Method for inducing differentiation into neural cells

Abstract

a) 60.0 to 90.0 vol% basal medium; 10.0 to 40.0 vol% physiological saline, and 1.0 to 100.0 ng / mL EGF, 0.2 to 20.0 ng / mL of FGF-2, 0.2 to 20.0 ng / mL PDGF, 0.5 to 8.0 mM magnesium ascorbyl phosphate; Cultivate mesenchymal stem cells for 1 to 10 days in a stem cell culture medium containing the following: b) Then, the protein components of the culture solution obtained by the above culture are precipitated with an organic solvent; c) The precipitate is separated and dissolved in physiological saline or a basal medium. Using a medium containing a cell-produced protein (CPPs) composition, 3 × 10 3 ~2×10 4 The present invention provides a method for inducing differentiation into nerve cells, in which mesenchymal stem cells are differentiated into nerve cells by culturing mesenchymal stem cells seeded at 1000 cells / mL for 1 to 3 days.

Description

[Technical field]

[0001] The present invention relates to a method for inducing differentiation into nerve cells, and in particular provides a method for inducing differentiation of mesenchymal stem cells into nerve cells using a cell-produced protein composition (CPPs composition) obtained by culturing mesenchymal stem cells in a specified stem cell culture medium. [Background technology]

[0002] In recent years, somatic stem cells, which can be extracted from adult tissues, have been attracting attention. Somatic stem cells exist in adult skin, bone marrow, fat, etc., and can proliferate in a test tube in an undifferentiated state. They can also differentiate into cells of specific tissues.

[0003] Among tissue regeneration, nerve regeneration is one of the most important. Nerve cells proliferate and distribute in the early stages of development to form the nervous system, but after this, they have little self-regenerative ability, and it is believed that once nerve tissue is damaged, it cannot be repaired. Therefore, there is a demand for a technology that can repair damaged nerve tissue by transplanting nerve cells differentiated from stem cells.

[0004] For example, Patent Document 1 describes inducing differentiation of adipose tissue stromal cells to express markers for immature neural cells, and describes a method of inducing differentiation that includes the steps of collecting adipose tissue stromal cells from adipose tissue and culturing the adipose tissue stromal cells in a medium containing dcAMP or forskolin.

[0005] Patent document 2 describes the differentiation of neural stem cells into neurons using TAT-VHL (157-171), a peptide synthesized by binding a VHL oligopeptide (amino acid sequence of SEQ ID NO: 1: TLKERCLQVVRSLVK), an oligopeptide having a partial amino acid sequence of the von Hippel-Lindau (VHL) protein, to a fusion protein TAT (amino acid sequence of SEQ ID NO: 2: YGRKKRRQRRRD), which has the ability to easily penetrate cell membranes. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2013-63088 A [Patent Document 2] JP 2005-330206 A Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, nerve cells do not divide or proliferate in principle, and therefore, there is a need for a technique to efficiently and safely obtain nerve cells from mesenchymal stem cells for treatments such as transplantation. In addition, in general, fetal bovine serum (FBS) is used in neural cell differentiation induction media, which has a strong effect of differentiating mesenchymal stem cells into neural cells and does not contain any cytotoxic components. FBS is a component derived from a different animal, and has the risk of antigenicity and zoonotic virus infection, making it difficult to use in the above-mentioned administration / transplantation destinations.

[0008] In view of the above circumstances, the present inventors have conducted further investigations and have discovered a new method for inducing differentiation into neurons using cell producing proteins (CPPs) containing neuronal differentiation-inducing components, which can be used in place of a neuronal differentiation-inducing medium and can be prepared from mesenchymal stem cells, thereby completing the present invention.

[0009] The present invention has been made in view of the above-mentioned circumstances, and aims to induce differentiation of mesenchymal stem cells into nerve cells. More specifically, the present invention aims to provide a method for inducing differentiation into nerve cells, which enables differentiation of mesenchymal stem cells into nerve cells using components obtained from a culture solution of mesenchymal stem cells. [Means for solving the problem]

[0010] The present invention has been made based on the above findings and has an object to advantageously solve the above problems. A first aspect of the present invention is a) 60.0 to 90.0 vol% basal medium; 10.0 to 40.0 vol% physiological saline, and 1.0 to 100.0 ng / mL EGF, 0.2 to 20.0 ng / mL of FGF-2, 0.2 to 20.0 ng / mL PDGF, 0.5 to 8.0 mM magnesium ascorbyl phosphate; Cultivate mesenchymal stem cells for 1 to 10 days in a stem cell culture medium containing the following: b) Then, the protein components of the culture solution obtained by the above culture are precipitated with an organic solvent; c) The precipitate is separated and dissolved in physiological saline or a basal medium. Using a medium containing a cell-produced protein (CPPs) composition, 3 × 10 3 ~2×10 4 This is a method for inducing differentiation into nerve cells, in which mesenchymal stem cells are differentiated into nerve cells by culturing mesenchymal stem cells seeded at 1000 cells / mL for 1 to 3 days. According to such a method for inducing differentiation into nerve cells, it is possible to induce differentiation of mesenchymal stem cells into nerve cells by using components obtained from mesenchymal stem cells, without using a nerve cell differentiation-inducing medium.

[0011] In the above aspect, the CPPs composition may include collagen. The present inventors have also newly discovered that collagen contained in the CPPs composition has properties that contribute to inducing differentiation of stem cells into nerve cells. The collagen may also include type I collagen and / or procollagen.

[0012] In the above aspect, The medium containing the CPPs composition may further contain TAT-VHL. In this way, it is possible to more efficiently induce the differentiation of mesenchymal stem cells into neural cells. Effect of the Invention

[0013] According to the present invention, it is possible to differentiate mesenchymal stem cells into nerve cells using components obtained from mesenchymal stem cells without using a nerve cell differentiation-inducing medium. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the results of Example 2. [Diagram 2] FIG. 2 shows the results of Example 2. [Diagram 3] FIG. 3 shows the results of Example 2. [Figure 4] FIG. 4 shows the results of Example 3. [Diagram 5] FIG. 5 shows the results of Example 3. [Figure 6] FIG. 6 shows the results of Example 4. [Figure 7] FIG. 7 shows the results of Example 5. [Figure 8] FIG. 8 shows the results of Example 6. [Figure 9] FIG. 9 shows the results of Example 6. [Figure 10] FIG. 10 shows the results of Example 6. [Figure 11] FIG. 11 shows the results of Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Hereinafter, an embodiment of the present invention will be described in detail. The method for inducing differentiation into nerve cells according to the first aspect of the present invention comprises: a) 60.0 to 90.0 vol% basal medium; 10.0 to 40.0 vol% physiological saline, and 1.0 to 100.0 ng / mL EGF, 0.2 to 20.0 ng / mL of FGF-2, 0.2 to 20.0 ng / mL PDGF, 0.5 to 8.0 mM magnesium ascorbyl phosphate; Cultivate mesenchymal stem cells for 1 to 10 days in a stem cell culture medium containing the following: b) Then, the protein components of the culture solution obtained by the above culture are precipitated with an organic solvent; c) The precipitate is separated and dissolved in physiological saline or a basal medium. Using a medium containing a cell-produced protein (CPPs) composition, 3 × 10 3 ~2×10 4 The method is characterized by differentiating mesenchymal stem cells into neural cells by culturing mesenchymal stem cells seeded at 1000 cells / mL for 1 to 3 days.

[0016] The present inventors have newly discovered that a cell-produced protein (CPPs) composition, which will be described later, can be obtained from mesenchymal stem cells cultured using the above-mentioned stem cell culture medium. As the basal medium, IMDM, DMEM, and α-MEM can be used. In the composition of the above-mentioned medium for stem cell culture, the purpose of adding physiological saline is to lower the Ca concentration in the above-mentioned basal medium to 10-40%. Therefore, when the Ca concentration of the basal medium used is 40-50 μg / mL, there is no need to dilute it with physiological saline.

[0017] Regarding the supplemental factors added to the above-mentioned stem cell culture medium, EGF is epidermal growth factor, FGF-2 is fibroblast growth factor 2, and PDGF is platelet-derived growth factor. The above-mentioned supplemental factors are generally used as supplemental factors to basal media for the purpose of cell proliferation, etc. In the medium for stem cell culture according to the first aspect of the present invention, EGF 1-10ng / mL, FGF-2, 0.5 to 5.0 ng / mL; PDGF 0.5-5.0ng / mL, When the amount of the CPPs added is within this range, the CPPs composition can be produced more efficiently.

[0018] In addition, in the above-mentioned stem cell culture medium, 0.1 to 10.0 μg / mL transferrin, and / or 0.2–20.0 μg / mL insulin, and / or 0.1-3.0ng / mL sodium selenite It is preferable to further add the above in terms of enhancing the proliferation ability of the cells.

[0019] Specific methods for preparing the stem cell culture medium include, for example, From the 500 mL of IMDM, remove 50 mL to 200 mL, add an equal amount of saline to make 500 mL, add 5 mL of antibiotics, moreover, EGF 1-10ng / mL, FGF-2, 0.5 to 5.0 ng / mL; PDGF 0.5-5.0ng / mL, Transferrin 0.1 to 10.0 μg / mL, Insulin 0.2-20.0μg / mL, Sodium selenite 0.1-3.0ng / mL, Magnesium ascorbyl phosphate, 0.5 to 8.0 mM; It can be prepared by adding in the range.

[0020] (Cell-Produced Proteins (CPPs) Composition) The CPPs composition is The mesenchymal stem cells are cultured in the above-mentioned stem cell culture medium for 1 to 10 days, Then, the protein components of the culture solution obtained by the above culture are precipitated with an organic solvent, The precipitate is separated and dissolved in physiological saline or basal medium. The present invention is characterized by being a CPPs composition for inducing differentiation of mesenchymal stem cells into neural cells, which is obtained by the above procedure.

[0021] The CPPs composition may contain type I procollagen and hyaluronic acid, and calcium may be bound to the type I procollagen. The CPPs composition contains, for example, 30 to 100 μg / mL of type I procollagen and 49 to 140 μg / mL of hyaluronic acid. The binding ratio of type I procollagen to calcium is, for example, 30 to 45%, and preferably 35 to 42%.

[0022] In the first embodiment, the mesenchymal stem cells used in the preparation of the CPPs composition may be bone marrow-derived mesenchymal stem cells, adipose-derived stem cells (ASC), peripheral blood-derived mesenchymal stem cells, umbilical cord Wharton's Jelly-derived mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, etc. As the basal medium, IMDM, DMEM, and α-MEM may be used. The culture period is 1 to 10 days, preferably 3 to 4 days.

[0023] In the first embodiment, in the step of "collecting the precipitated portion and dissolving it in physiological saline or a basal medium" following the step of "precipitating the protein component with an organic solvent", the protein component precipitated with the organic solvent may be suspended in hydrochloric acid (e.g., 1N HCl) at pH 1, vortexed (e.g., for about 10 minutes), and then neutralized (e.g., with 1.2N NaOH) to simultaneously perform virus inactivation treatment. In this case, the neutralized solution is centrifuged, and the resulting supernatant is used as the CPPs composition.

[0024] (Method for inducing differentiation of mesenchymal stem cells into neural cells) The present inventors have newly discovered that mesenchymal stem cells can be induced to differentiate into neural cells by culturing them under specific conditions using the above-mentioned CPPs composition. The differentiation induction method according to the present invention involves using a medium containing a CPPs composition to incubate 3×103 ~2×10 4 The mesenchymal stem cells are seeded at 5×10 cells / mL and cultured for 1-3 days to differentiate into neural cells. 3 ~1×10 4 It is preferable to culture mesenchymal stem cells seeded at 1000 cells / mL for 1 to 3 days, as this provides a higher efficiency of differentiation induction. According to such a method for inducing differentiation into nerve cells, it is possible to induce differentiation of mesenchymal stem cells into nerve cells by using components obtained from mesenchymal stem cells, without using a nerve cell differentiation-inducing medium. The CPPs composition preferably has a protein concentration of 80 to 120 μg / mL, and is contained in a ratio of medium:CPPs composition=1:1 / 8 to 1, that is, 12.5 to 50.0 vol % relative to the medium. In the first aspect, the mesenchymal stem cells may be derived from bone marrow, fat, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

[0025] In the first aspect, The CPPs composition may include collagen. The collagen may also include type I collagen and / or procollagen. Type I collagen is a type of collagen that is found in large amounts in skin and bones, and is a heterotrimer consisting of two α1(I) chains (α1 collagen chain) and one α2(I) chain (α2 collagen chain). Procollagen (collagen precursor) is a precursor in the collagen synthesis process, and has procollagen peptides at the N-terminus and C-terminus of the peptide chain. In the past, atelocollagen (collagen with cleaved telopeptides) has been used to coat culture vessels in order to increase cell adhesion to the vessels, but collagen has never been used as a differentiation inducer. Thus, it was not known that collagen has the properties of a differentiation-inducing factor that contributes to the induction of neural differentiation of stem cells under certain conditions, and this property was newly discovered by the present inventors. Furthermore, since the collagen contained in the CPPs composition has not been subjected to a treatment that would cleave the telopeptides (e.g., pepsin treatment) in the production process of the CPPs composition, it is highly likely that the collagen contained in the CPPs composition is mature collagen (native collagen / topocollagen) and procollagen.

[0026] In the first aspect, The medium containing the CPPs composition may further contain TAT-VHL. As described in JP 2005-330206 A, TAT-VHL is a peptide synthesized by binding a VHL oligopeptide (amino acid sequence of SEQ ID NO: 1: TLKERCLQVVRSLVK), an oligopeptide having a partial amino acid sequence of the von Hippel-Lindau (VHL) protein, to the fusion protein TAT (amino acid sequence of SEQ ID NO: 2: YGRKKRRQRRRD), which has the ability to easily penetrate cell membranes. By using a CPPs composition in combination with TAT-VHL, it is possible to induce differentiation of mesenchymal stem cells into neural cells with higher efficiency. TAT-VHL is preferably contained at 0.34 to 3.40 ng / mL in the medium containing the CPPs composition. EXAMPLES

[0027] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0028] Example 1 Preparation of stem cell culture medium and CPPs composition 5 mL of Antibiotic-Antimycotic Mixed Stock Solution (Nacalai Tesque, product number: 02892-54) was added as an antibiotic to 500 mL of BSCM-PL2 medium for stem cell proliferation (Bio Mirai Kobo) to prepare a stem cell culture medium for creating a CPPs composition. Next, 4 × 10 adipose-derived stem cells (ASCs) (manufactured by Lonza, product number: PT-2501) belonging to mesenchymal stem cells (MSCs) were 5 2 × 10 cells / mL were cultured in a T-75 flask (SARSTEDT, product number: 83.3911.002) using BMCM-PL1 medium (Bio Mirai Kobo) containing 2% human serum until the cells reached 90% confluence, and then detached with trypsin-EDTA solution (Nacalai Tesque, product number: 32777-44). Then, 2 × 10 cells / mL were cultured in a Hyper Flask (Corning, Inc.) until the cells reached 90% confluence. 6 The cells were seeded at 200 cells / mL and then cultured again in 560 mL of BMCM-PL1 medium (Bio Mirai Kobo) containing 1% human serum until the cells became confluent.

[0029] The medium was then replaced with BSCM-PL2 medium for stem cell proliferation (manufactured by Bio Mirai Kobo Co., Ltd.) and cultured for 5 days. The entire medium was collected and 560 mL of new medium for stem cell culture was added, and cultured for another 5 days. The entire medium was collected and added until the cells were detached, and the culture supernatant thus collected was centrifuged (3000 rpm, 5 minutes) to remove cell debris. The protein fraction was then separated by organic solvent precipitation using 80% ethanol or acetone. The protein fraction was dissolved in PBS(-) (manufactured by Nacalai Tesque, product number: 07269-84), the insoluble fraction was removed by centrifugation (8000 rpm, 10 minutes), and precipitated again with 80% ethanol or acetone.

[0030] The precipitate was dissolved in 10 mL of 1N hydrochloric acid (Nacalai Tesque, product number: 18320-15), stirred at room temperature at 2,500 rpm for 10 minutes on a vortex, neutralized with 1.2N caustic soda (Nacalai Tesque, product number: 31511-05) to inactivate the virus, and then centrifuged to remove insoluble matter. Finally, the solution was sterilized through a 0.45 μm filter (Cytiva, product number: 6900-2504) to obtain a CPPs composition.

[0031] (Example 2) Analysis of differentiation induction into nerve cells 1 (CPPs composition) The ability to induce differentiation into neural cells was analyzed using a medium containing the CPPs composition. Adipose-derived stem cells (ASCs) belonging to mesenchymal stem cells (MSCs) (Lonza, product number: PT-5006) 4 x 10 5 Cells / mL were cultured in a T-75 flask (SARSTEDT, product number: 83.3911.002) using BMCM-PL1 medium (Bio Mirai Kobo) containing 2% human serum until the cells became 90% confluent, and then detached with trypsin-EDTA solution (Nacalai Tesque, product number: 32777-44).

[0032] The ASCs were suspended in IMDM medium containing 5% FBS (manufactured by Cytiva, product number: SH30910.03) and plated at 1 × 10 4 The next day, the medium was replaced with 1 μM TAT-VHL and CPPs composition (protein concentration: 80 to 120 μg / mL solution):IMDM (1:1) and cultured for 3 days.

[0033] First, Nissl staining was performed according to the following procedure. After adding the CPPs composition and culturing the cells for 3 days as described above, 10% neutral formaldehyde solution (manufactured by Nacalai Tesque, product number: 37152-51) was added and fixed for 10 minutes, and then washed with water. Next, Crisil Violet solution (manufactured by Muto Chemical Co., Ltd., product number: 41022) was added dropwise, and the cells were left for 30 minutes, washed with water, and photographed. The results are shown in Figure 1. No staining was observed in the cells cultured in IMDM as a control (Figure 1A). In contrast, in the cells cultured in a medium containing the CPPs composition, granular blue staining was observed in the cytoplasm (Nissl bodies) as indicated by the arrows (Figure 1B). This confirmed from a morphological point of view that mesenchymal stem cells were induced to differentiate into nerve cells by culturing in a medium containing the CPPs composition.

[0034] Next, staining with anti-βIII-tubulin antibody was performed as follows. The cells were fixed in the same manner as in Nissl staining, blocked by adding 1% human albumin aqueous solution, and washed with water. Then, the cells were reacted with anti-βIII-tubulin antibody Alexa Fluor 488 (Merck Millipore, product number: AB15708A) in a refrigerator for 1 hour, washed with water, and photographed under a fluorescence microscope (Keyence, BZ-X800). After staining the cytoplasm with anti-neuron specific anti-βIII-tubulin antibody-Alexa Fluor 488 as described above, 5μg / mL Hoechst 33342 (ThermoFisher, product number: H3570) aqueous solution was added and incubated for 3-5 minutes in the dark to stain the nucleus. The aqueous solution was removed, the cells were washed with water, and photographed under a fluorescent microscope. The results are shown in Figure 2. No process extension was observed in IMDM (Figure 2A). In contrast, cells cultured in a medium containing the CPPs composition showed process extension and network formation, and were stained green with anti-βIII-tubulin antibody-Alexa Fluor 488 (Figure 2B). This confirmed from a morphological standpoint that mesenchymal stem cells were induced to differentiate into neural cells by culturing in a medium containing the CPPs composition.

[0035] Next, for genetic analysis, RNA was extracted from the cells cultured as described above using Maxwell RSC simplyRNA (Promega, product number: AS1390), and qPCR was performed using StepOne PLUS (ThermoFisher) and the following TaqMan primers (ThermoFisher) to analyze the expression of the following genes, which are said to be specifically expressed in neural progenitor cells, neurons, and neural stem cells: Oct3 / 4:Hs00999632_g1, Nestin:Hs04187831_g1, MAP2:Hs00258900_m1. The expression of neural-specific genes was compared between cells cultured in IMDM (control), cells cultured in a medium containing TAT-VHL, and cells cultured in a medium containing the CPPs composition. The results are shown in Figure 3. The vertical axis of the graph represents the mRNA expression ratio based on IMDM. From Figure 3, it can be seen that only cells induced to differentiate with the CPPs composition showed significantly higher expression of Nestin, which is one of the indicators for evaluating the induction of differentiation into neural cells. This confirmed from a genetic perspective that mesenchymal stem cells were induced to differentiate into neural cells by culturing in a medium containing the CPPs composition. The above results demonstrate that cells cultured in a medium containing the CPPs composition were induced to differentiate into nerve cells.

[0036] (Example 3) Analysis of differentiation induction into neural cells 2 (CPPs composition + TAT-VHL) Using a medium containing the CPPs composition and TAT-VHL, the ability to induce differentiation into neural cells was analyzed. As mesenchymal stem cells, a different lot of ASCs (manufactured by Lonza, product number: PT-5006) was used as in Example 2, and analysis was performed using the same procedures as in Example 2, except that TAT-VHL was added to the culture medium. Specifically, the ASCs were suspended in IMDM medium containing 5% FBS (manufactured by Cytiva, product number: SH30910.03) and plated at 1 × 10 4The next day, (A) 2 μM TAT-VHL:IMDM (1:1); (B) 1 μM TAT-VHL and CPPs composition (protein concentration: 80 to 120 μg / mL solution): IMDM (1:1), The medium was replaced with IMDM and cultured for 3 days. The medium was left unchanged as a control. The results are shown in Figure 4.

[0037] As in Example 2, the cytoplasm was stained with anti-neuron specific anti-βIII-tubulin antibody-Alexa Fluor 488, and the nucleus was stained with Hoechst 33342. As shown in FIG. 4, when comparing TAT-VHL alone (FIG. 4A) with CPPs composition + TAT-VHL (FIG. 4B), it is clear that the cells induced with CPPs composition + TAT-VHL (B) have more process extension and longer processes. From the morphological viewpoint, it was confirmed that mesenchymal stem cells were differentiated into nerve cells more efficiently by culturing in a medium containing TAT-VHL in addition to the CPPs composition.

[0038] Next, as in Example 2, RNA was extracted from the cells cultured as described above, and qPCR was performed to analyze the expression of the following genes, which are believed to be specifically expressed in neural progenitor cells, neural cells, neural stem cells, and the like: Oct3 / 4:Hs00999632_g1, Nestin:Hs04187831_g1, MAP2:Hs00258900_m1. The results are shown in Figure 5. The vertical axis of the graph represents the mRNA expression ratio based on IMDM. 5 shows that the expression of nestin and MAP2 was significantly higher in cells induced to differentiate with the CPPs composition + TAT-VHL than in cells induced to differentiate with TAT-VHL, the CPPs composition, or either alone, compared to the control (IMDM), TAT-VHL alone, or the CPPs composition alone. This confirmed from a genetic perspective that mesenchymal stem cells were induced to differentiate into neural cells more efficiently by culturing in a medium containing the CPPs composition in addition to TAT-VHL. These results demonstrate that cells induced in a medium containing the TAT-VHL+CPPs composition were induced to differentiate into nerve cells more efficiently.

[0039] (Example 4) Analysis of CPPs composition 1 The CPPs composition was electrophoresed on a 10% gel of SDS-PAGE together with type I atelocollagen (manufactured by Daiichi Fine Chemical Co., Ltd., product number: Y-1), then transferred to a nitrocellulose membrane and subjected to Western blotting (performed according to Protein Experimental Notebook, revised 4th edition, Yodosha Co., Ltd.). The results are shown in Figure 6. Figure 6A shows the results of CBB staining (manufactured by Nacalai Tesque, product number: 04543-51), and Figure 6B shows the electrophoretic pattern obtained by reacting with anti-Procollagen 1C-Terminal Propeptide (anti-PICP) antibody (manufactured by Cloud-Clone Crop, product number: PAA570Hu08). As shown in Figure 6A(b), type I atelocollagen was shown as a two-chain structure of approximately 120 kDa (mass) by CBB staining, but did not react with anti-PICP antibody that labels the C-terminus of the propeptide, and no band was detected, as shown in Figure 6B(b). On the other hand, in the CPPs compositions shown in Figure 6 (c) and (d), at least four bands (four-stranded) were detected at approximately 120 to 200 kDa. This result indicates that the collagen in the CPPs composition contains type I procollagen in addition to type I collagen. The arrow in Figure 6 (B) indicates the procollagen band.

[0040] (Example 5) Analysis of CPPs composition 2 To analyze the small amount of collagen contained in the CPPs composition, it was purified separately. The ASC used in Example 2 was grown in BSCM-PL1 medium in a T-175 flask until it reached 100% confluence, and then cultured in 50 mL of medium supplemented with 5 mM magnesium ascorbyl phosphate for 4 days, and the culture supernatant was collected. The culture was repeated several times until the cells were detached, and cell debris was removed from the collected culture supernatant by centrifugation, followed by salting out. This salting out was repeated eight times to purify collagen. Figure 7 shows the results of SDS-PAGE stained with CBB (manufactured by Nacalai Tesque, product number: 04543-51). From the positions of the observed bands, it can be seen that the purified collagen in (c) contains α1 chain collagen and α2 chain collagen in a ratio of 2:1. This further confirmed that the CPPs composition contains type I collagen.

[0041] (Example 6) Analysis of differentiation induction into nerve cells 3 (purified collagen) (c) of Figure 7 The ability to induce differentiation into neural cells was analyzed using a medium containing purified collagen (0.1 mg / mL). As mesenchymal stem cells, ASCs (manufactured by Lonza, product number: PT-5006) similar to those used in Example 3 were used, and analysis was performed using the same procedures as in Example 3, except that purified collagen (0.1 mg / mL) was added to the culture medium. Specifically, the ASCs were suspended in IMDM medium containing 5% FBS (manufactured by Cytiva, product number: SH30910.03) and plated at 1 × 10 4 The next day, (A) IMDM, (B) IMDM containing 50 μg / mL purified collagen; (C) 1 μM TAT-VHL; (D) IMDM containing 50 μg / mL purified collagen: 2 μM TAT-VHL:IMDM (1:1); The medium was replaced with IMDM and cultured for 3 days. The medium was left unchanged in IMDM as a control. The results are shown in Figures 8 to 10.

[0042] In the cells cultured in IMDM containing 50 μg / mL purified collagen (B) above, granular areas stained blue in the cytoplasm were observed (Nissl bodies), as indicated by the arrows in Figure 8. This confirmed from a morphological point of view that the collagen component contained in the CPPs composition contributes to the effect of inducing differentiation of mesenchymal stem cells into nerve cells.

[0043] As in Example 2, the cytoplasm was stained with anti-neuron specific anti-βIII-tubulin antibody-Alexa Fluor 488, and the nucleus was stained with Hoechst 33342, as shown in FIG. 9. Comparing the control cells (A) cultured in IMDM (FIG. 9A) with the control cells (D) cultured in IMDM containing 50 μg / mL purified collagen (FIG. 9B), it is clear that the cells in (B) have more process extension. This confirmed that the collagen component contained in the CPPs composition contributes to the differentiation of mesenchymal stem cells into nerve cells from the viewpoint of morphology.

[0044] Next, as in Example 2, RNA was extracted from the cells cultured as described above, and qPCR was performed to analyze the expression of the following genes, which are believed to be specifically expressed in neural progenitor cells, neural cells, neural stem cells, and the like: Sox2:Hs00415716_m1, Oct3 / 4:Hs00999632_g1, Nestin:Hs04187831_g1, MAP2: Hs00258900_m1, NF(Neurofilament):Hs00196245_m1. The results are shown in Figure 10. The vertical axis indicates the mRNA expression ratio based on IMDM. Figure 10 shows that (B) cells cultured in IMDM containing 50 μg / mL purified collagen expressed neurofilament (NF) at a level equivalent to (C) cells cultured in 1 μM TAT-VHL, and (D) cells cultured in IMDM containing 50 μg / mL purified collagen: 2 μM TAT-VHL: IMDM (1:1) showed significantly higher expression of nestin in addition to NF. This confirmed from a genetic point of view that the collagen component contained in the CPPs composition contributes to the effect of inducing differentiation of mesenchymal stem cells into neural cells. These results demonstrate that the collagen component contained in the CPPs composition contributes to the effect of inducing differentiation of mesenchymal stem cells into nerve cells.

[0045] (Example 7) Analysis of differentiation induction into neural cells 4 (bone marrow-derived stem cells) Bone marrow-derived stem cells (BM-MSC) (Lonza, product number: PT-2501) 4 × 10 5 Cells / mL were cultured in a T-75 flask (SARSTEDT, product number: 83.3911.002) using BMCM-PL1 medium (Bio Mirai Kobo) containing 2% human serum until the cells became 90% confluent, and then detached with trypsin-EDTA solution (Nacalai Tesque, product number: 32777-44). Using these cells, BM-MSCs were suspended in IMDM medium containing 5% FBS (manufactured by Cytiva, catalogue no. SH30910.03) and plated at 1 × 10 4 The next day, culture medium (A) to (D): (A)IMDM (B) 1 μM TAT-VHL:IMDM (1:1); (C) CPPs composition: IMDM (1:1), (D) 2 μM TAT-VHL:CPPs composition 1:1) The medium was replaced with IMDM and cultured for 3 days. The medium in IMDM was used as the control. As in Example 2, the cytoplasm was stained with anti-neuron specific anti-βIII-tubulin antibody-Alexa Fluor 488 and the nucleus was stained with Hoechst 33342, as shown in Figure 11. The vertical axis of the graph is the mRNA expression ratio based on IMDM. As shown in Figure 11, staining was faint in medium (A) (Figure 11A), and little process extension was observed in medium (B) (Figure 11B). In contrast, process extension was observed in medium (C) (Figure 11C), and process extension was more prominent in medium (D) (Figure 11D). These results demonstrated that bone marrow-derived stem cells can be induced to differentiate into neural cells by culturing them using the CPPs composition. In addition, it was demonstrated that mesenchymal stem cells can be induced to differentiate into neural cells more efficiently by combining the CPPs composition with TAT-VHL.

[0046] According to the culture method of the present invention, it is possible to induce differentiation of mesenchymal stem cells into neurons using components obtained from mesenchymal stem cells, and further, components derived from autologous cells, without using a neuronal differentiation-inducing medium.

Claims

1. a) 60.0 to 90.0 vol% of a basal medium; 10.0 to 40.0 vol% physiological saline; and 1.0 to 100.0 ng / mL of EGF; 0.2 to 20.0 ng / mL of FGF-2; 0.2 to 20.0 ng / mL of PDGF; 0.5 to 8.0 mM magnesium ascorbyl phosphate; Cultivating mesenchymal stem cells for 1 to 10 days in a stem cell culture medium comprising the above-mentioned b) thereafter, precipitating protein components of the culture solution obtained by the above-mentioned culture with an organic solvent; c) The precipitate is separated and dissolved in physiological saline or a basal medium. Using a medium containing a cell-produced protein (CPPs) composition, ３ ~2×10 ４ A method for inducing differentiation into nerve cells, comprising culturing mesenchymal stem cells seeded at 1000 ng / mL for 1 to 3 days to differentiate the mesenchymal stem cells into nerve cells.

2. The method for inducing differentiation into nerve cells according to claim 1 , wherein the CPP composition contains collagen.

3. The method for inducing differentiation into nerve cells according to claim 2 , wherein the collagen comprises type I collagen and / or procollagen.

4. The method for inducing differentiation into nerve cells according to claim 1 , wherein the medium containing the CPPs composition further contains TAT-VHL.