COMPOSITION FOR TREATING NEURODEGENERATIVE DISORDERS COMPRISING MESENCHYMAL STEM CELLS

Abstract

The present invention relates to a method or a composition for treating neurodegenerative diseases, and particularly, the present invention relates to a method for treating neurodegenerative diseases of the cerebellum, comprising administering a composition comprising stem cells as an active ingredient to a patient having a neurodegenerative disease of the cerebellum, or to a composition for treating neurodegenerative diseases of the cerebellum comprising mesenchymal stem cells as an active ingredient. The treatment method or the treatment composition using mesenchymal stem cells, according to the present invention, has remarkable effects in reducing neuroinflammation, inhibiting M1 microglia, activating M2 microglia, inhibiting apoptosis of Purkinje cells, inhibiting death of neurons, improving motor ability, and the like, and therefore may be effectively utilized in alleviating and treating neurodegenerative diseases including cerebellar ataxia and multiple system atrophy.

Claims

1. A method for treating neurodegenerative diseases of the cerebellum, comprising: administering a composition comprising stem cells as an active ingredient to a patient having a neurodegenerative disease of the cerebellum.

2. The method for treating neurodegenerative diseases of the cerebellum according to claim 1, wherein the stem cell is an adult stem cell.

3. The method for treating neurodegenerative diseases of the cerebellum according to claim 2, wherein the adult stem cell is at least one selected from the group consisting of a mesenchymal stem cell (MSC), a mesenchymal stromal cell, and a multipotent stem cell.

4. The method for treating neurodegenerative diseases of the cerebellum according to claim 1, wherein the stem cells induce anti-inflammatory activity of the cerebellum.

5. The method for treating neurodegenerative diseases of the cerebellum according to claim 1, wherein the stem cells reduce the expression of inflammatory cytokines or inflammatory chemokines.

6. The method for treating neurodegenerative diseases of the cerebellum according to claim 1, wherein the stem cells inhibit the activation of inflammatory neuroglia and activate anti-inflammatory neuroglia.

7. The method for treating neurodegenerative diseases of the cerebellum according to claim 1, wherein the stem cells inhibit damage of a Purkinje cell.

8. The method for treating neurodegenerative diseases of the cerebellum according to claim 1, wherein the neurodegenerative disease of the cerebellum is at least one selected from the group consisting of a neurodegenerative disease induced by inflammation, a neurodegenerative disease induced by toxins, and a neurodegenerative disease caused by genetic modification.

9. The method for treating neurodegenerative diseases of the cerebellum according to claim 1, wherein the neurodegenerative disease of the cerebellum includes cerebellar ataxia or multiple system atrophy.

10. The method for treating neurodegenerative diseases of the cerebellum according to claim 1, wherein 90% or more of the stem cells express a positive surface marker including CD29, CD44, CD73, CD90 or CD105, or 5% or less of the stem cells express a negative surface marker including CD34 or CD45.

11. The method for treating neurodegenerative diseases of the cerebellum according to claim 1, wherein the administration is performed via at least one route of administration selected from the group consisting of intrathecal, intravenous, intramuscular, intraarterial, intramedullary, intradural, intraneural, intraventricular and intracerebrovascular.

12-21. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0044] FIG. 1A shows a result obtained by confirming the construction of an animal model of a disease induced by LPS administration. It is a result obtained by confirming the expression levels of Iba1 (microglia), inflammatory cytokines (IL-1β and TNFα), and inflammatory chemokines (MCP-1 and MIP-1α) in the cerebellum on day 1 and day 7 after LPS administration. Iba1: **p<0.01 and ***p<0.001 vs. CON, .sup.$$$p<0.001 vs. PBS (day 7), .sup.##p<0.01 vs. PBS (day 1). IL-1β **p<0.01 vs. CON, .sup.$p<0.05 vs. PBS (day 7), .sup.##p<0.01 vs. PBS (1 d). TNFα: **p<0.01 and *p<0.05 vs. CON. MCP-1: **p<0.01 vs. CON (t-test analysis; in each experimental group, n=3). MIP-1α: ** p<0.01 vs. CON, .sup.#p<0.05 vs. PBS (day 1) (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=3).

[0045] FIG. 1B shows a result obtained by confirming the construction of an animal model of a disease induced by LPS administration. It is a result obtained by confirming the expression level of Purkinje cells in the cerebellum and rotarod test for 4 weeks after LPS administration, and it is a result obtained by confirming the establishment of an animal model of a neurodegenerative disease induced by inflammation. Rotarod test for 4 weeks: **p<0.01 and ***p<0.001 vs. CON (t-test analysis; CON, n=6; PBS, n=4; LPS, n=3). Cal-D28K: ***p<0.001 vs. CON (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=4).

[0046] FIG. 2A shows a result obtained by confirming the construction of an animal model of a disease induced by Ara-C. It is a result obtained by confirming the adult immature cerebellum for NISSL, immunostaining, anatomical formation state, and brain weight after injection of Ara-C from day 1 to day 3 after birth. *p<0.05 (t-test analysis; CON, n=4; Ara-C, n=4).

[0047] FIG. 2B shows a result obtained by confirming the construction of an animal model of a disease induced by Ara-C. It is a result obtained by evaluating the improvement of body weight and behavioral disorder of an animal of a disease induced by Ara-C, and it is a result obtained by confirming the establishment of an animal model of a neurodegenerative disease. ***p<0.001 (t-test analysis; CON, n=4; Ara-C, n=4).

[0048] FIG. 3A shows a result obtained by establishing an animal model of a disease in which genetic stability is secured by backcrossing up to the fifth generation, which is a neurodegenerative disease SCA2 animal model through genetic modification.

[0049] FIG. 3B shows a result obtained by performing the rotarod test to evaluate the behavioral motor of a neurodegenerative disease SCA2 animal model through genetic modification of the fifth generation.

[0050] FIG. 4A shows a result obtained by observing the spindle-shaped morphology of the initial cultured hMSCs (within P5) by microscope.

[0051] FIG. 4B shows a result obtained by analyzing the population doubling time (PDT) of hMSCs within 10 passages. **p<0.01 and ***p<0.001 vs. P6 (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=4).

[0052] FIG. 4C shows a result obtained by analyzing the immunophenotyping for the expression profile of the cell surface markers of early stage hMSCs.

[0053] FIG. 4D shows a result obtained by confirming the multilineage differentiation ability of hMSCs.

[0054] FIG. 4E shows a result obtained by confirming the neurotropic factors expressed in hMSCs in the culture solution, and it is a result obtained by confirming the expression of ANG, BDNF, and IGF, which are well known neuroprotective and growth factors.

[0055] FIG. 5A shows a result obtained by performing the rotarod test for 4 weeks after transplanting hMSCs into a mouse in which the cerebellar inflammatory response is induced by LPS. Significant differences between control group (CON) or LPS+hMSCs group and LPS alone group or LPS+HTS group began to appear from 11 weeks of age throughout the experimental period (***p<0.001, one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=5).

[0056] FIG. 5B shows a result obtained by performing the evaluation of behavioral disorder improvement for 4 weeks after transplanting hMSCs. **p<0.01 vs. CON, .sup.##p<0.01 vs. LPS (t-test analysis; in each experimental group, n=5).

[0057] FIG. 5C shows a result obtained by confirming the expression level of Iba1 (microglia-related) in the cerebellum exposed to LPS at 7 days after transplantation of hMSCs. *p<0.05 vs. CON, .sup.#p<0.05 and .sup.##p<0.01 vs. LPS (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=3).

[0058] FIG. 5D shows a result obtained by confirming the expression level of pro-inflammatory cytokines (IL-1β and TNFα) in the cerebellum at 7 days after transplantation of hMSCs. IL-1β **p<0.01 vs. CON, .sup.##p<0.01 and .sup.###p<0.001 vs. LPS (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=3). TNFα: *p<0.05 vs. CON, .sup.#p<0.05 and .sup.##p<0.01 vs. LPS (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=3).

[0059] FIG. 5E shows a result obtained by confirming the expression level of Cal-D28K (Purkinje cell-related) in the cerebellum at 7 days after transplantation of hMSCs. *p<0.05 vs. CON, .sup.#p<0.05 vs. LPS (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=4).

[0060] FIG. 5F shows a result obtained by confirming the expression level of CD86 and CD206 in the cerebellum at 7 days after transplantation of hMSCs. CD86: *p<0.05 vs. CON, .sup.#p<0.05 and .sup.###p<0.001 vs. LPS (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=3). CD206: .sup.##p<0.01 vs. LPS (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=3).

[0061] FIG. 6A shows a result obtained by confirming the effect of improving motor ability according to the concentration and frequency of administration of hMSCs in an animal model of a neurotoxicity disease induced by Ara-C (rotarod test).

[0062] FIG. 6B shows a result obtained by confirming the effect of improving the size of the cerebellum according to the frequency of administration of hMSCs in an animal model of a neurotoxicity disease induced by Ara-C. ***p<0.001 vs. CON (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=3).

[0063] FIG. 6C shows a result obtained by comparing the effect of improving general motor activity according to the concentration and frequency of administration of hMSCs in an animal model of a neurotoxicity disease induced by Ara-C. *p<0.05 vs. WT, .sup.#p<0.05 and .sup.##p<0.01 vs. Ara-C (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=3).

[0064] FIG. 6D shows a result obtained by confirming the increase in the amount of protein in neurons according to the frequency of administration of hMSCs in an animal model of a neurotoxicity disease induced by Ara-C. ***p<0.001 vs. con, .sup.###p<0.01 vs. Ara-C (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=3).

[0065] FIG. 6E shows a result obtained by confirming the improvement of neuromotor disorder according to the dose and frequency of administration of hMSCs in an animal model of a neurotoxicity disease induced by Ara-C. ***p<0.001 vs. WT, .sup.#p<0.05 vs. Ara-C (one-way ANOVA and Tukey's post-hoc analysis; in each experimental group, n=3).

[0066] FIG. 7 shows a result obtained by confirming the effect of improving motor ability according to the dose of administration of hMSCs in an animal model of a SCA2 genetic disease caused by genetic modification.

BEST MODE FOR CARRYING OUT THE INVENTION

Experimental Materials and Methods

Animal

[0067] Male C57BL/6 mice (Daehan Biolink, Republic of Korea) and B6D2-Tg(Pcp2-SCA2)11Plt/J mice were bred in a controlled environment with a 12-hour photoperiod, and food was distributed ad libitum. All animal experimental procedures were performed in accordance with the regulations of the Animal Experimental Ethics Committee of Kyungpook National University (No. KNU 2016-42).

Animal Model of Inflammatory Degenerative Disease Induced by LPS Injection and Transplantation of Human Mesenchymal Stem Cells (hMSCs)

[0068] Inflammation-related cerebellar ataxia was induced by directly injecting lipopolyssacharide (LPS, 5 μg/5 μL) into the cerebellum of mice. Specifically, 10-week-old mice were anesthetized by injecting 115 mg/kg of ketamine (Yuhan, Republic of Korea) and 23 mg/kg of Rompun (Bayer Korea, Republic of Korea) into the abdominal cavity of 10-week-old mice, and then fixed on a stereotaxic device (David Kopf Instruments, Tujunga, Calif., USA). For injection into the cerebellum, the skull was exposed through a central sagittal incision, and then burr hole opening was performed. A total of 5 μL of LPS (1 mg/mL) or phosphate buffered saline (PBS) was injected into the cerebellum through an injection pump (KD Scientific, New Hope, Pa., USA) connected to a Hamilton syringe (10 μL, 30 G) (AP: −0.25 cm; DV: −0.25 cm, relative to the lambda). The needle was left in place for 5 minutes to control back flow along the injection tube.

[0069] Next, the head of the mouse injected with LPS was turned about 90° toward the body in the stereotaxic device. The underlying dura mater was exposed, and then hMSCs (1×10.sup.5 cells/20 μL or 1×10.sup.6 cells/20 μL) were transplanted into the cisterna magna using a Hamilton syringe (25 μL, 30 G) connected to an injection pump. The needle was removed after 10 minutes, and the incision site was sutured with a silk suture.

Animal Model of Cerebellar Ataxia Induced by Ara-C Injection and Transplantation of Human Mesenchymal Stem Cells (hMSCs)

[0070] Ara-C, an anti-miotic agent, was used to induce cerebellar ataxia independent of inflammation in mice. Specifically, an animal model of neurotoxicity induced cerebellar ataxia was constructed by intraperitoneal administration of Ara-C at a concentration of 40 mg/kg daily to mice from day 1 to day 3 after birth.

[0071] Next, in the same manner as above, hMSCs (1×10.sup.5 cells/20 μL or 1×10.sup.6 cells/20 μL) were transplanted into the cisterna magna of the mice injected with Ara-C.

Isolation of hMSCs and Analysis of Properties of Stem Cells

[0072] Human bone marrow samples were secured from 8 healthy donors according to the procedure approved by the Clinical Research Review Board (IRB: 2017-11-015-007) of Kyungpook National University Chilgok Hospital.

[0073] Mononuclear cells were isolated from the bone marrow by density gradient centrifugation using Ficoll (Ficoll-Paque Premium; GE Healthcare Bio-Sciences AB), plated in CSBM-A06 medium (Corestem, Republic of Korea) at a density of about 1×10.sup.5 cells/cm.sup.2, and then cultured with 10% FBS (fetal bovine serum; Life Technologies, Grand Island, N.Y., USA), 2.5 mM L-alanyl-L-glutamine (Biochrom AG, Berlin, Germany), and 1% penicillin-streptomycin (Biochrom AG). Cells not attached were removed with a fresh medium, and then the medium was changed once every 3 to 4 days. Cells grown to 70-80% confluency were defined as passage 0 (passage zero, P0). Before subsequent experiments, cells were subcultured until passage 10 (P10).

[0074] Population doubling time (PDT): PTD was measured in successive subculture and calculated using the following equation:

[00001]$\frac{\left(T-T0\right).Math.\log 2}{\log \left(N-N0\right)}$

[0075] where T0 is the cell transplantation time, T is the cell harvest time, N0 is the initial number of cells, and N is the number of harvested cells.

[0076] Multilineage differentiation assays: Cell differentiation was induced according to the prior art. Specifically, hMSCs were cultured in a 24-well plate and stimulated to be differentiated according to adipogenic lineage, osteogenic lineage, and chondrogenic lineage using an hMSC functional identification kit (R&D Systems, Minneapolis, Minn., USA). After differentiation, fat droplets of adipocytes were visualized using an oil red O staining reagent (Sigma, Saint Louis, Mo., USA). Osteogenic differentiation was confirmed by staining calcium accumulation with alizarin red (Sigma, Saint Louis, Mo., USA), and chondrogenic differentiation was confirmed by an alcian blue staining (Sigma, Saint Louis, Mo., USA).

[0077] Immunophenotyping: In order to confirm the characteristics of hMSCs, cells within five passages were stained with the cell surface markers CD29, CD73, CD90, CD105, CD34, CD45 (BD Pharmingen, Heidelberg, Germany), and CD44 (BD Biosciences, San Diego, Calif.). The expression of the cell surface markers was measured using a flow cytometer (BD FACS Canto⊚II), and hMSCs were identified as CD29/CD44/CD73/CD105-positive and CD34/CD45-negative cells.

Behavior Test

[0078] For animal model behavior test, a rotarod test was performed one day before hMSC transplantation and weekly for 4 weeks after hMSC transplantation, and a simple composite phenotype scoring system was performed once every four weeks after hMSC transplantation.

[0079] Rotarod test: The rotarod test was used to evaluate motor coordination and balance according to the method disclosed in the prior literature (Zhang M J, Sun J J, Qian L, Liu Z, Zhang Z, Cao W, Li W, Xu Y: Human umbilical mesenchymal stem cells enhance the expression of neurotrophic factors and protect ataxic mice. Brain Res 2011, 1402:122-131). Experimental mice were carefully placed on a rotating rod weekly for 4 weeks. The rotation speed was increased linearly from 4 rpm to 40 rpm for 5 minutes, and the same speed (40 rpm) was maintained for 5 minutes. The time (latency) it takes for the mouse to lose balance and fall off the rod, i.e., the total time the mouse could remain on the rotating rod was recorded. In order to prevent muscle fatigue, each mouse was allowed to rest for 10 minutes between each experiment.

[0080] Simple composite phenotype scoring system: In order to quantitatively analyze the severity of the disease in the LPS-induced cerebellar ataxia mouse model, a conventionally known scoring system was used in combination with the ledge test and the hindlimb clasping test (Guyenet S J, Furrer S A, Damian V M, Baughan T D, La Spada A R, Garden G A: A simple composite phenotype scoring system for evaluating mouse models of cerebellar ataxia. J Vis Exp 2010). The scoring system was done in 14-week-old mice, all tests were rated on a 0-3 point scale, and a composite phenotype score of 0-6 was added: A score of 0 means no relevant phenotype, and a score of 3 means the most severe symptom. Each test was performed three times.

[0081] Ledge test: Motor imbalance and ataxia were evaluated through the ledge test. A score of 0 was applied when the mouse walked along a ledge without losing overall balance, a score of 1 was applied when the mouse walked in an unbalanced position along a ledge, a score of 2 was applied when the mouse stumbled while walking along a ledge, and a score of 3 was applied when the mouse was unable to use its hindlimbs effectively.

[0082] Hindlimb clasping test: This test was used as a marker of disease progression in a mouse model of cerebellar ataxia (Chou A H, Yeh T H, Ouyang P, Chen Y L, Chen S Y, Wang H L: Polyglutamine-expanded ataxin-3 causes cerebellar dysfunction of SCA3 transgenic mice by inducing transcriptional dysregulation. Neurobiol Dis 2008, 31:89-101). A score of 0 was applied when all of the mouse's hindlimbs were consistently spread out from the abdomen, a score of 1 was applied when one hindlimb was retracted toward the abdomen for more than 50% of the measurement time, a score of 2 was applied when both hindlimbs were partially retracted toward the abdomen for more than 50% of the measurement time, and a score of 3 was applied when both hindlimbs were completely retracted toward the abdomen or touched the abdomen for more than 50% of the measurement time.

[0083] General motility test (open field test): After a mouse was placed in the center of a white acrylic box (40×40×40 cm) and then allowed to move freely for 5 minutes, the movement of the mouse was measured using Smart video tracking software (Panlab Harvard Apparatus).

Western Blot Analysis

[0084] For Western blotting, total cell lysates were prepared from cerebellar vermis for the mice injected with LPS and from the entire cerebellum for the mice injected with Ara-C. The cerebellar vermis was isolated, and each tissue was homogenized with a protease inhibitor cocktail (1:100, Millipore, Burlington, Mass., USA) and a phosphatase inhibitor cocktail (1:100, Cell Signaling Technology) in a lysis buffer (58 mM Tris-HCl, pH 6.8; 10% glycerol; and 2% SDS). The lysate was centrifuged, and then the protein concentration was quantified using a BCA kit (Bio-Rad Laboratories, Hercules, Calif., USA). Proteins were separated using gel electrophoresis and then transferred onto a membrane using an electrophoresis transfer system (Bio-Rad Laboratories). The membrane was incubated overnight at 4° C. with the following primary antibodies: anti-Iba1 (anti-ionized calcium-binding adapter molecule 1, 1:1500, Wako, Osaka, Japan), anti-GFAP (anti-glial fibrillary acidic protein, 1:2000, Millipore, Billerica, Mass., USA), anti-TNFα (anti-tumor necrosis factor alpha, 1:1000, Abcam, Cambridge, UK), anti-IL-1β (1 beta, 1:1000, Abcam, Cambridge, Mass., USA), anti-MCP-1 (anti-monocyte chemoattractant protein 1, 1:500, Abcam, Cambridge, UK), anti-MIP-1α (anti-macrophage inflammatory protein 1 alpha, 1:1000, R&D Systems, Minneapolis, Minn., USA), anti-Cal-D28K (anti-calbindin-D-28K, 1:2000, Sigma, St. Louis, Mo., USA), anti-cleaved caspase-3 (1:1000, Cell Signaling, Beverly, Mass., USA), anti-caspase-3 (1:1000, Cell Signaling, Beverly, Mass., USA), anti-CD86 (anti-cluster of differentiation 86, 1:1000, Invitrogen, Carlsbad, Calif., USA), anti-CD206 (anti-cluster of differentiation 206, 1:1000, R&D Systems, Minneapolis, Minn., USA), anti-iNOS (anti-inducible nitric oxide synthase, 1:1000, Abcam, Cambridge, UK), anti-IL-10 (anti-interleukin 10, 1:1000, Abcam, Cambridge, Mass., USA), anti-TSG-6 (anti-TNFα stimulated gene-6, 1:1000, GeneTex, Irvine, Calif., USA)), and anti-beta actin (anti-(31:2000, Santa Cruz, Calif., USA).

[0085] Next, it was incubated with HRP-conjugated secondary antibody (Amersham Biosciences, Piscataway, N.J., USA), and then Western blotting was performed with a chemiluminescence Western blot detection reagent (Amersham Biosciences). For semi-quantitative analysis, the band density was measured using an ImageQuant LAS 500 imager (GE Healthcare Life Science). The intensity of each protein band was calculated with Multi Gauge V3.0 software (Fuji Film, Tokyo, Japan) and normalized to the intensity of the corresponding beta actin (β band).

Statistical Analysis

[0086] All statistical analyses were performed using SigmaPlot software 12.0 (Systat Software, San Leandro, Calif.). Data is expressed as mean±standard error (standard error of the mean, SEM). Statistical significance was determined using ANOVA (one-way analysis of variance), followed by Tukey's post hoc test for multiple comparisons between groups or Student's t-test for comparison between two groups.

Experiment Result

1. Animal Model of Neurodegenerative Disease Induced by Inflammation-Inducing Substance (LPS)

[0087] In order to establish an animal model of a neurodegenerative disease induced by inflammation, LPS was directly injected into the cerebellum as an effective substance for inflammatory neurodegeneration.

[0088] As shown in the Western blot results of FIG. 1A, the expression of Iba1 (microglia) was significantly increased over time by LPS injection (on day 1 after LPS-injection **p<0.01 and ***p<0.001, on day 7 after LPS-injection ***p<0.001 vs. control group, CON). As shown in the Iba1 expression results, the expression levels of inflammatory cytokines such as IL-1α and TNFα were also significantly increased by 2.4 times and 2.6 times compared to the intact control group on day 7 after LPS injection (FIG. 1A; **p<0.01, *p<0.05 vs. CON). On the other hand, the expression levels of gliacytes and inflammatory cytokines in mice treated with PBS were not different from those of the control group.

[0089] In addition, in this example, the production of inflammatory chemokines such as MIP-1α and MCP-1, known as chemoattractants for inflammatory cells and mesenchymal stem cells, was confirmed in the cerebellum injected with LPS. As shown in the Western blot analysis results (FIG. 1A), the expression of MIP-1α and MCP-1 in the cerebellum was significantly increased at 1 day after LPS injection compared to the control group (**p<0.01 vs. CON) and decreased therefrom after 7 days.

[0090] In order to confirm the suitability of the final animal model, the motility defect and Purkinje cell loss of the mouse were confirmed through the rotarod test. As a result, the residence time on the rotating rod was significantly reduced at 1 week after LPS injection compared to the normal control group, and the decreased value was maintained for 4 weeks (FIG. 1B; ***p<0.001 2 weeks and 3 weeks after LPS injection vs. CON, **p<0.01 4 weeks after LPS injection vs. CON). In addition, calbindin protein was significantly reduced in the cerebellum on day 7 after LPS injection in the mice injected with LPS compared to the normal control group, indicating a loss of Purkinje cells (FIG. 1B; ***p<0.001 vs. CON).

2. Animal Model of Neurodegenerative Disease Induced by Anti-Miotic Agent

[0091] In order to establish an animal model of a neurodegenerative disease induced by an anti-miotic agent, Ara-C was intraperitoneally injected daily for 1 to 3 days after birth.

[0092] As shown in the results of FIG. 2A, the structure of the cerebellum, neurons, and Purkinje cells were confirmed through histopathological staining. As a result, it was confirmed that the development of the cerebellum of the animals administered with Ara-C was inhibited, and it was confirmed that neurons and Purkinje cells of the cerebellum were not formed.

[0093] Additionally, the disease animal model was evaluated through behavioral evaluation. As a result, it was confirmed that there was a behavioral imbalance compared to the control group, such as parallel, hanging, and gait (FIG. 2B).

3. Animal Model of Genetic Neurodegenerative Disease through Genetic Modification

[0094] In order to establish an animal model of a genetic neurodegenerative disease through genetic modification, B6D2-Tg(Pcp2-SCA2)11Plt/J mouse was established by inserting and overexpressing the human SCA2 gene into the mouse Pcp2 promoter. However, the secured B6D2-Tg(Pcp2-SCA2)11Plt/J mouse was an animal model established by fertilization of C57BL/6J ovum and DBA/2J sperm. Because the genetic background was not uniform, crossing was carried out until the fifth generation, which showed 94% genetic stability through repeated backcrossing with C57BL/6J mice.

[0095] Therefore, as shown in FIG. 3A, genetic stabilization was performed through backcrossing, and then the SCA2 gene expression was confirmed to establish an animal model of a genetic neurodegenerative disease.

[0096] Additionally, as shown in the results of FIG. 3B, it was confirmed that there was a behavioral imbalance compared to the control group (Wild type, WT) animals through the rotarod test, which is a behavioral evaluation.

4. Properties of Mesenchymal Stem Cell Therapeutic Agent (hMSC)

[0097] In order to confirm the properties of the mesenchymal stem cell therapeutic agent isolated from the bone marrow, the properties of the stem cell therapeutic agent were confirmed through analysis of shape, marker, and differentiation ability, and the functional properties of the stem cells were also confirmed through the secreted cytokines.

[0098] First, the characteristics of hMSCs were determined by the morphology of fibroblastoid, the expression pattern of hMSC-related surface markers, and differentiation possibility according to the criteria of the International Society for Cellular Therapy (ISCT).

[0099] In hMSCs isolated from the bone marrow, the original shape of spindle-shaped fibroblast-shaped stem cells could be visually confirmed (FIG. 4A). In addition, the PDT of hMSCs for each passage was evaluated to confirm the proliferative capacity of stem cells. Although hMSCs at the early passage stage showed high proliferative capacity (37-51 hours), it was confirmed that the proliferative capacity of hMSCs was poor in the late passage stage (after Passage 7) (FIG. 4B).

[0100] As a result of flow cytometry, 95% or more of the early hMSCs were positive for hMSC-related CD markers such as CD29, CD44, CD73, CD90, and CD105, whereas they were negative for hematopoietic stem cell-related CD markers such as CD34 and CD45 (FIG. 4C).

[0101] In order to confirm the differentiation ability of bone marrow-derived hMSCs, stem cells at the early stage (Passage 2 to 4) were cultured in adipogenic medium, osteogenic medium and chondrogenic medium for 2 to 4 weeks to confirm differentiation into adipocytes, osteoblasts and chondrocytes. Through this, it was found that the mesenchymal stem cell therapeutic agent isolated/produced with the unique properties of mesenchymal stem cells well maintained the properties of stem cells until the 7th passage (FIG. 4D).

[0102] In order to predict the therapeutic effect in a neurodegenerative disease, the neurotropic factor was identified to identify the secreted protein of the mesenchymal stem cell therapeutic agent. As a result, the expression of ANG, BDNF, and IGF, which are closely related to neuroprotective factors and growth and differentiation of neurons, was confirmed. It is thought that the mesenchymal stem cell therapeutic agent may be beneficial for a neurodegenerative disease (FIG. 4E).

[0103] Based on the above results, only hMSCs within passage 5 (P1 to P5) were used in subsequent experiments.

5. Therapeutic Effect of hMSCs in Animal Model of Neurodegenerative Disease Induced by Inflammation

[0104] In order to evaluate whether intrathecal transplanted hMSCs have beneficial effects in an animal model of a neurodegenerative disease induced by LPS, motor coordination and balance were evaluated using the rotarod test weekly for 4 weeks after hMSC transplantation using 10-week-old mice. The control group mice were administered with HTS (HypoThermosol), an optimal preservative used to store stem cells at low temperature. As a result, before LPS administration, mice in all groups showed a similar level of motor coordination, but after LPS administration, it was confirmed that the disease animal had gradually decreased motility compared to the control group. When the stem cell therapeutic agent was transplanted at a low dose (1×10.sup.5 cells/mouse) and at a high dose (1×10.sup.6 cells/mouse), it was confirmed that the motor performance capability of the disease animal was significantly enhanced compared to the control group (FIG. 5A). When behavioral disorders were evaluated through the composite ataxia phenotype scoring system, it was confirmed that a behavioral disorder was significantly improved in a group administered with the mesenchymal stem cell therapeutic agent (FIG. 5B).

[0105] Next, by confirming not only behavioral indicators but also the reduction of inflammatory response, inhibition of apoptosis of Purkinje cells, and the expression level of M1- and M2-type microglia, the effect of a mesenchymal stem cell therapeutic agent in various mechanisms was confirmed.

[0106] In order to confirm the reduction of the inflammatory response of the mesenchymal stem cell therapeutic agent, the activation of gliacytes induced by the LPS inflammatory response was confirmed. It was confirmed that Iba1 was significantly reduced in a group administered with the mesenchymal stem cell therapeutic agent compared to the control group (FIG. 5C), and it was also confirmed that the inflammatory cytokines IL-1α and TNF-α were also reduced in the cerebellum (FIG. 5D). This is confirmed by anti-inflammatory and immune-modulating effects of the mesenchymal stem cell therapeutic agent.

[0107] In addition, the protective effect of a stem cell therapeutic agent capable of inhibiting and protecting the apoptosis of Purkinje cells induced by inflammatory response was confirmed. The Purkinje cell marker Cal-D28K was reduced in the cerebellum of the disease animal, but it was confirmed that the expression of the marker was higher in a group administered with the mesenchymal stem cell therapeutic agent compared to the control group (FIG. 5E). In addition, in order to confirm the change in polarization of M1- and M2-type microglia, when CD86 as an M1-type marker and CD206 as an M2-type marker were identified, it was confirmed that M1-type microglia of CD86 were reduced and M2-type microglia of CD206 were increased (FIG. 5F). This indicates that M1-type microglia are polarized into M2-type microglia related to tissue regeneration, and this can represent the secondary therapeutic improvement effect of stem cells.

6. Therapeutic Effect of hMSCs in Animal Model of Neurodegenerative Disease Induced by Anti-Miotic Agent

[0108] In order to compare the effect of improving motor ability according to the administration method of a stem cell therapeutic agent at a low dose (2×10.sup.4 or 6×10.sup.4 cells) in an animal model of cerebellar ataxia induced by Ara-C, an anti-miotic agent, the rotarod test was performed every 2 weeks for 12 weeks from the time of stem cell administration (10 weeks of age), and motor coordination and balance were evaluated. In this example, stem cells were administered in the following four ways:

[0109] {circle around (1)} once administration of 2×10.sup.4 cells (n=2)

[0110] {circle around (2)} 3 times repeated administration of 2×10.sup.4 cells at a 4-week interval (10 w, 14 w, 18 w) (n=3)

[0111] {circle around (3)} once administration of 6×10.sup.4 cells (n=3)

[0112] {circle around (4)} 3 times repeated administration of 6×10.sup.4 cells at a 4-week interval (10 w, 14 w, 18 w) (n=3)

[0113] As a result, the cerebellar ataxia model (Ara-C) mice administered with Ara-C failed to maintain balance and fell off in the rotarod test compared to the normal control group (WT). On the other hand, when the Ara-C model mice were administered with hMSCs, there was a difference in the enhancement of motor performance capability according to the administration dose and method. Specifically, when the mice were administered with 2×10.sup.4 cells, there was no significant difference compared to the mice which were not administered with the stem cells. When 2×10.sup.4 cells were repeatedly administered and when 6×10.sup.4 cells were administered once, the balance ability was gradually increased until 16 weeks of age, but after that, it was reduced again. Finally, it was confirmed that the balance ability was continuously increased when 6×10.sup.4 cells were repeatedly administered (FIG. 6A). Through this, it was confirmed that, in relation to the effect of mesenchymal stem cells in the disease animal, the behavioral improvement effect appeared when at least 6×10.sup.4 cells were administered once or repeatedly.

[0114] In addition, in order to confirm anatomical changes, changes in the size and weight of the cerebellum after administration of the mesenchymal stem cell therapeutic agent were confirmed. As a result, it was confirmed that the size of the cerebellum was significantly reduced in the cerebellar ataxia model (Ara-C) mice administered with Ara-C compared to the normal control group (WT), but the size of the cerebellum was slightly increased after administration of stem cells (FIG. 6B).

[0115] In addition, in order to measure general motor activity, the open field test was performed. In this example, stem cells were administered once or repeatedly at two concentrations (1×10.sup.5 or 1×10.sup.6 cells), and the results were compared. Specifically, stem cells at each concentration were administered once at 10 weeks of age or administered repeatedly 3 times at a 4-week interval, and then at 22 weeks of age, general motility was measured through the open field test. As a result, it was confirmed that the motility was significantly reduced in the cerebellar ataxia model (Ara-C) mice administered with Ara-C compared to the normal control group (WT), but the motility was enhanced after administration of stem cells. The effect of improving motor activity was more pronounced when the stem cells were administered repeatedly than when the stem cells were administered once, and in both the single administration and the repeated administration, when the stem cells were administered at a concentration of 1×10.sup.5 cells, the effect of improving motor activity was higher (FIG. 6C).

[0116] The effect of the therapeutic agent was also confirmed by differences in the protein. After administration of mesenchymal stem cells at the concentration of 1×10.sup.5 cells in the disease animal, it was confirmed that the expression of the neuron protein NeuN and the neuroblast and neuron progenitor cell marker DCX (doublecortin) was significantly increased. This is considered to be a therapeutic effect due to improvement of maturation disorder of the cerebellum (FIG. 6D).

[0117] In the composite phenotype scoring system, which is a neurobehavioral test, the mesenchymal stem cell therapeutic agent was administered once or repeatedly at two concentrations (1×10.sup.5 or 1×10.sup.6 cells), and the results were compared. Specifically, stem cells at each concentration were administered once at 10 weeks of age or administered repeatedly 3 times at a 4-week interval, and then at 22 weeks of age, the effect of improving behavioral disorders was measured through the composite phenotype scoring system. As a result, it was confirmed that the total mean score was significantly reduced after administration of stem cells compared to the cerebellar ataxia model (Ara-C) mice administered with Ara-C, and neurobehavioral symptoms were more alleviated when administered repeatedly than when administered once, and when administered at a concentration of 1×10.sup.5 cells than when administered at a concentration of 1×10.sup.6 cells (FIG. 6E).

7. Therapeutic Effect of hMSCs in Animal Model of SCA2 Neurodegenerative Disease through Genetic Modification

[0118] In order to evaluate whether intrathecal transplanted hMSCs have therapeutic effects in an animal model of a genetic modification disease (SCA2), motor coordination and balance were evaluated using the rotarod test weekly for 4 weeks after transplantation of hMSCs into 10-week-old mice. As a result, behavioral imbalance was confirmed in the SCA2 gene mutation animal model. In addition, when the mesenchymal stem cell therapeutic agent was transplanted at a low dose (6×10.sup.4 cells/mouse) and at a high dose (1×10.sup.5 cells/mouse), it was confirmed that the motor performance capability of the disease animal was enhanced compared to the control group. This is the result of confirming the therapeutic effect of mesenchymal stem cells in the animal model of genetic diseases as well as in the animal model of diseases artificially induced by substances, and it was confirmed that the mesenchymal stem cell therapeutic agent has a therapeutic effect on various pathological mechanisms (FIG. 7).

INDUSTRIAL APPLICABILITY

[0119] The treatment method or the treatment composition using mesenchymal stem cells, according to the present invention, has remarkable effects in reducing neuroinflammation, inhibiting M1 microglia, activating M2 microglia, inhibiting apoptosis of Purkinje cells, inhibiting death of neurons, improving motor ability, and the like, and therefore may be effectively utilized in alleviating and treating neurodegenerative diseases including cerebellar ataxia and multiple system atrophy, and thus has high industrial applicability.