Pharmaceutical composition containing stem cell in which vascular endothelial growth factor is overexpressed as effective ingredient for preventing or treating neurodegenerative disease

Abstract

The present invention relates to a pharmaceutical composition containing stem cells in which vascular endothelial growth factor (VEGF) is overexpressed as an effective ingredient for preventing or treating neurodegenerative diseases. The stem cells in which VEGF is overexpressed according to the present invention effectively act against the neurodegenerative disease-induced VEGF reduction of neural stem cells in the SVC to inhibit the abnormal migration of neural stem cells, suppress inflammatory responses, and restrain the accumulation of cholesterol and spingolipids in the cerebral cortex, thus restoring the behavior exercise capacity of animal models, whereby the pharmaceutical composition can be used as an effective therapeutic agent for neurodegenerative diseases.

Claims

1. A method for treating a neurodegenerative disease in a subject in need thereof, the method comprising administering an effective amount of a composition comprising stem cells overexpressing vascular endothelial growth factor (VEGF) as an active ingredient to a subventricular zone (SVZ) of the subject, wherein the neurodegenerative disease is selected from the group consisting of Niemann-Pick disease, Gaucher disease, Fabry disease, Tay-Sachs disease, and Sandhoff disease.

2. The method of claim 1, wherein the stem cells are at least any one selected from the group consisting of adult stem cells, embryonic stem cells, mesenchymal stem cells, tumor stem cells, and induced pluripotent stem cells.

3. The method of claim 2, wherein the adult stem cells are neural stem cells or neural progenitor cells.

4. The method of claim 1, wherein the stem cells overexpressing the vascular endothelial growth factor (VEGF) reduce a cerebral inflammation and inhibit cholesterol or sphingolipid accumulation.

5. The method of claim 1, wherein the composition is a pharmaceutical composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A confirms the proportions of neural stem cells existing in SVZ of normal (WT) and NP-C mice (n=6 per group). *p<0.05. Data were expressed as the mean±SEM.

(2) FIG. 1B confirms the expression of VEGF in neural stem cells isolated from SVZ tissue of normal (WT) and NP-C mice (n=4 per group). **p<0.01. Data were expressed as the mean±SEM.

(3) FIG. 1C confirms VEGF expressed in neural stem cells through immunofluorescent staining of normal and NP-C mouse SVZ (left) and shows a graph of quantification therefor (right) (n=4 per group). ***p<0.001. Data were expressed as the mean±SEM.

(4) FIG. 2A shows an experimental design for investigating the migration of SVZ neural stem cells by injection of the neural stem cell labeling fluorescent substance MPIO into SVZ of normal and NP-C mice.

(5) FIG. 2B confirms the presence or absence of MPIO fluorescent substance-labeled neural stem cells in different brain regions on days 7 and 21 after the injection of MPIO into normal and NP-C mouse SVZ, and shows a graph of quantification therefor (n=6 per group, {circle around (1)}=LV, {circle around (2)}=RMS, {circle around (3)}=OB, {circle around (4)}=LLV, {circle around (5)}=thalamus). *p<0.05, **p<0.01, ***p<0.001. Data were expressed as the mean±SEM.

(6) FIG. 3A shows an experimental design for investigating the effects of SVZ damage on the entire brain when, for the induction of the damage to neural stem cells existing in the SVZ environment, GFAP/CD133 split-cre was injected into SVZ of NPCf.sup.f1/f1 or VEGF.sup.f1/f1 mice to reduce the expression of NPC or VEGF specifically to neural stem cells.

(7) FIG. 3B confirms the proportions of neural stem cells in SVZ of NPC.sup.f1/f1 or VEGF.sup.f1/f1 when SVZ was injected with GFAP/CD133 split-cre for four weeks (n=6 per group). **p<0.01. Data were expressed as the mean±SEM.

(8) FIG. 3C confirms the inflammation responses of the SVZ environment of the NPC.sup.f1/f1 or VEGF.sup.f1/f1 mice when SVZ was injected with GFAP/CD133 split-cre for four weeks (GFAP: astrocyte, Iba-1: microglia. n=6 per group). *p<0.05, ***p<0.001. Data were expressed as the mean±SEM.

(9) FIG. 3D confirms the presence or absence of MPIO fluorescent substance-labeled neural stem cells in different brain regions in NPC.sup.f1/f1 or VEGF.sup.f1/f1 mice when SVZ was injected with GFAP/CD133 split-cre and MPIO (n=6 per group). **p<0.01, ***p<0.001. Data were expressed as the mean±SEM.

(10) FIG. 3E confirms, through neurosphere (NS) cultures, the presence or absence of neural stem cells in SVZ and thalamus tissues of NPCf.sup.f1/f1 or VEGF.sup.f1/f1 mice when SVZ was injected with GFAP/CD133 split-cre for four weeks (n=6 per group). **p<0.01. Data were expressed as the mean±SEM.

(11) FIG. 3F confirms the inflammation responses in the thalamus and cortex of NPCf.sup.f1/f1 or VEGF.sup.f1/f1 when SVZ was injected with GFAP/CD133 split-cre for 4 weeks (GFAP: astrocyte, Iba-1: microglia. n=6 per group). *p<0.05, **p<0.01. Data were expressed as the mean±SEM.

(12) FIG. 3G confirms the cholesterol and lipid accumulation in the cortex of NPCf.sup.f1/f1 or VEGFf.sup.f1/f1 when SVZ was injected with GFAP/CD133 split-cre for 4 weeks (n=6 per group). p<0.05. Data were expressed as the mean±SEM.

(13) FIGS. 4A and 4B confirm the presence or absence of the impairments of sensory function (FIG. 4A) and motor function (FIG. 4B) of NPC.sup.f1/f1 or VEGF.sup.f1/f1 with SVZ injected with GFAP/CD133 split-cre for 4 weeks (n=10-13 per group). *p<0.05, **p<0.01. Data were expressed as the mean±SEM.

(14) FIG. 5A shows an experimental design for injection of normal neural stem cells (WT NSCs), VEGF-overexpressing neural stem cells (VEGFtg NSCs), and VEGF-overexpressing vector (VEGF OX vector) in order to improve the damaged SVZ environment of NP-C mice.

(15) FIG. 5B confirms the percentages of neural stem cells in SVZ after NP-C mice were injected with normal neural stem cells, VEGF-overexpressing neural stem cells, and VEGF-overexpressing vector twice a week for a total of 4 weeks (n=6 per group). *p<0.05, ***p<0.001. Data were expressed as the mean±SEM.

(16) FIGS. 5C and 5D confirm the inflammation responses in the SVZ, thalamus, and cortex after SVZ of NP-C mice was injected with normal neural stem cells, VEGF-overexpressing neural stem cells, and VEGF-overexpressing vector twice a week for a total of 4 weeks (GFAP: astrocyte, Iba-1: microglia. n=6 per group). **p<0.01, ***p<0.001. Data were expressed as the mean±SEM.

(17) FIG. 5E confirms the existence proportions (%) of MPIO fluorescent substance-labeled neural stem cells in different brain regions in NP-C mice with SVZ injected with VEGF-overexpressing neural stem cells twice a week for a total of four weeks (n=6 per group). *p<0.05, **p<0.01, ***p<0.001. Data were expressed as the mean±SEM.

(18) FIG. 5F confirms, through neurosphere (NS) cultures, the presence or absence of neural stem cells in SVZ and thalamus tissues of NP-C mice with SVZ injected with VEGF-overexpressing neural stem cells twice a week for a total of four weeks (n=6 per group). **p<0.01, ***p<0.001. Data were expressed as the mean±SEM.

(19) FIG. 6A confirms, through filipin immunostaining, the cholesterol levels accumulated in the cerebrum after SVZ of NP-C mice was injected with normal neural stem cells, VEGF-overexpressing neural stem cells, and VEGF-overexpressing vector twice a week for a total of 4 weeks, and shows a graph of quantification therefor (n=6 per group). p<0.05. Data were expressed as the mean±SEM.

(20) FIG. 6B confirms the lipid levels accumulated in the cerebrum after SVZ of NPC mice was injected with VEGF-overexpressing neural stem cells twice a week for a total of 4 weeks (n=6 per group). **p<0.01, ***p<0.001. Data were expressed as the mean±SEM.

(21) FIGS. 7A, 7B, and 7C confirm the presence or absence of the improvement of sensory function (FIG. 7A) and motor function (FIG. 7B) and the increase or decrease in survival rate (FIG. 7C) after SVZ of NP-C mice was injected with VEGF-overexpressing neural stem cells twice a week for a total of 4 weeks (n=10-12 per group). *p<0.05, **p<0.01, ***p<0.001. Data were expressed as the mean±SEM.

(22) FIG. 8A confirms the decrease of neurons existing in the Purkinje cell layer (PCL) by staining the cerebellar tissues composed of molecular layer (ML), PCL, and granular cell layer (GCL) after SVZ of NP-C mice was injected with VEGF-overexpressing neural stem cells (NP-C/VEGFtg NSCs) or after the cerebellum was directly injected with VEGF-loaded microspheres (NP-C/VEGF microsphere) (left), and shows a graph of the quantification of the reduction of neurons in each of cerebellar lobe Nos. III to VIII (right) (n=3 per group). *p<0.05. Data were expressed as the mean±SEM.

(23) FIGS. 8B to 8D confirm the inflammation responses (FIG. 8B), lipid accumulation (FIG. 8C), and the presence or absence of the improvement of motor function (FIG. 8D) after SVZ of NP-C mice was injected with VEGF-overexpressing neural stem cells (NP-C/VEGFtg NSCs) or after the cerebellum was directly injected with VEGF-loaded microspheres (NP-C/VEGF microsphere) (FIGS. 8B & 8C; n=3 per group, FIG. 8D; n=7 per group). *p<0.05. Data were expressed as the mean±SEM.

MODE FOR CARRYING OUT INVENTION

(24) Hereinafter, examples of the present invention will be described in detail.

Example 1

Materials and Methods

(25) 1-1. Preparation of Mice

(26) A strain of NPC mutant mice deficient in Balb/C and NPC1 genes (NP-C mice: weighing less than normal mice of the same week age, showing limb jerking and seizure due to severe motor function impairment from 4-6 weeks of age, and being approximately 9-10 weeks in life-expectancy) was maintained through genotyping through PCR, and each strain of NPCf.sup.f1/f1 and VEGFf.sup.f1/f1 mice was maintained through strain cross. The mice were allocated to experimental groups using the block randomization method. In order to eliminate prejudice, there was no involvement in data collection and data analysis. All mice experiments were approved by the Kyungpook National University Institutional Animal Care and Use Committee (IACUC).

(27) 1-2. Injection of Medicine and Neural Cells

(28) For injection of NPC.sup.f1/f1 or VEGF.sup.f1/f1 mice with normal neural stem cells, VEGF-overexpressing neural stem cells, VEGF-overexpressing vector, or GFAP/CD133 spilt-cre, the mice were anesthetize with a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine. The mice were injected with normal neural stem cells (˜1×10.sup.6 cells/3 ul), VEGF-overexpressing neural stem cells (˜1×10.sup.6 cells/3 ul), VEGF-overexpressing vector (MGC 12075 clone, Thermo Scientific) (3 ul), and NPC.sup.f1/f1 or VEGF.sup.f1/f1 mice were daily injected with GFAP/CD133 spilt-cre (1:1.5, 2 ul for each).

(29) In addition, 1.63 um-diameter polystyrene/divinylbenzene-coated fluorescent iron oxide particles (MPIOs, 3.00 mg Fe/ml, green fluorescent dye: 480 nm excitation, 520 nm emission; Bangs Laboratories, Fishers, Ind., USA) were used for investigating the migration of neural stem cells, and a total volume of 1.5 ul of MPIO (0.67 mg Fe/ml) mixed with 0.05 mg/ml PLL (Sigma) was injected to SVZ of each mouse. The injection rate of cells and medicines was a flow rate of 0.3 μl/min.

(30) For the direct injection of VEGF microspheres into the cerebellum, a glass capillary (1.2 mm×0.6 mm) was used for transplantation of VEGF microspheres into the cerebellum. The injection coordinates were+5.52 mm anterior relative to the bregma and 2.50 mm in the injection depth. Regarding the injection rate, 3 mg was transplanted at a flow rate of 0.15 μl/min.

(31) 1-3. Preparation of VEGF-Containing Microspheres

(32) For the preparation of VEGF-containing microspheres, human recombinant vascular endothelial growth factor (VEGF) was used after purchase from R&D System (Catalog No. 293-VE-010). A water-in-oil-in-water emulsification method was used to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres containing the human recombinant vascular endothelial growth factor (VEGF). The human recombinant vascular endothelial growth factor-A (VEGF-A) was encapsulated in PLGA microspheres (1:1 of lactic acid and glycolic acid in PGLA and a molecular weight of 40,000-75,000).

(33) 1-4. Immunofluorescent Staining

(34) The brain tissue of the mice was cut into a thickness of 50 um using a vibratome, and then cultured together with anti-VEGF (rabbit, 1:500, abcam), anti-SOX (mouse, 1:100, R&D), anti-GFAP (rabbit, 1:500, DAKO), anti-Iba-1 (rabbit, 1:500, DAKO), and anti-Calbindin (rabbit, 1:100, Millipore). For the investigation of the accumulation degree of cholesterol, the tissue of the mice was subjected to filipin staining. The SVZ, thalamus, cortex, and cerebellum were analyzed using a laser scanning confocal microscope equipped with Fluoview SV1000 imaging software (Olympus FV1000) or with an Olympus BX51 microscope. Metamorph software (Molecular Devices) was used to quantify the percentage of the stained part relative to the area of the whole tissue. The number of cells in the stained part was quantified by Visiomorph software (VisioMorph) using Stereology.

(35) 1-4. Culture of Neurospheres and Culture of Normal Cells and VEGF-Overexpressing Neural Stem Cells

(36) For the investigation of the abnormal migration, into the thalamus, of neural stem cells of mice with damaged SVZ or NP-C mice injected with VEGF-overexpressing neural stem cells, the SVZ and thalamus parts were extracted from the mouse brains. Each tissue was extracted in ice-cold Hibernate A/B27/Glutamax medium (HABG) (Invitrogen), and then dissociated by reaction at 37° C. for 30 minutes through immersion in papain solution (Worthington). Thereafter, the dissociated tissue was centrifuged using Optiprep (Sigma) density gradient solution, and a layer containing neural stem cells was isolated, and cultured in Neurobasal A (Invitrogen)/B27 medium containing glutamax (0.5 mM), gentamycin (10 ug/ml, Invitrogen), mouse fibroblast growth factor 2 (mFGF2, 5 ng/ml, Invitrogen), and mouse platelet-derived growth factor-bb (mPDGFbb, 5 ng/ml, Invitrogen) for one week. The cultured neural stem cells grew to spherical neurospheres, and after one week, the cells were collected and dissociated, and then cultured in 24-well plate at 1×10.sup.4 cells. The neurospheres generated after 1-week culture were counted on a microscope, and the minimum cut-off size of neurospheres was limited to 50 um. For culture of neural stem cells for injection of normal and VEGF-overexpressing neural stem cells into SVZ of NP-C mice, when the neurospheres were injected after the culture by the above-described method, the cells were separated by Triple select (Gibco) solution and single cells were injected.

(37) 1-6. Lipid Measurement

(38) The cerebrum and cerebellum of each mouse were extracted, and a homogeneous buffer containing 50 mM HEPES (Invitrogen), 150 mM sodium chloride (NaCl) aqueous solution (Sigma), 0.2% Igepal CA-630 (Sigma), and protease inhibitor (Milipore) was added, and the mixture was homogenized using a homogenizer, and centrifuged at 13,000 rpm for 10 minutes. After 10 minutes, additional centrifugation was conducted at 13,000 rpm for 30 minutes. After 30 minutes, the supernatant was taken, and dichloromethane (DCM, Sigma), DCM:M (dichloromethane:methanol=1:3, Sigma) and 10% sodium hypochlorite (NaHCl) were sequentially added thereto, and the mixture was centrifuged at 13,000 rpm for 1 minute, and then only organic dissolution layer was separated. The separated sample was dried using a rotary vacuum evaporator. As described above, the dried lipid extract was re-suspended in 0.2% Igepal CA-630 (Sigma), and then the concentration of each lipid was measured using UPLC system.

(39) 1-7. Cholesterol Measurement

(40) The cerebrum of each mouse was extracted, and a homogenous buffer containing 50 mM phosphate buffer, 500 mM sodium chloride (NaCl) aqueous solution, 25 mM cholic acid, and 0.5% Triron X-100 was added thereto, and the mixture was homogenized using a homogenizer, and centrifuged at 13,000 rpm for 10 minutes. After 10 minutes, only the organic dissolution layer was separated, and the cholesterol level was measured using Amplex Red Cholesterol Assay Kit (Molecular Probes).

(41) 1-8. Sensory Ability Tests

(42) For investigation of sensory ability of mice with damaged SVZ or NP-C mice injected with VEGF-overexpressing neural stem cells, hot-plate test and tail-flick test were performed. For the hot-plate test, a heating apparatus (Panlab/Harvard Apparatus, Spain) was maintained at 50° C., and respective mice were located at the end of the heated surface, and the first nociception, that is, the time to jump or paw licking was measured. The cut-off time was 60 seconds. Between the measurements, the heated surface was washed with a detergent and ethanol, and the temperature thereof was maintained at 50° C. For the tail-flick test, the mouse was wrapped in a dark cloth, and then the tail was placed on a heated instrument surface (Panlab/Harvard Apparatus, Spain), and the time taken for the tail to respond was measured.

(43) 1-9. Motor Ability Tests (Beam Walking Test)

(44) For investigation whether motor ability of mice with damaged SVZ or NP-C mice injected with VEGF-overexpressing neural stem cells is improved, open field, rota-rod, and beam tests were performed. The mice with the cerebellum directly injected with VEGF microspheres was subjected to rota-rod test. In the open field test, the mouse was placed in a square box for 10 minutes to measure overall activity and the time spent wandering the wall and center. For the rota-rod test (Ugo Basile, Comerio, VA, Italy), a machine equipped with a 3-cm diameter rod that was properly machined to provide grip was rotated at a rotation velocity of 4 rpm, and rota-rod exercise was conducted three times or more to measure the endurance time of a test animal in seconds, and the mean value thereof was recorded. Each rota-rod exercise test should not exceed 5 minutes in each time. In the beam test, a mouse was placed on the start point on a 6 mm or 12 mm-width rod, and then the time taken for the mouse to move to the end point was measured.

(45) 1-10. Real-Time Quantitative PCR

(46) For investigation of expression level in SVZ neurons of NP-C mice, the SVZ of the mice was isolated, and then neuron stem cells were isolated therefrom. The expression level of VEGF in the isolated neuron stem cells was measured using real-time quantitative PCR. Total RNA was extracted from the neurons using RNeasy Plus mini kit (Qiagen, Korea, Ltd), and a kit of Clontech (Mountain View, Calif.) was used to synthesize 5 μg of cDNA from the total RNA. In addition, Corbett research RG-6000 real-time PCR instrument was used to perform real-time quantitative PCR by repeating 40 cycles each composed of 95° C. for 10 min, 95° C. for 10 sec, 58° C. for 15 sec, and 72° C. for 20 sec.

(47) 1-11. Statistical Analysis

(48) Comparisons of respective groups were performed with Student's t-test. For comparison between another group and two or more groups, one-way analysis of variance (ANOVA) and Tukey's HSD test were performed. The comparison of the overall survival rate was performed using Log-rank test. SPSS statistical software was used for all statistical analyses. Data were considered to be significant at p<0.05, p<0.01, and p<0.001.

Example 2

Confirmation of Proportions of Neural Stem cells and VEGF expression levels in SVZ of NPC mice

(49) For investigation of the proportions of neural stem cells existing in SVZ of 6-week-old NP-C mice, SVZ of each of normal and NP-C mice was immunofluorescent stained by the method described in Example 1-3 above. As a result, FIG. 1A confirmed reductions of neural stem cells (SOX2+/GFAP+, Mash1+, and DCX+ cells) (*p<0.05. n=6 per group).

(50) For investigation of the expression level of VEGF in SVZ of 6-week-old NP-C mice, SVZ region were extracted from brains of normal mice (WT) and NP-C mice, and then neural stem cells were isolated. Total RNA was extracted from the isolated neural stem cells by the method described in Example 1-9 above, and then cDNA was synthesized, and the expression level of VEGF expressed in neurons were analyzed by real-time quantitative PCR. The results are shown in FIG. 1B (**p<0.01. n=4 per group).

(51) SVZ of each mouse was immunofluorescent stained by the method described in Example 1-3 above using neural stem cell labeling antibody and VEGF antibody. The results are shown in FIG. 1C (**p<0.01. n=4 per group).

(52) As shown in FIG. 1, it could be confirmed that the survival rates of the neural stem cells existing in the SVZ environment were decreased and the expression of VEGF in neurons was reduced in NP-C mice compared with normal mice (WT).

Example 3

Confirmation of Abnormal Migration of Neural Stem Cells in SVZ Environment

(53) It has been known that neural stem cells existing in normal SVZ environment can migrate to the olfactory bulb (OB) through the rostral migratory stream (RMS), and especially, VEGF expressed in neural stem cells is involved in such migration (See Neuron. 70, 687-702, 2011; J Neurosci. 29, 8704-8714, 2009; J Neurosci Res. 88, 248-257, 2010).

(54) For investigation of migration patterns of VEGF expression-reduced neural stem cells in SVZ of NP-C mice, the neural stem cell labeling fluorescent substance MPIO was injected into SVZ of normal and NP-C mice (FIG. 2A). It was confirmed that after 7 days, the MPIO labeled neural stem cells of normal mice existed a lot in RMS and OB, whereas the neural stem cells of NP-C mice existed a lot in the thalamus, and after 21 days, the neural stem cells of NP-C mice existed a lot in the thalamus (FIG. 2B, *p<0.05, **p<0.01, ***p<0.001. n=6 per group).

(55) That is, it could be confirmed that in the normal state, the neural stem cells in SVZ migrate a lot to RMS and OB but rarely migrate to the thalamus, whereas the VEFG expression-reduced neural stem cells do not migrate a lot and abnormally migrate to the thalamus.

Example 4

Effect of SVZ Environment Damage on Inflammation Responses and Cholesterol and Lipid Accumulation of the Entire Brain

(56) For investigation of the effect of the damage of neural stem cells existing in SVZ environment on the inflammation responses of the entire brain, a cannula was inserted in SVZ of 4-week-old NPCf.sup.f1/f1 or VEGF.sup.f1/f1 mice, and as shown in FIG. 3A, GFAP/CD133 split-cre was daily injected into SVZ for a total of 4 weeks. That is, pathological changes shown after (i) GFAP/CD133 split-cre was injected into SVZ of NPCf.sup.f1/f1 mice to inhibit the expression of NPC1 in neural stem cells existing in SVZ, or (ii) GFAP/CD133 split-cre was injected into SVZ of VEGFf.sup.f1/f1 mice to inhibit the expression of VEGF in neural stem cells existing in SVZ were observed.

(57) As a result, as shown in FIGS. 3B and 3C, it was confirmed that the survival rates of neural stem cells existing in the SVZ environment of the NPC1 or VEGF expression-inhibited mice were decreased and the inflammation responses were significantly increased (GFAP: astrocyte, Iba-1: microglia. *p<0.05, ***p<0.001, n=6 per group),

(58) The NPC or VEGF expression-reduced neural stem cells caused by such problems of SVZ showed abnormal migration to the thalamus (FIGS. 3D and 3E, **p<0.01, ***p<0.001, n=6 per group).

(59) It was confirmed that the abnormal migration of NPC or VEGF expression-reduced neural stem cells to the thalamus also increased the inflammation responses in the thalamus and cortex (FIG. 3E), and also increased the cholesterol and lipid accumulation, which is a major pathological pattern in NP-C mice (FIGS. 3F and 3G, GFAP: Iba-1: microglia. *p<0.05, **p<0.01. n=6 per group).

(60) It could be seen from the results above that the damage to SVZ environment reduces the survival rates of neural stem cells existing in SVZ environment and causes abnormal migration of NPC or VEGF expression-reduced neural stem cells to the thalamus, thereby increasing the inflammation responses in the thalamus and, furthermore, inducing the inflammation responses and the cholesterol and lipid accumulation in the cortex. This indicates that the damage to SVZ environment can affect the entire brain.

Example 5

Effects of SVZ Environment Damage on Sensory and Motor Abilities

(61) For investigation whether inflammation responses and cholesterol and lipid accumulation in the entire brain due to SVZ environment damage affect sensory and motor abilities, NPC.sup.f1/f1 or VEGF.sup.f1/f1 mice with SVZ injected with GFAP/CD133 split-cre for a total of 4 weeks were subjected to hot-plate and tail-flick tests as sensory ability test, and open field, rota-rod, and beam tests as motor ability tests.

(62) As a result, the mice with damaged SVZ environment showed reduced sensory and motor abilities (FIGS. 4A and 4B, *p<0.05, **p<0.01. n=10-13 per group).

(63) It could be seen from the above results that the inflammation responses and the cholesterol and lipid accumulation in the entire brain due to SVZ environment damage reduce sensory and motor abilities.

Example 6

Mitigation of Inflammation Responses Through Improvement of Damaged SVZ Environment of NP-C Mice

(64) In cases of NP-C mice, the expression of VEGF has been reduced in neural stem cells existing in SVZ, and therefore, it was investigated whether the inflammation responses in the entire brain can be mitigated through VEGF increase in the damaged SVZ environment. Specifically, as shown in FIG. 5A, SVZ of NP-C mice was injected with normal neural stem cells, VEGF-overexpressing neural stem cells, and VEGF-overexpressing vector twice a week for a total of 4 weeks.

(65) It was confirmed from the results in FIG. 5B that the reduced survival rates of neural stem cells were increased only when VEGF-overexpressing neural stem cells were injected into SVZ of NP-C mice (*p<0.05, ***p<0.001. n=6 per group).

(66) It was confirmed from the results in FIGS. 5C and 5D that the SVZ environment and the inflammation responses in the thalamus and cortex of NP-C mice were reduced only when VEGF-overexpressing neural stem cells were injected (GFAP: astrocyte, Iba-1: microglia. **n=6 per group). It can be seen from the above results that the injection of normal neural stem cells is insufficient to increase the reduced VEGF in SVZ of NP-C mice, and VEGF-overexpressing vector is also insufficient to mitigate the inflammation responses since the vector cannot increase VEGF specifically to neural stem cells.

(67) Furthermore, the VEGF-overexpressing neural stem cells mitigated the abnormal migration of neural stem cells in SVZ of NP-C mice to the thalamus (FIGS. 5E and 5F, ***n=6 per group).

(68) It can be therefore seen that when VEGF-overexpressing neural stem cells capable of sufficiently restoring reduced VEGF were injected into SVZ in order to improve the SVZ environment of NP-C mice, the abnormal migration of neural stem cells can be reduced and the inflammation responses in the entire brain can be mitigated.

Example 7

Reduction of Cholesterol Accumulation Through Improvement of Damaged SVZ Environment of NP-C Mice

(69) For investigation whether the improvement of SVZ environment by the injection of VEGF-overexpressing neural stem cells into NP-C mice can reduce cholesterol and lipid accumulation, which is a major pathological pattern of NP-C mice, the cortex region of NP-C mice, which was injected with normal neural stem cells, VEGF-overexpressing neural stem cells, and VEGF-overexpressing vector was subjected filipin staining to investigate the cholesterol accumulation levels.

(70) As shown in FIGS. 6A and 6B, it was confirmed that the cholesterol accumulated in the cortex of NP-C mice with the SVZ environment injected with VEGF-overexpressing neural stem cells were reduced, and the lipid accumulation was also reduced (*p<0.05, **p<0.01, ***p<0.001, n=6 per group).

(71) It can be seen from the above results that the improvement of SVZ environment by the injection of VEGF-overexpressing neural stem cells can reduce the cholesterol and lipid accumulation in the entire brain.

Example 8

Improvement of Sensory and Motor Abilities Through Improvement of Damaged SVZ Environment of NP-C Mice

(72) It was investigated whether the improvement of SVZ environment by the injection of VEGF-overexpressing neural stem cells into NP-C mice can improve the reduced sensory and motor abilities of NP-C mice.

(73) Through hot-plate and tail-flick tests as sensory ability test, and open field, rota-rod, and beam tests as motor ability tests, shown in FIGS. 7A and 7B, it can be seen that the sensory and motor abilities of NP-C mice with SVZ environment injected with VEGF-overexpressing neural stem cells were improved (*p<0.05. **p<0.01, ***p<0.001, n=10-12 per group). Furthermore, it was confirmed that the survival rate of NP-C mice was increased (FIG. 7C **p<0.01, n=10-12 per group).

(74) It can be seen from the above results that the improvement of SVZ environment by the injection of VEGF-overexpressing neural stem cells can reduce the cholesterol and lipid accumulation in the entire brain, thereby improving sensory and motor abilities and increasing the survival rate.

(75) It can be therefore seen from the above results that the improvement of SVZ environment can affect the entire brain; especially regarding the reduced VEGF in SVZ in NP-C mice, the SVZ environment can be improved only when VEGF is increased specifically to neural stem cells; and such the improvement of SVZ environment can improve the inflammation responses, cholesterol and lipid accumulation, and sensory and motor abilities in the entire brain.

Example 9

Improvement of Cerebellar Environment Through Improvement of Damaged SVZ Environment of NP-C Mice

(76) It was investigated whether the improvement of SVZ environment by the injection of VEGF-overexpressing neural stem cells into NP-C mice can also improve the cerebellar environment of NP-C mice. In addition, the improvement effects of cerebellar environment by such SVZ correction was compared with effects of direct transplant of VEGF microspheres into the cerebellum.

(77) It was confirmed from FIG. 8A that Purkinje neurons (PNs) reduction was mitigated in NP-C mice with SVZ environment injected with VEGF-overexpressing neural stem cells. Such the injection of VEGF-overexpressing neural stem cells showed an excellent mitigation effect in more regions of the cerebellum than the direct transplant injection of VEGF microspheres into the cerebellum. As shown in FIG. 8B, the cerebellar inflammation responses were reduced more effectively in NP-C mice injected with VEGF-overexpressing neural stem cells, and as shown in FIG. 8C, there was also a more excellent effect in the reduction of cerebellar lipid accumulation (*p<0.05. n=3 per group).

(78) As shown in FIG. 8D, the SVZ correction by the injection of VEGF-overexpressing neural stem cells improved the motor function more effectively than the direct transplant of VEGF microspheres (*p<0.05. n=7 per group).

(79) Therefore, the increase of VEGF specific to neural stem cells in SVZ of NP-C mice can also improve the cerebellar environment through the improvement of SVZ environment, and this indicates that the improvement of SVZ environment is important in alleviating the pathogenesis of NP-C mice. It could be especially confirmed that the improvement of SVZ environment can alleviate pathogenesis more effectively than the direct increase of cerebellar VEGF.