cell number Peptide concentration Prep volume EV particle no. / ml EV particle no. / cell Example 1 ghMSC 3.4E+610 μM 0.2 ml 3.8E+112.235E+4 Example 2 ghMSC 3.2E+610 μM 0.2 ml 2.6E+111.625E+4 Comparative example 1 ghMSC 3.4E+6-0.2 ml 3.2E+101.882E+3 Comparative example 2 ghMSC 3.2E+6-0.2 ml 6.2E+103.750E+3

[0163] Looking at Table 1 above, it can be seen that Comparative Example 1 produced about 1,882 exosomes per cell, while Example 1 produced about 22,350 exosomes per cell, confirming that the production per cell increased by about 11 times. In addition, Comparative Example 2 produced about 3,750 exosomes per cell, while Example 2 produced about 16,250 exosomes per cell, confirming that the production per cell increased by about 4 times or more. In addition, as a result of the characteristic analysis of Example 1, it was confirmed that it showed the same aspects as Comparative Example 1 produced without peptide treatment in terms of diameter and protein expression.

[0164] The above results indicate that pretreatment of ghMSCs with the peptide of the present invention can significantly improve the exosome production per cell (Figures 1a, 1b, 1c, 1d, and 1e).

[0165] 1-2: Confirming the expression of exosome-related markers

[0166] As a result of Western blotting of exosomes according to the above Example 1, expression of CD9 and CD63, which are positive markers of exosomes, was observed, confirming specifically that the separated material was an exosome (Fig. 1f).

[0167] 1-3: Morphological analysis of exosomes

[0168] As a result of negative staining of exosomes according to Example 1 and Comparative Example 1, it was confirmed that Comparative Example 1 and Example 1 showed the same morphological appearance (Fig. 1g).

[0169]

[0170] Experimental Example 2: Neuroprotective Effect of ghMSC-Derived Exosomes in a Nerve Injury Model

[0171] hMSC (human bone marrow-derived mesenchymal stem cells), ghMSC (glial cells, positive control), Natural-EV(N) (Comparative Example 1), and Pep-EV(N) (Example 1) were each treated in the above nerve injury model, and cultured for 7 days, after which cell death was observed (Fig. 2 and Table 2).

[0172] ClassificationNormalNegative control group(LPC+Vehicle)LPC+hMSCPositive control group(LPC+ghMSC)Comparative example 1Example 1Apoptosis(%)6.963.050.120.736.625.4Relative ratio-10079.532.958.140.3Increase / decrease--20.5% decrease67.1% decrease41.9% decrease59.7% decrease

[0173] Looking at Figure 2 and Table 2, it can be confirmed that the ghMSC treatment group (positive control group) showed a 67.1% decrease in cell death compared to the LPC+Vehicle group (nerve damage-induced group, negative control group). In contrast, the Natural-EV(N) treatment group (Comparative Example 1) showed a 41.9% decrease in cell death compared to the negative control group, whereas the Pep-EV(N) treatment group of the present invention (Example 1) showed a 59.7% decrease in cell death compared to the negative control group, confirming that the neuroprotective effect is significantly improved compared to Comparative Example 1.

[0174]

[0175] Experimental Example 3: Nerve regeneration effect of ghMSC-derived exosomes in a nerve injury model.

[0176] The above nerve injury model was treated with hMSC (human bone marrow-derived mesenchymal stem cells), ghMSC (glial cells, positive control), Natural-EV(N) (Comparative Example 1), and Pep-EV(N) (Example 1), respectively, and cultured for 7 days. Then, NF-M, a nerve regeneration marker, was stained and observed (Fig. 3 and Table 3).

[0177] ClassificationNormalNegative control group(LPC+Vehicle)LPC+hMSCPositive control group(LPC+ghMSC)Comparative example 1Example 1NF-M fluorescence intensity(%)100.012.359.788.178.794.5Relative ratio to negative control group--4.85 times7.16 times6.40 times7.68 times

[0178] Looking at Figure 3 and Table 3, it can be confirmed that the ghMSC treatment group (positive control group) showed a 7.16-fold increase in nerve regeneration compared to the LPC+Vehicle group (nerve damage-induced group, negative control group). In contrast, the Natural-EV(N) treatment group (Comparative Example 1) showed only a 6.4-fold increase in nerve regeneration compared to the negative control group, whereas the Pep-EV(N) treatment group of the present invention (Example 1) showed a 7.68-fold increase in nerve regeneration compared to the negative control group, confirming that the nerve regeneration effect was significantly improved compared to Comparative Example 1. In particular, the Pep-EV(N) treatment group of Example 1 showed a more excellent nerve regeneration effect than the ghMSC cell treatment group, which is the positive control group. The above results indicate that when ghMSCs are pretreated with the peptide of the present invention, exosomes with excellent nerve regeneration effects can be produced. Specifically, it was confirmed that the nerve regeneration effect of exosomes isolated from ghMSCs pretreated with the peptide of the present invention was more superior than that of exosomes derived from ghMSCs not pretreated with the peptide.

[0179] Experimental Example 4: Anti-inflammatory effect of ghMSC-derived exosomes in a neuroinflammation model.

[0180] The BV2 microglial neuroinflammation model was treated with Natural-EV(N) (Comparative Example 1) and Pep-EV(N) (Example 1), respectively, and cultured for 6 hours. LPS was then treated and cultured again for 24 hours. Thereafter, nitric oxide (NO) production and gene expression levels of inflammatory factors TNF-α and IL-6 were observed (Fig. 4 and Table 4).

[0181] ClassificationNormalNegative control group(LPS+Vehicle)Comparative example 1(LPS+Natural-EV(N))Example 1(LPS+Pep-EV(N))NO production (μM)0.0912.014.954.63TNF-α expression level (relative ratio to negative control group)-10010086IL-6 expression level (relative ratio to negative control group)-1009082

[0182] Looking at Figure 4 and Table 4, both the Natural-EV(N) treatment group (Comparative Example 1) and the Pep-EV(N) treatment group (Example 1) showed a significant decrease in NO production compared to the negative control group. In addition, as a result of comparing the expression levels of inflammatory factor genes in microglia, the gene expression level of TNF-α in Comparative Example 1 decreased by 10% compared to the negative control group, and the gene expression level of IL-6 did not decrease, whereas the gene expression level of TNF-α in Example 1 decreased by about 20% and the gene expression level of IL-6 decreased by about 15% compared to the negative control group. Therefore, it can be confirmed that the neuroinflammation inhibitory effect of Example 1 is significantly improved compared to Comparative Example 1.

[0183] Although the present invention has been described with reference to the preferred embodiments mentioned above, various modifications and variations are possible without departing from the spirit and scope of the invention. Furthermore, the appended claims encompass such modifications and variations as fall within the spirit of the present invention.

[0184] The present invention is an invention carried out through the following tasks.

[0185] [National Research and Development Project Supporting This Invention]

[0186] [Project ID] 2710003628

[0187] [Assignment Number] 24A0203L1

[0188] [Ministry Name] Ministry of Science and ICT

[0189] [Name of Project Management (Specialist) Agency] Inter-Ministry Regenerative Medicine Technology Development Project Group

[0190] [Research Project Name] Inter-Ministry Regenerative Medicine Technology Development Project

[0191] [Research Project Name] Advancing an Endogenous Stem Cell Activation Platform Based on Immunomodulation and Enhanced Tissue Regeneration for the Treatment of Intractable Neuropathy and Development of Source Technology for Induced Factor Delivery

[0192] [Name of the project performing organization] Konkuk University Industry-Academic Cooperation Foundation

[0193] Research Period: April 1, 2024 - December 31, 2024

Claims

1. A pharmaceutical composition for preventing or treating a nervous system disease caused by nerve damage, comprising an extracellular vesicle derived from glial-like cells (ghMSC) differentiated from human mesenchymal stem cells (hMSC) as an active ingredient, A pharmaceutical composition for preventing or treating a nervous system disease caused by nerve damage, characterized in that the above-mentioned pseudo-glial cells are pretreated with a peptide containing the amino acid sequence of sequence number 1.

2. In paragraph 1, A composition characterized in that the above mesenchymal stem cells are bone marrow-derived mesenchymal stem cells.

3. In paragraph 1, A composition characterized in that the extracellular vesicles are obtained by a method comprising the following steps: a) A step of differentiating human bone marrow-derived mesenchymal stem cells to obtain glioma cells; b) culturing the above-described cells in a medium composition containing a peptide comprising the amino acid sequence of sequence number 1; and c) A step of collecting the medium composition of step b) including the extracellular vesicles and removing the stem cells through centrifugation.

4. In paragraph 1, A composition characterized in that the extracellular vesicles have an average diameter of 50 to 300 nm.

5. In paragraph 1, The above composition comprises 10 extracellular vesicles 6 10 inland 14 A composition characterized by comprising a concentration of dog / mL.

6. In paragraph 1, A composition characterized in that the neurological disease caused by the above nerve damage is a neurological disease caused by nerve damage in the central nervous system or nerve damage in the peripheral nervous system.

7. In paragraph 6, A composition characterized in that the neurological disease caused by nerve damage of the central nervous system is at least one selected from organic diseases and dysfunction of the central nervous system, epilepsy, multiple sclerosis, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Alzheimer's, dementia with Lewy bodies, Huntington's disease, Parkinson's disease, schizophrenia, traumatic brain injury, stroke, Pick's disease, Creutzfeldt-Jakob disease, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, spinocerebellar degeneration, cerebellar atrophy, post-traumatic stress disorder, amnesia, vascular dementia, and cerebral infarction.

8. In paragraph 6, The above-mentioned nervous system diseases caused by nerve damage of the peripheral nervous system include peripheral neuropathy, diabetic neuropathy, peripheral neuropathic pain, peripheral neuropathy due to chemotherapy, complex regional pain syndrome, optic neuropathy, mononeuropathy, multiple mononeuropathy (mononeuritis multiplex), polyneuropathy, Guillain-Barré syndrome (acute inflammatory demyelinating polyneuropathy), chronic inflammatory demyelinating polyneuropathy, hereditary neuropathy, plexus disorder, glaucoma, macular degeneration, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), progressive muscular atrophy, progressive bulbar palsy, polio, post-polio syndrome, spastic-person syndrome, Isaacs syndrome, myasthenia gravis, neonatal myasthenia, botulism, Eaton-Lambert syndrome, thoracic outlet syndrome, A composition characterized by at least one selected from Charcot-Marie-Tooth disease and spinal muscular atrophy.

9. A method for producing stem cell-derived extracellular vesicles with improved nerve regeneration efficacy and yield, comprising the following steps: a) A step of differentiating human bone marrow-derived mesenchymal stem cells to obtain glial-like cells (ghMSCs); b) culturing the above-described cells in a medium composition containing a peptide comprising the amino acid sequence of sequence number 1; and c) A step of collecting the medium composition of step b) containing the extracellular vesicles and removing the pseudoglia through centrifugation.

10. A method for preventing, improving or treating a nervous system disease caused by nerve damage, comprising administering to a subject in need thereof an effective amount of a composition containing extracellular vesicles derived from glia-like cells (ghMSCs) differentiated from human mesenchymal stem cells (hMSCs) as an effective ingredient. A method for preventing, improving or treating a nervous system disease caused by nerve damage, characterized in that the above-mentioned pseudo-glial cells are pretreated with a peptide containing the amino acid sequence of sequence number 1.

11. In paragraph 10, A method characterized in that the above mesenchymal stem cells are bone marrow-derived mesenchymal stem cells.

12. In paragraph 10, A method characterized in that the extracellular vesicles are obtained by a method comprising the following steps: a) A step of differentiating human bone marrow-derived mesenchymal stem cells to obtain glioma cells; b) culturing the above-described cells in a medium composition containing a peptide comprising the amino acid sequence of sequence number 1; and c) A step of collecting the medium composition of step b) including the extracellular vesicles and removing the stem cells through centrifugation.

13. In paragraph 10, A method characterized in that the extracellular vesicles have an average diameter of 50 to 300 nm.

14. In paragraph 10, The above composition comprises 10 extracellular vesicles 6 10 inland 14 A method characterized by comprising a concentration of dog / mL.

15. In paragraph 10, A method characterized in that the neurological disease caused by the above nerve damage is a neurological disease caused by nerve damage in the central nervous system or nerve damage in the peripheral nervous system.

16. In paragraph 15, A method characterized in that the neurological disease caused by nerve damage of the central nervous system is at least one selected from organic diseases and dysfunction of the central nervous system, epilepsy, multiple sclerosis, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Alzheimer's, dementia with Lewy bodies, Huntington's disease, Parkinson's disease, schizophrenia, traumatic brain injury, stroke, Pick's disease, Creutzfeldt-Jakob disease, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, spinocerebellar degeneration, cerebellar atrophy, post-traumatic stress disorder, amnesia, vascular dementia, and cerebral infarction.

17. In paragraph 15, The above-mentioned nervous system diseases caused by nerve damage of the peripheral nervous system include peripheral neuropathy, diabetic neuropathy, peripheral neuropathic pain, peripheral neuropathy due to chemotherapy, complex regional pain syndrome, optic neuropathy, mononeuropathy, multiple mononeuropathy (mononeuritis multiplex), polyneuropathy, Guillain-Barré syndrome (acute inflammatory demyelinating polyneuropathy), chronic inflammatory demyelinating polyneuropathy, hereditary neuropathy, plexus disorder, glaucoma, macular degeneration, amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), progressive muscular atrophy, progressive bulbar palsy, polio, post-polio syndrome, spastic-person syndrome, Isaacs syndrome, myasthenia gravis, neonatal myasthenia, botulism, Eaton-Lambert syndrome, thoracic outlet syndrome, A method characterized by at least one selected from Charcot-Marie-Tooth disease and spinal muscular atrophy.

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