KR20170024873A - Pharmaceutical composition for prevention or treatment of Gaucher disease containing mesenchymal stem cells - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a Gaucher disease, comprising mesenchymal stem cells, or a stem cell therapeutic agent for treating a Gaucher disease. The mesenchymal stem cell or a mesenchymal stem cell-derived M-CSF of the present invention has an effect of promoting autoreplication, proliferation and differentiation of neural stem cells, thereby being able to be usefully used for preventing or treating the Gaucher disease.

Description

[0001] The present invention relates to a pharmaceutical composition for prevention or treatment of Gaucher disease comprising mesenchymal stem cells,

The present invention relates to a pharmaceutical composition for preventing or treating a Gaucher disease comprising mesenchymal stem cells, or a stem cell therapeutic agent for treating Gaucher disease.

Gaucher disease is an autosomal recessive lysosomal disease caused by a mutation of glucocerebrosidase gene (GBA). Gaucher disease is caused by a genetic deficiency of enzyme GCase. The mutation of GBA, a gene encoding GCase, deficiency of glucosylceramide (GluCer) degradation, and then GluCer accumulates in liver, spleen, bone marrow, CNS, And fatal neurological symptoms. Clinically, the two main categories of Gaucher disease are non-neuropathic and neuropathic Gaucher disease, which can be further subdivided into three types. Type 1 Gaucher disease is the most common form of disease and includes non-neuropathic Gaucher disease. Type 2 and Type 3 Gaucher disease include acute and subacute neuropathic Gaucher disease. In patients with neuropathic Gaucher disease, the toxic effects of GluCer cause nervous necrosis of the nervous system and neurological deterioration. However, little is known about the molecular response to these nerve necrosis. As a treatment method for type 1 Gaucher disease, enzyme replacement therapy is widely practiced. However, there is no treatment method for type 2 and type 3 Gaucher disease, which is more fatal and mortality rate than type 1 Gaucher disease, and a new therapeutic agent .

On the other hand, mesenchymal stem cells are cells of stromal origin and have self-renewal characteristics and can be differentiated into various cell lines. These mesenchymal stem cells can be isolated from umbilical cord blood, Wharton's jelly, bone marrow, placenta, or adipose tissue and are therefore more suitable for cell therapy than other types of stem cells that can cause ethical problems Do. Another characteristic of mesenchymal stem cells is their homing ability to the site of inflammation. It is known that the regression mechanism of mesenchymal stem cells arises from the migration of stem cells expressing its receptor CXCR4 into damaged tissue by SDF-1 (CXCL12) expressed in damaged tissue or bone marrow. Due to this regression, researches on the use of mesenchymal stem cells as gene therapy and drug delivery systems themselves have been actively carried out. In particular, it is known that when the drug is transferred using the regression to the inflammation site, the drug is delivered only to the inflammation site, thereby minimizing adverse effects such as damage to other organs.

Currently, the efficacy of mesenchymal stem cells in inflammatory diseases such as acute and chronic bacterial inflammatory diseases, cystic fibrosis and septicemia has been proved and its utilization has attracted attention, but there is no relation with the Gaucher disease.

Thus, there is an urgent need for new therapies and stem cell therapies that effectively treat Gaucher's disease and do not cause resistance.

The present inventors confirmed that mesenchymal stem cells have an effect of promoting autoreplication, differentiation and proliferation of neuroblastoma stem cells, and have found that M-CSF (macrophage colony stimulating factor), which is a soluble factor secreted from mesenchymal stem cells, The present invention has been completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating a Gaucher disease comprising mesenchymal stem cells.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating Gaucher's disease, which comprises an mesenchymal stem cell-derived macrophage colony stimulating factor (M-CSF) as an active ingredient.

Another object of the present invention is to provide a stem cell treatment agent for treating Gaucher disease comprising mesenchymal stem cells.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating a Gaucher disease comprising mesenchymal stem cells.

In addition, the present invention provides a pharmaceutical composition for preventing or treating Gaucher disease, which comprises an mesenchymal stem cell-derived macrophage colony stimulating factor (M-CSF) as an active ingredient.

The present invention also provides a stem cell treatment agent for treating Gaucher disease comprising mesenchymal stem cells.

The mesenchymal stem cell or mesenchymal stem cell-derived M-CSF of the present invention has an effect of promoting autoreplication, proliferation and differentiation of neural stem cells, and thus can be usefully used for prevention or treatment of Gaucher disease.

FIG. 1 is a graph showing the results of measurement of GCase activity in NSC (neuronal stem cell) of a Gaucher mouse model. All data are expressed as mean ± SEM.
2 is a graph showing the results of confirming the GluCer level in the NSC of the Gaucher mouse model. All data were expressed as mean ± SEM (* P < 0.05).
FIG. 3 is an experimental design for verifying the effect of BM-MSC (bone marrow-mesenchymal stem cell) on the self-renewal and proliferation of NSC.
Fig. 4 is a graph showing the results of analysis of NS (neurosphere) formation in an indirect co-culture of BM-MSC and NSC (scale bar, 1 mm).
FIG. 5 is a chart (scale bar, 20 mm) showing the effect of NSC proliferation enhancement upon indirect co-culture of BM-MSC and NSC by immunofluorescence detection.
FIG. 6 shows the results of confirming the relationship between the effect of enhancing NSC proliferation of BM-MSC and GCase activity. All data were expressed as mean ± SEM (* P <0.05, ** P <0.01, *** P <0.005).
FIG. 7 shows an experimental design for analyzing the effect of BM-MSC on NSC differentiation.
FIG. 8 is a graph showing quantitative analysis of fluorescence images and cells (scale bar, 50 mm) of βIII-tubulin-positive cells, which are markers of neurons, for analyzing the effect of BM-MSC on NSC differentiation.
FIG. 9 is a fluorescence image (scale bar, 50 mm) of βIII-tubulin-positive cells, which is a marker of neurons, for the analysis of the effect of BM-MSC on NSC differentiation.
FIG. 10 is a graph showing quantitative analysis of fluorescence images and cells (scale bar, 50 mm) of MBP-positive cells, which are markers of astrocytic cells, for the purpose of analyzing the effect of BM-MSC on NSC differentiation.
FIG. 11 is a graph showing quantitative analysis of fluorescence images and cells (scale bar, 50 mm) of GFAP-positive cells, which are markers of spiral cells, for the analysis of the effect of BM-MSC on NSC differentiation.
Fig. 12 is a view showing the influence of the BM-MSC on the morphogenesis of neurons. All data were expressed as mean ± SEM (* P <0.05, *** P <0.005).
Figure 13 is an analysis of the relative expression patterns of 50 different BM-MSC-derived cytokines.
FIG. 14 shows qRT-PCR results of M-CSF, IL-1ra, MCP-1, MMP-2 and MMP-3 which are BM-MSC derived cytokines in NSC.
FIG. 15 shows the results of ELISA analysis of the expression of M-CSF in indirectly co-cultured NSC with BM-MSC (* P <0.05).
FIG. 16 is a chart for confirming NS-forming ability of M-CSF (§§P <0.005, #P <0.05).
FIG. 17 is a graph showing the effect of promoting NSC proliferation of M-CSF (§P <0.05, #P <0.05).
FIG. 18 is a chart for confirming the NSC differentiation promoting effect of M-CSF. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating a Gaucher disease comprising mesenchymal stem cells.

In the present invention, "Gaucher disease" is a genetic disease caused by mutation of glucocerebrosidase gene (GBA), and Gaucher disease is caused by genetic deficiency of enzyme GCase. In the present invention, the Gaucher disease includes both non-neuropathic and neuropathic Gaucher disease, but preferably refers to a neuropathic Gaucher disease.

The term "mesenchymal stem cell" in the present invention refers to an allogeneic precursor cell before differentiation into specific organs such as bone, cartilage, fat, tendon, nerve tissue, fibroblast and muscle cell. In the present invention, the mesenchymal stem cells are contained in the composition in an undifferentiated state. The mesenchymal stem cells of the present invention may be derived from bone marrow, umbilical cord blood, Wharton's jelly, placenta and adipose tissue, but are preferably derived from bone marrow.

The mesenchymal stem cells of the present invention include macrophage colony stimulating factor (M-CSF). M-CSF is a cytokine that is a soluble factor secreted from mesenchymal stem cells.

The mesenchymal stem cell or mesenchymal stem cell-derived M-CSF of the present invention is characterized by restoring the self-renewal ability of neuronal stem cells (NSC) and promoting proliferation and differentiation .

The term "self-renewal ability" in the present invention means that a stem cell undergoes cell division to produce another identical stem cell. The stem cells produced in this case have the same self-renewal and differentiation ability as the inherent characteristics of the stem cells.

In the present invention, the term "proliferation" means that cells undergo cell division resulting in an increase in the number of cells.

The term "differentiation " in the present invention means that a stem cell develops into a specific cell.

In the present invention, the term "neuronal stem cell" refers to a cell having a self-renewal ability and capable of differentiating into neurons, astrocytes, and oligodendrogens.

In addition, the present invention provides a pharmaceutical composition for preventing or treating Gaucher's disease, comprising an mesenchymal stem cell-derived macrophage colony stimulating factor (M-CSF) as an active ingredient.

The mesenchymal stem cells according to the present invention are not limited as long as they have pluripotency, and may be derived from adult cells such as mammals including humans, all known tissues derived from humans, cells and the like. For example, can be derived from bone marrow, umbilical cord blood, placenta (or placental tissue cells), fat (or adipose tissue cells), wharton's jelly and the like, preferably from bone marrow.

Methods for the isolation of mesenchymal stem cells are known in the art. For example, bone marrow, peripheral nerve blood, umbilical cord blood, periosteum, dermis, mesenchymal tissue and the like, and the isolated mesenchymal stem cells may be cultured as needed.

The mesenchymal stem cells of the present invention can be injected into a patient's body in a state where the cells are cultured alone or in an incubator. For example, Lyndal et al. (1989, Arch. Neurol. 46: 615-31) or Douglas conchiolca (Douglas Kondziolka, Pittsburgh, 1998). In addition to the mesenchymal stem cells, the formulation may contain conventional pharmaceutically acceptable carriers. In the case of injections, preservatives, analgesics, solubilizers or stabilizers may be included. In the case of topical preparations, bases, excipients, Preservatives, and the like.

The pharmaceutically acceptable carriers to be contained in the composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, no. The composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, preservatives, etc. in addition to the above components.

The composition of the present invention may be prepared in a unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in an oil or aqueous medium, and may additionally contain dispersing or stabilizing agents.

The composition of the present invention can be administered parenterally, intravenously, subcutaneously, intraperitoneally, or topically, and preferably can be administered directly to the affected site. Compositions for parenteral administration (e. G., Injections) of the compositions according to the present invention can be prepared by dispersing and / or dissolving a pharmaceutically acceptable carrier, e. G., Sterile purified water, buffer at about pH 7, or physiological saline, And if necessary, may contain conventional additives such as preservatives, stabilizers and the like.

In addition, the amount of mesenchymal stem cells injected in the present invention may be 10 ⁴ -10 ¹⁰ cells / sec, preferably 10 ⁵ -10 ⁹ cells / sec, and most preferably 5 × 10 ⁸ cells / cells / circuit, but is not limited thereto. In addition, the mesenchymal stem cell-derived M-CSF injected into the present invention may be administered at 50ng-500mg / cycle, preferably 10mg-200mg / cycle, but is not limited thereto. The dose may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient.

The present invention also provides a stem cell treatment agent for treating Gaucher disease comprising mesenchymal stem cells.

The term "cell therapeutic agent" in the present invention refers to a medicament (US FDA regulation) used for the purpose of treatment, diagnosis and prevention of cells and tissues prepared by separation, culture and special manipulation from a human, Diagnosis, and prevention through a series of actions such as alive, homologous or xenogeneic cell propagation, screening, or otherwise altering the biological characteristics of a cell.

As used herein, the term "prophylactic " means any action that inhibits or slows the progression of Gaucher disease by administration of the composition of the present invention.

As used herein, the term "treatment" refers to any action in which the administration of the composition of the present invention improves or alleviates the Gaucher disease.

Terms not otherwise defined herein have meanings as commonly used in the art to which the present invention belongs.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention and treatment of tuberculosis.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the following.

Manufacturing example  1 - Manufacture of Gaucher Mouse Model

Gba ^{flox / flox} ; nestin-Cre mice (hereinafter referred to as 'Gba ^{- / -} mice') were used as neuropathic Gaucher disease models (Enquist et al., 2007). To prepare Gba ^{- / -} mice, Gba ^{flox / flox} mice were ^treated with normal control Gba ^{flox / +} ; nestin-Cre mice (hereinafter referred to as &quot; Gba ^+/- mice &quot;). Genomic DNA was extracted from the tail for genotyping of mice after mating and PCR was performed. All the procedures were followed by protocols approved by the Kyungpook National University Institutional Animal Care and Use Committee. The manufactured Gaucher mouse model was temperature-regulated and housed in a room that was repeated 12 hours a day and night.

Manufacturing example  2 - Isolation and culture of neuronal stem cells (NSCs)

NSC was isolated from the cerebral cortex of the neonatal Gba ^+/- or Gba ^{- / -} mice that were prepared in the above-mentioned Preparation Example 1. After removing the cortex and held in a cooler with ice ^{Ca 2 + / Mg 2 + -free} Hank's balanced salt solution (Invitrogen, Carlsbad, CA, USA). Subsequently, the cortex was cultured in NSC culture medium (Dulbeccomcomodified Eaglemedium (DMEM) / F12 (Invitrogen), 1% N2 supplement (Invitrogen), 20 ng / ml epidermal growth factor (Peprotech, Rocky Hill, NJ, USA) Basic fibroblast growth factor (Peprotech)]. The cell suspension were inoculated with 40-m cell strainer 2 × 10 5 cells / well in each well of a filter made via (BDBiosciences, SanJose, CA, USA ) 6- well tissue culture plates (BD Biosciences). Inoculated NSCs proliferated in NSC culture medium and formed aggregates known as NS (neurosphere). Twice a day, half of the medium in each well was replaced with fresh NSC culture medium.

Manufacturing example  3 - BM-MCS (Bone-marrow mesenchymal  stem cell, Bone marrow origin Mesenchymal stem cells ), Culture

Four to six weeks old normal mice were dissected to obtain bone marrow of tibia and femur. A single-cell suspension was obtained from the bone marrow of the tibia and femur of the obtained mice using a 40-m cell strainer. Approximately 1 x 10 &lt; ⁶ &gt; cells were seeded at 25 cm ² containing MesenCult MSC Basal Medium and Mesenchymal Stem Cell Stimulatory Supplements (Stem Cell Technologies, Vancouver, BC, Canada) Sprayed in a flask. Cell culture was performed for one week and cells attached to the bottom surface were determined by BM-MSC and used in subsequent experiments.

Example  1 - NSC of manufactured mouse model GCase Identification of activity

In order to confirm whether deletion of the nestin-Cre-mediated GCase gene interferes with the activity of GCase, the activity of GCase was measured in NSC of Gba ^+/- mice and Gba ^{- / -} mice obtained in Preparation Example 2 and GluCer levels Respectively.

1.1 of NSC GCase Identification of activity

Enzyme activity assay was performed to measure GCase activity of NSC. NSC was dissolved in a homogenization buffer (50 mM HEPES (Invitrogen), 150 mM NaCl (Sigma), 0.2% Igepal CA-630 (Sigma), protease inhibitor (Calbiochem, San Diego, CA, USA). Since NSCs have been shown to promote the conversion of (7-nitro-2-1,3-benzoxadiazol-4-yl) -D-erythro (NBD) -C12-GluCer (AvantiPolarLipids, Alabaster, AL, USA) to NBD- The activity of Gcase was measured by the degree of action, which is shown in FIG. 1 (n = 5 / group).

As shown in Figure 1, GCase activity was reduced in Gba ^{- / -} NSC compared to Gba ^+/- NSC. Thus, it was confirmed that the NSC and Gba ^{- / -} mice obtained in the above Preparation Example were produced in accordance with the purpose of the present invention.

1.2 From NSC GluCer  Confirmation of the level

Lipid extraction and GluCer quantitation were performed to measure GluCer levels in NSC. 2 μl of the lipid extract dissolved in 0.2% Igepal CA-630 was dissolved in 2 μl of GluCer lysis buffer [0.2 M citrate-phosphate buffer, pH 4.5, 0.3 M NaCl, And incubated at 37 ° C for one hour. The cells were cultured in the presence of 50 ng / μl rhAC (human recombinant acid ceramidase), 2.5% GCase (Cerezyme, Genzyme Corporation, Cambridge, Mass., USA) The reaction was stopped by treating with 20 μl of NDA derivatization reaction mixture (25 mM borate buffer, pH 9.0, 2.5 mM NDA, 2.5 mM sodium cyanide). The reaction mixture was diluted 1: 3 with ethanol, incubated at 50 DEG C for 10 minutes, and then centrifuged at 13,000 x g for 5 minutes. After centrifugation, 30 μl of the supernatant was transferred to a glass sample vial and UPLC system was performed with 5 μl of the supernatant. A fluorescent sphingosine derivative was observed using a model 474 scanning fluorescence detector (Waters). The sphingosine peak was quantified by calculating the sphingosine standard curve using Waters Millennium software. Similar measurements were made in the GCase-deficient reaction mixture to calculate the final GluCer content of the sample, the intrinsic sphingosine and the ceramice signal, and corrected by subtracting it from the quantification results. The final quantification results are shown in Fig.

As shown in FIG. 2, it was confirmed that the level of GluCer in Gba ^{- / -} NSC was higher than that of Gba ^+/- NSC in the control group. From these results, it can be seen that GluCer accumulates in NSC due to the reduced activity of GCase identified in 1.1 above, thereby increasing the level of GluCer.

Example  2 - BM- MSC of Self-playing ability  And effects on proliferation

An experimental design to confirm whether BM-MSC has the effect of improving the self-renewal ability and proliferation of Gba ^{- / -} NSC is shown in FIG. As shown in FIG. 3, the numbers of NS formed and NSC proliferation were measured after indirect co-culture with BM-MSC at day 12 after NSC culture.

2.1 NSC and BM- MSC (Indirect co-culture)

After placing Millicell Hanging Cell Culture Inserts (Millipore, Billerica, MA, USA) with a pore size of 1.0 μm on the NCS, the BM-MSC prepared in Preparation Example 3 was impregnated at a density of 3 × 10 ⁴ cells / insert. In this process, there was no direct contact between the NSC and the BM-MSC. During the incubation period of 7 days after inoculation, half of the medium was replaced with fresh medium every 72 hours.

2.2 NS formation assay

To determine the effect of indirect co-culture of BM-MSC and NSC on NS formation, NS formation in NSC was analyzed. Specifically, the surviving cells were obtained after mechanically separating NS, and the viable cells were inoculated into 24-well plates (BDBiosciences) at a concentration of 1 × 10 ⁴ . The inoculated cells were indirectly co-cultured with BM-MSC. After indirect co-cultivation, newly formed NSs were counted in each well using an IX71 microscope (Olympus Co., Tokyo, Japan). The minimum diameter of the counted NS was 50 mm. The results of counting the number of formed NS are shown in Fig. 4 (n = 6 / group).

As, BM-MSC and Gba the indirect co-culture is not the control shown in Fig. 4 ^{- / -} the number of the NS in the NSC culture medium was low in NSC ^+/- Gba significantly as compared to the number of the NS in the culture medium, Gba ^{+ / -} The indirect co-culture of NSC and BM-MSC resulted in increased NS formation, thus confirming that BM-MSC induces NS formation in Gba ^+/- NSC culture (n = 6 / group).

2.3 NSC Proliferation Assay

The proliferation activity of NSCs was measured by immunocytochemistry using BrdU (5-bromo-2-deoxyuridine) to determine whether BM-MSC enhances NSC proliferation. NS-derived single-cell suspension to poly-l-ornithine (Sigma, St. Louis, MO, USA) and a density of 1 × 10 ⁴ cells / cm ² on glass coverslips coated with laminin (Invitrogen) Inoculated. After inoculation, cells were incubated with BM-MSC for 7 days, labeled with 10 μM BrdU (Sigma) and further cultured for an additional 12 hours. After removal of the labeling medium, cells were fixed with phosphate-buffered 4% (w / v) paraformaldehyde (Sigma) for 20 minutes at room temperature. For denaturation of nuclear DNA, the cells were incubated in 2 N HCl (Sigma) for 1 hour and then treated with 0.15 M sodium borate (Sigma) for 15 minutes. The treated cells were washed with PBS (phosphate-buffered saline, Invitrogen) and cultured with DAPI (4 ', 6-diamidino-2-phenylindole, Vector Laboratories Inc., Burlingame, CA, USA) Immunofluorescence was detected and the detection results are shown in Fig.

As shown in Figure 5, in both Gba ^{+ / -} and Gba ^{- / -} NSC cultures indirectly co-cultured with BM-MSC as compared to control cultures that were not incubated with BM-MSC, BrdU- Respectively. Therefore, it was confirmed that BM-MSC promotes NSC proliferation (n = 7 / group).

2.4 BM- MSC Of NSC proliferation and GCase  Identify the relationship of activity

The GCase activity was analyzed to confirm whether the effect of enhancing the NSC proliferation of the BM-MSC identified in 2.3 was mediated by the activity of GCase. The results are shown in FIG.

NS-derived single-cell suspension to poly-l-ornithine (Sigma, St. Louis, MO, USA) and a density of 1 × 10 ⁴ cells / cm ² on glass coverslips coated with laminin (Invitrogen) Inoculated. After inoculation, cells were incubated with BM-MSC for 7 days, and NSCs attached to cover slips were collected by dissolving in TrypLE ™ Select (ThermoFisher). The collected NSC was dissolved in a homogenization buffer (50 mM HEPES (Invitrogen), 150 mM NaCl (Sigma), 0.2% Igepal CA-630 (Sigma), protease inhibitor (Calbiochem, San Diego, CA, USA) . (NBD) -C12-GluCer (AvantiPolarLipids, Alabaster, AL, USA) to NBD-C12-ceramide The activity of GCase was measured according to the degree of action, which is shown in Fig. 6 (n = 3 / group).

As shown in FIG. 6, there was no change in GCase activity in both Gba ^{+ / -} and Gba ^{- / -} NSC indirectly co-cultured with BM-MSC compared to the control group not cultured with BM-MSC .

In other words, BM-MSC promotes the self-renewal and proliferation of Gba ^{- / -} NSC and this effect is secreted from BM-MSC in view of the fact that this effect was achieved by indirect co-culture without direct contact between BM-MSC and NSC Neurogenic &lt; / RTI &gt; water soluble factor.

Example  3 - BM- MSC Of NSC Differentiation

In order to confirm the effect of additional BM-MSC on NSC differentiation, differentiation analysis was carried out, and the experimental design for this was briefly shown in FIG.

3.1 Differentiation assay

Immunocytochemistry was used to determine the effect of BM-MSC on the differentiation of NSC into the major nervous system. Specifically, single-cell suspensions from NS were incubated in vitro To a glass cover slip coated with poly-1-ornithine and laminin, 1 x 10 ⁴ cells / cm ² At a density of And cultured for 7 days. The cover slip was incubated in Neurobasal-A medium supplemented with 100 U / ml penicillin / streptomycin, 2 mM l-glutamine, 10 mg / ml heparin, 2% B-27 supplement, and 3% fetal bovine serum All samples were purchased from Invitrogen. The culture was plated. The cells were then fixed with 0.1 M PBS containing 4% para-formaldehyde at room temperature for 15 minutes and then treated with 0.1% Triton X-100 (Sigma) in PBS for 5 minutes. Cells treated to block background signals were preincubated with PBS containing 0.4% Triton X-100 with 3% normal goat serum (Vector Laboratories Inc.) and 2% BSA (bovine serum albumin, Invitrogen) . For NSC differentiation analysis, the differentiated cultures were incubated with various primary antibodies overnight at 4 &lt; 0 &gt; C. Anti-GFP (Dako, Glostrup, Denmark) rabbit polyclonal antibody diluted 1: 1,000 as a marker of neurons was immunoprecipitated with anti-βIII-tubulin (Chemicon, Temecula, CA, USA) mouse monoclonal antibody diluted 1: Anti-MBP (Abcam, Cambridge, UK) rabbit polyclonal antibody diluted 1: 500 as a marker for astrocyte was used as a marker for oligodendrocytes. To visualize the primary antibody, an appropriate Alexa Fluor 488-conjugated secondary antibody (Molecular Probes, Carlsbad, Calif., USA) diluted 1: 1,000 was added to the culture and incubated at room temperature for 1 hour. Immunofluorescence in the cells was analyzed using a laser-scanning confocal microscope of the luoView SV1000 imaging software (Olympus FV1000, Olympus Co., Tokyo, Japan) or BX51 microscope (Olympus Co.) Representative fluorescence images and quantitative analysis are shown in Figures 8-11. Quantitative analysis results were expressed as the average ratio of marker-positive cells to DAPI-stained cells in each group.

As shown in FIGS. 8-11, three neural gene markers (? III-tubulin, MBP and GFAP) were detected in NSC cells, with or without BM-MSC, In the indirect co-cultured group, the expression of neural cell markers was significantly increased, confirming that BM-MSC improves the neural differentiation efficiency of NSC. On the other hand, as shown in FIG. 10, the differentiation rate of MBP-positive cells was decreased in the culture of Gba ^{- / -} NSC compared to the culture of Gba ^+/- NSC. In the case of indirect co-culture with BM-MSC, Respectively. However, as shown in Fig. 11, there was no significant change in the number of GFAP-positive cells in the Gba ^{- / -} NSC culture as compared to Gba ^+/- NSC. Therefore, it was confirmed that BM-MSC selectively promoted NSC differentiation and neuronal morphogenesis and proliferation.

3.2 Morphological analysis of differentiated neurons

The total outgrowth of differentiated neurons and the dendritic process of neurons were measured using MetaMorph software (Universal Imaging Corp., Downingtown, Pa., USA) for morphological analysis of differentiated neurons. I wrote. All recordings and MetaMorph analysis were performed in a blinded manner and the results are shown in FIG. At the time of recording, randomly selected cells were recorded at a minimum of 60 cells / disc (n = 5 different mouse-derived discs). As shown in FIG. 12, it was confirmed that the BM-MSC significantly increased the numerical increase and the total length of the dendrites of the differentiated neurons. In other words, it was confirmed that BM-MSC promotes morphogenesis of neurons.

Example  4 - BM- MSC  Identification of derived factors

In order to identify the water-soluble biologically active factor derived from BM-MSC, which is an effective ingredient for promoting self-renewal, proliferation and differentiation of NSCs in the indirect co-culture of BM-MSC and NSC,

4.1 Antibody-Based Mouse Cytokine Analysis

In order to identify the active components of BM-MSC, the relative expression patterns of 50 different cytokines were analyzed. Specifically, RayBio Mouse custom Cytokine Antibody Arrays (Raybiotech, Norcross, GA, USA) were used according to manufacturer's instructions for cell culture supernatant analysis obtained from co-culture experiments. The membranes were treated with 2 ml blocking buffer for 30 minutes at room temperature. (Conditioned) to samples co-cultured indirectly with BM-MSC (Gba ^+/- BM-MSC and Gba ^{- / -} + BM-MSC) or uncoagulated samples (Gba ^+/- and Gba ^{- / -} medium) was added at about 1 ml, followed by overnight incubation at 4 ° C. After decanting the sample, the membrane was washed with Buffer I and II with shaking at 120 rpm at room temperature. The membrane was then incubated overnight at 4 ° C with 1 ml of biotin-conjugated antibody (1: 250), washed, and incubated with 2 ml of horseradish peroxidase-conjugated streptavidin (1: 1000) Lt; / RTI &gt; and then further washed. Detection buffers C and D were used to visualize the spot. The membrane was wrapped in a plastic wrap and exposed to a Kodak X-Omat radiographic film (Kodak, Rochester, NY, USA) for 20 minutes and then the signal was detected using a film developer. Each film was scanned into an image processor and densitometric measurements were performed using an imaging densitometer (Bio-Rad, Hercules, Calif., USA). And then quantified with Bio-Rad analysis software. Densitometry measurements and statistical analyzes were performed on immunoblot and the results are shown in FIG.

As shown in Fig. 13, the cytokines that were increased in the GBA ^{- / -} NSC conditioned medium indirectly co-cultured with BM-MSC compared with those not indirectly co-cultured with BM-MSC were M-CSF, IL-1ra , MCP-1, MMP-2 and MMP-3. Therefore, it can be seen that M-CSF, IL-1ra, MCP-1, MMP-2 and MMP-3 are associated with the self-renewal ability of Gba ^{- / -} NSC.

4.2 qPCR  analysis

Analysis of mRNA levels for BM-MSC-derived cytokines M-CSF, IL-1ra, MCP-1, MMP-2 and MMP-3 confirmed to be related to the autoregulatory ability of Gba ^{- / -} QPCR was performed.

Specifically, RNA was extracted from the cell lysates of Gba ^+/- and Gba ^{- / -} NSC using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). cDNA (complementary DNA) was synthesized from 5 mg of total RNA using cDNA Synthesis Kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's instructions. The reaction was allowed to proceed for 1 hour at 42 DEG C, and cDNA synthesis was stopped at 70 DEG C for 10 minutes. QPCR analysis of the synthesized cDNA was performed using Corbett research RG-6000 thermal cycler (Corbett Life Science, Sydney, Australia). The amplification was repeated 40 times at 95 ° C for 10 seconds, 58 ° C for 15 seconds, and 72 ° C for 20 seconds. The primers used for amplification are as follows: Mcsf (5'-GCA CAG GCT GGT GAA TGA C-3 'and 5'-CAC CTG TCT GTC CTC AT-3'), genes (5'-CTG TCT TCC ACC TGC TG- -CCC CGT GGA TGC CCA AG-3 '), MCP-1 gene (5'-CAG TTA ATG CCC CAC TC-3 'and 5'-CTT ATT GGG GTC AGC AC-3'), Mmp2 gene (5'-CGA TGC TGA TAC TGA- (5'-AAG GAG GCA GCA GAG AA-3 'and 5'-TAG GAT GAG CAC ACA AC-3') and the GAPDH gene (5'-AGC CTC AAG ATC ATC AGC-3 'And 5'-GCA GGT TTT TCT AGA CGG -3'). The results of qPCR execution are shown in Fig. 14 (n = 4 / group).

And as compared with the control group ^+/- Gba NSC as shown in 14, Gba ^{- / -} expression of the gene in Mcsf NSC had significantly decreased as compared with in the ^{Gba +/- NSC Gba - / - IL} -1ra in NSC , MCP-1, Mmp2, and Mmp3 mRNA levels were increased. The decrease in the expression of M-CSF may predict the impairment of autoregulatory ability in Gba ^{- / -} NSC. Thus, M-CSF secreted by BM-MSC is a potential candidate molecule for mitigating autoregulation deficiency.

4.3 ELISA analysis

The concentration of M-CSF was measured by an enzyme-linked immunosorbent assay (ELISA) in order to confirm the secretion of M-CSF identified as a potential candidate molecule in Section 4.2 above. Specifically, the Quantikine Mouse M-CSF ELISA Kit (R & D Systems) and the licensed M-CSF standard were used to measure the concentration of M-CSF. Follow manufacturer's instructions. ELISA analysis was repeated twice for each standard and experimental sample and the average of the results was shown in FIG.

As shown in FIG. 15, the M-CSF protein level was significantly improved as compared with the group not co-cultured with Gba ^{- / -} NSC indirectly co-cultured with BM-MSC, and Gba ^{+ / -} NSC, the level of M-CSF protein was significantly decreased in Gba ^{- / -} NSC not co-cultured with BM-MSC. Therefore, M-CSF, a water-soluble factor derived from BM-MSC, was identified as a candidate molecule capable of improving the self-renewal ability of NSC.

Example  5-M- CSF Check the effect of

To confirm the neurogenic potential of M-CSF identified in Examples 3 and 4, the recombinant murine M-CSF (R & D Systems, Minneapolis, MN, USA) The effect was confirmed.

In order to confirm the self-renewal ability of M-CSF, NS formation ability was measured at different concentrations and the same procedure as in 2.2 was performed. The results are shown in Fig. If the self-regenerative capacity of NS was significantly decreased, M-CSF is treated with 10 and 50 ng / ml concentration of Gba ^{- -} even compared to the Gba ^+/- NS as shown in 16, Gba ^{- / / -} NSC showed an increase in NS formation. M-CSF at a concentration of 10 ng / ml was used for the subsequent experiments.

To confirm the proliferation promoting effect of M-CSF, the ratio of BrdU-positive cells was measured by the proliferation assay as described in 2.3 above, and the results are shown in FIG. As shown in Fig. 17, it was confirmed that the proportion of BrdU-positive cells in Gba ^{- / -} NSC was increased by M-CSF.

In order to confirm the effect of M-CSF on NSC differentiation, NSC differentiation was observed by the same method as in Example 3, and the results are shown in FIG. When treated with M-CSF as shown in FIG. 18 Gba ^{- / -} has been NSC differentiation is promoted and the total length of the projection is significantly increased, compared to ^+/- Gba NSC, Gba ^{- / -} NSC of neurons Showed significant decrease in the total length and branching process.

Therefore, as shown in FIG. 16 to FIG. 18, M-CSF promotes the self-renewal, proliferation and differentiation of NSCs and plays an important role in the morphogenesis of NSCs. Therefore, M-CSF is usefully used as a pharmaceutical composition for treating Gaucher disease Respectively.

Formulation example  - Manufacture of medicines

Sanje  Produce

5 x 10 &lt; ⁸ &gt; cells of mesenchymal stem cells or 100 mg of M-CSF

Lactose 100mg

10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Injection preparation

5 x 10 &lt; ⁸ &gt; cells of mesenchymal stem cells or 100 mg of M-CSF

Sterile sterilized water for injection

pH adjuster

(2 ml) per 1 ampoule in accordance with the usual injection method.

Liquid  Produce

5 x 10 &lt; ⁸ &gt; cells of mesenchymal stem cells or 100 mg of M-CSF

Sugar 20g

20g per isomer

Lemon incense quantity

Purified water was added to the total volume of 100 ml. The above components are mixed according to a usual method for producing a liquid agent, and then filled in a brown bottle and sterilized to prepare a liquid agent.

Claims (10)

A pharmaceutical composition for preventing or treating a Gaucher disease comprising mesenchymal stem cells.
The pharmaceutical composition according to claim 1, wherein the mesenchymal stem cell comprises a macrophage colony stimulating factor (M-CSF).
The pharmaceutical composition for preventing or treating Gaucher disease according to claim 1, wherein the mesenchymal stem cells are derived from bone marrow.
The pharmaceutical composition for preventing or treating Gaucher disease according to claim 1, wherein the Gaucher disease is neuropathic Gaucher disease.
The pharmaceutical composition for preventing or treating Gaucher disease according to claim 1, wherein the mesenchymal stem cell regenerates the self regenerating ability of a neuronal stem cell (NSC).
The pharmaceutical composition for preventing or treating Gaucher disease according to claim 1, wherein the mesenchymal stem cell promotes proliferation of neuronal stem cells (NSCs).
The pharmaceutical composition for preventing or treating Gaucher disease according to claim 1, wherein the mesenchymal stem cells promote the differentiation of neuronal stem cells (NSCs).
A pharmaceutical composition for preventing or treating Gaucher's disease, comprising an mesenchymal stem cell-derived macrophage colony stimulating factor (M-CSF) as an active ingredient.
A stem cell therapeutic agent for treating Gaucher disease comprising mesenchymal stem cells.
10. The stem cell treatment agent for treating Gaucher disease according to claim 9, wherein the mesenchymal stem cell comprises a macrophage colony stimulating factor (M-CSF).

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