Differentiation induction method into neural cells

Abstract

Provided is a method for inducing differentiation into neural cells by culturing mesenchymal stem cells seeded at 3 x 10 3 ~ 2 x 10 4 cells / mL for 1 to 3 days using a medium containing a cell production protein (CPPs) composition obtained by the following steps: a) culturing mesenchymal stem cells in a stem cell culture medium composed of 60.0 to 90.0 vol% of a basal medium and 10.0 to 40.0 vol% of physiological saline, further added with 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, and 0.5 to 8.0 mM of magnesium ascorbyl phosphate for 1 to 10 days; and b) thereafter, precipitating protein components in a culture solution obtained by the culturing using an organic solvent; and c) isolating and collecting the precipitated portion and dissolving in physiological saline or a basal medium.

Description

Technical Field

[0001] This invention relates to a method for inducing differentiation into nerve cells, and more particularly, to a method for inducing mesenchymal stem cells to differentiate into nerve cells using a cell-producing protein composition (CPPs composition) obtained by culturing mesenchymal stem cells in a predetermined stem cell culture medium. Background Technology

[0002] In recent years, adult stem cells, which can be collected from adult tissues, have attracted much attention. Adult stem cells exist in the skin, bone marrow, and fat of adults and can proliferate in an undifferentiated state in vitro. In addition, they can differentiate into cells of specific tissues.

[0003] Among tissue regeneration, nerve regeneration stands out as one of the most important. After nerve cells proliferate and distribute to build the nervous system during early development, their self-regeneration capacity is poor, leading to the belief that damaged nerve tissue cannot be repaired. Therefore, there is a need for a technology that repairs damaged nerve tissue by transplanting nerve cells differentiated from stem cells.

[0004] For example, Patent Document 1 describes a method for inducing the differentiation of adipose tissue mesenchymal cells to express immature neural cell markers, and describes a differentiation induction method having the following steps: collecting adipose tissue mesenchymal cells from adipose tissue, and culturing adipose tissue mesenchymal cells in a culture medium containing dcAMP or trichodin.

[0005] Furthermore, Patent Document 2 describes the use of TAT-VHL (157-171) to differentiate neural stem cells into neural cells. The TAT-VHL (157-171) is a peptide synthesized by combining a fusion protein TAT (amino acid sequence of sequence number 2: YGRKKRRQRRRD) which has the ability to easily penetrate the cell membrane with a VHL oligopeptide (amino acid sequence of sequence number 1: TLKERCLQVVRSLVK), wherein the VHL oligopeptide is an oligopeptide having a portion of the amino acid sequence of the von Hippel-Lindau (VHL) protein.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Application Publication No. 2013-63088

[0009] Patent Document 2: Japanese Patent Application Publication No. 2005-330206 Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] As mentioned above, since nerve cells do not divide or proliferate in principle, it is desirable to develop a technology that can efficiently and safely obtain nerve cells from mesenchymal stem cells for use in treatments such as transplantation.

[0012] Furthermore, while neural cell differentiation induction media generally have a strong effect on differentiating mesenchymal stem cells into neural cells and do not contain components that exhibit cytotoxicity, they use fetal bovine serum (FBS). FBS is a xenobiotic-derived component, which carries antigenicity and the risk of zoonotic viral infection, making it difficult to use in the aforementioned drug administration / transplantation subjects.

[0013] In view of the above, the inventors conducted further research and discovered for the first time a method for inducing differentiation into neural cells using cell-producing proteins (CPPs) containing neural cell differentiation-inducing components. These cell-producing proteins can replace neural cell differentiation-inducing culture media and can be prepared from mesenchymal stem cells, thus completing the present invention.

[0014] This invention was made in view of the above-mentioned circumstances, and its object is to induce mesenchymal stem cells to differentiate into nerve cells. More specifically, its object is to provide a method for inducing differentiation into nerve cells using components obtained from the culture medium of mesenchymal stem cells.

[0015] Methods for solving problems

[0016] This invention is based on the above-mentioned findings, and its purpose is to advantageously solve the aforementioned problems. A first aspect of this invention is a method for inducing differentiation into nerve cells, which involves using a culture medium containing a composition of cell-producing proteins (CPPs) at a concentration of 3 × 10⁻⁶... 3 ~2×10 4 Mesenchymal stem cells seeded at a concentration of cells / mL were cultured for 1–3 days to differentiate into nerve cells. The culture medium containing a cell-derived protein (CPP) composition was obtained through the following steps:

[0017] a) Mesenchymal stem cells were cultured for 1 to 10 days in a stem cell culture medium consisting of 60.0–90.0 vol% basal medium and 10.0–40.0 vol% physiological saline, with the addition of 1.0–100.0 ng / mL EGF, 0.2–20.0 ng / mL FGF-2, 0.2–20.0 ng / mL PDGF and 0.5–8.0 mM magnesium ascorbate phosphate.

[0018] b) Subsequently, an organic solvent was used to precipitate the protein components in the culture medium obtained through the above culture.

[0019] c) Separate and collect the precipitate and dissolve it in physiological saline or basal culture medium.

[0020] Based on this method of inducing differentiation into nerve cells, it is possible to induce mesenchymal stem cells to differentiate into nerve cells using components obtained from mesenchymal stem cells, without using a nerve cell differentiation induction culture medium.

[0021] In the above manner, the CPPs composition may contain collagen.

[0022] The inventors of this application have also discovered for the first time that the collagen contained in the CPPs composition has properties that help induce differentiation from stem cells into nerve cells.

[0023] In addition, the aforementioned collagen may contain type I collagen and / or procollagen.

[0024] In the above manner, the culture medium containing the above CPPs composition may further contain TAT-VHL.

[0025] This method allows mesenchymal stem cells to be induced to differentiate into nerve cells with greater efficiency.

[0026] Invention Effects

[0027] According to the present invention, mesenchymal stem cells can be differentiated into nerve cells using components obtained from mesenchymal stem cells, without the use of a neural cell differentiation induction culture medium. Attached Figure Description

[0028] Figure 1 This is a graph showing the results of Example 2.

[0029] Figure 2 This is a graph showing the results of Example 2.

[0030] Figure 3 This is a graph showing the results of Example 2.

[0031] Figure 4 This is a graph showing the results of Example 3.

[0032] Figure 5 This is a graph showing the results of Example 3.

[0033] Figure 6 This is a graph showing the results of Example 4.

[0034] Figure 7 This is a graph showing the results of Example 5.

[0035] Figure 8This is a graph showing the results of Example 6.

[0036] Figure 9 This is a graph showing the results of Example 6.

[0037] Figure 10 This is a graph showing the results of Example 6.

[0038] Figure 11 This is a graph showing the results of Example 7. Detailed Implementation

[0039] The embodiments of the present invention will now be described in detail.

[0040] The first aspect of the present invention's method for inducing differentiation into neural cells is characterized by using a culture medium containing a composition of cell-producing proteins (CPPs) at a concentration of 3 × 10⁻⁶. 3 ~2×10 4 Mesenchymal stem cells seeded at a concentration of cells / mL were cultured for 1–3 days to differentiate into nerve cells. The culture medium containing a cell-derived protein (CPP) composition was obtained through the following steps:

[0041] a) Mesenchymal stem cells were cultured for 1 to 10 days in a stem cell culture medium consisting of 60.0–90.0 vol% basal medium and 10.0–40.0 vol% physiological saline, with the addition of 1.0–100.0 ng / mL EGF, 0.2–20.0 ng / mL FGF-2, 0.2–20.0 ng / mL PDGF and 0.5–8.0 mM magnesium ascorbate phosphate.

[0042] b) Subsequently, an organic solvent was used to precipitate the protein components in the culture medium obtained through the above culture.

[0043] c) Separate and collect the precipitate and dissolve it in physiological saline or basal culture medium.

[0044] The inventors have discovered for the first time that the cell-producing protein (CPP) composition described below can be obtained from mesenchymal stem cells cultured using the above-described stem cell culture medium.

[0045] IMDM, DMEM, and α-MEM can be used as the basal culture medium. The purpose of adding physiological saline to the above-mentioned stem cell culture medium is to reduce the Ca concentration in the basal medium to 10–40%. Therefore, if the Ca concentration of the basal medium used is 40–50 μg / mL, dilution with physiological saline is not necessary.

[0046] Regarding the additives added to the aforementioned stem cell culture media, EGF stands for Epidermal Growth Factor, FGF-2 for Fibroblast Growth Factor 2, and PDGF for Platelet-Derived Growth Factor. These additives are typically used individually as basal medium additives for purposes such as promoting cell proliferation.

[0047] In the stem cell culture medium of the first embodiment of the present invention described above, when EGF in the range of 1 to 10 ng / mL, FGF-2 in the range of 0.5 to 5.0 ng / mL, and PDGF in the range of 0.5 to 5.0 ng / mL are added, the CPPs composition can be generated more efficiently.

[0048] Furthermore, from the viewpoint of improving cell proliferation performance, it is preferable to further add 0.1–10.0 μg / mL of transferrin, and / or 0.2–20.0 μg / mL of insulin, and / or 0.1–3.0 ng / mL of sodium selenite to the above-mentioned stem cell culture medium.

[0049] For example, a specific method for preparing a culture medium for stem cell culture can be used to remove 50 mL to 200 mL from a 500 mL container of IMDM, add an equal volume of physiological saline to make 500 mL, add 5 mL of antibiotics, and further add EGF (1–10 ng / mL), FGF-2 (0.5–5.0 ng / mL), PDGF (0.5–5.0 ng / mL), transferrin (0.1–10.0 μg / mL), insulin (0.2–20.0 μg / mL), sodium selenite (0.1–3.0 ng / mL), and L-ascorbic acid phosphate magnesium (0.5–8.0 mM).

[0050] (A composition of cell-produced proteins (CPPs))

[0051] The above-mentioned CPPs composition is characterized in that it is a CPPs composition obtained through the following steps for inducing mesenchymal stem cells to differentiate into neural cells:

[0052] Mesenchymal stem cells were cultured for 1–10 days in the aforementioned stem cell culture medium.

[0053] Subsequently, an organic solvent was used to precipitate the protein components in the culture medium obtained through the above culture.

[0054] Separate and collect the precipitate and dissolve it in physiological saline or basal culture medium.

[0055] CPPs compositions may contain type I procollagen and hyaluronic acid, with calcium bound to the type I procollagen.

[0056] In the CPPs composition, for example, the content of type I procollagen is 30–100 μg / mL, and the content of hyaluronic acid is 49–140 μg / mL. The binding ratio of type I procollagen to calcium is, for example, 30–45%, preferably 35–42%.

[0057] In the first method described above, the mesenchymal stem cells used to prepare the CPPs composition can be bone marrow-derived mesenchymal stem cells, adipose-derived stem cells (ASCs), peripheral blood-derived mesenchymal stem cells, Wharton's jelly-derived mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, etc. As the basal culture medium, IMDM, DMEM, or α-MEM can be used. The culture time is 1 to 10 days, preferably 3 to 4 days.

[0058] Furthermore, in the first method described above, in the step of "separating and collecting the precipitate and dissolving it in physiological saline or basal culture medium" following the step of "precipitating the protein components with organic solvents," the protein components precipitated with organic solvents can be suspended in hydrochloric acid (e.g., 1N HCl) at pH 1 and vortexed (e.g., for about 10 minutes), and then neutralized (e.g., using 1.2N NaOH), thereby simultaneously inactivating the virus. In this case, the neutralized solution is centrifuged, and the resulting supernatant is used as the CPPs composition.

[0059] (Methods for inducing the differentiation of mesenchymal stem cells into nerve cells)

[0060] The inventors have discovered for the first time that by using the above-mentioned CPPs composition and culturing under predetermined conditions, mesenchymal stem cells can be induced to differentiate into nerve cells.

[0061] The differentiation induction method of this invention is characterized by using a culture medium containing a CPPs composition, wherein the differentiation is induced at a concentration of 3 × 10⁻⁶ CPPs. 3 ~2×10 4 Mesenchymal stem cells seeded at 5 × 10⁶ cells / mL were cultured for 1–3 days to differentiate into nerve cells. 3 ~1×10 4 The differentiation induction efficiency is higher when mesenchymal stem cells seeded at a rate of 1-3 cells / mL are cultured for 1-3 days.

[0062] Based on this method of inducing differentiation into nerve cells, it is possible to induce mesenchymal stem cells to differentiate into nerve cells using components obtained from mesenchymal stem cells, without using a nerve cell differentiation induction culture medium.

[0063] Regarding the CPPs composition, a CPPs composition of 80–120 μg / mL based on protein concentration is used, preferably in a ratio of culture medium:CPPs composition = 1:1 / 8–1, i.e., containing 12.5–50.0 vol relative to the culture medium.

[0064] Furthermore, in the first approach described above, the mesenchymal stem cells can also be derived from bone marrow, fat, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

[0065] In the first embodiment described above, the CPPs composition may contain collagen.

[0066] In addition, the aforementioned collagen may contain type I collagen and / or procollagen.

[0067] Type I collagen is a type of collagen that is abundant in the skin and bones. It is a heterotrimer composed of two α1(I) chains (α1 chain collagen) and one α2(I) chain (α2 chain collagen).

[0068] Procollagen (collagen precursor) is a precursor in the collagen synthesis process, and it has procollagen peptides at the N-terminus and C-terminus of the peptide chain.

[0069] To date, in order to improve cell adhesion to culture containers, telopeptide-removed collagen (collagen with telopeptides removed) has sometimes been used in the coating of culture containers, but collagen has never been used as a differentiation-inducing factor.

[0070] Thus, the property of collagen as a differentiation-inducing factor that facilitates the induction of neural cell differentiation in stem cells under predetermined conditions is previously unknown, and this property is a first discovery by the inventors of this application. Furthermore, since the collagen contained in the CPPs composition was not subjected to treatment such as telopeptide removal (e.g., pepsin treatment) during the formation of the CPPs composition, it is highly likely that the collagen contained in the CPPs composition is mature collagen (natural collagen / procollagen) and procollagen.

[0071] In the first embodiment described above, the culture medium containing the above-described CPPs composition may further contain TAT-VHL.

[0072] As described in Japanese Patent Application Publication No. 2005-330206, TAT-VHL is a peptide synthesized by combining a fusion protein TAT (amino acid sequence of sequence number 2: YGRKKRRQRRRD) which has the function of easily penetrating cell membranes with a VHL oligopeptide (amino acid sequence of sequence number 1: TLKERCLQVVRSLVK), wherein the VHL oligopeptide is an oligopeptide having a portion of the amino acid sequence of the von Hippel-Lindau (VHL) protein.

[0073] By combining TAT-VHL with CPPs, mesenchymal stem cells can be induced to differentiate into nerve cells with greater efficiency.

[0074] In a culture medium containing CPPs composition, the TAT-VHL content is preferably 0.34–3.40 ng / mL.

[0075] Example

[0076] The present invention will be specifically described below with reference to specific embodiments, but the present invention is not limited to these embodiments.

[0077] (Example 1) Preparation of culture medium for stem cell culture and CPPs composition

[0078] 5 mL of an antibacterial-antimycotic mixed stock solution (manufactured by Nacalai Tesque, product number: 02892-54) was added to a 500 mL container of BSCM-PL2 medium (manufactured by Bio Mirai Kobo Co., Ltd.) for stem cell proliferation to prepare a culture medium for the preparation of CPPs compositions.

[0079] Next, using BMCM-PL1 medium (manufactured by BioMirai Kobo) containing 2% human serum, 4 × 10⁴ cells were added to a T-75 culture flask (manufactured by SARSTEDT, product number: 83.3911.002). 5 Adipose-derived stem cells (ASCs) belonging to mesenchymal stem cells (MSCs) (manufactured by Lonza, product number: PT-2501) were cultured at 90% confluence and then dissected using trypsin-EDTA solution (manufactured by Hanjing Reagent Co., Ltd., product number: 32777-44). Subsequently, they were cultured at 2 × 10⁶ cells / mL. 6 Cells were seeded at HYPERFlask (Corning) and cultured again in 560 mL of BMCM-PL1 medium (Bio Future Works) containing 1% human serum until confluence was achieved.

[0080] Next, the culture medium was replaced with BSCM-PL2 medium for stem cell proliferation (manufactured by BioMirai Kobo Co., Ltd.), and cultured for 5 days. The medium was then completely recovered, and 560 mL of fresh stem cell culture medium was added, followed by another 5 days of culture. This process of completely recovering and adding the medium continued until cell detachment occurred. The recovered culture supernatant was centrifuged (3000 rpm, 5 minutes) to remove cell debris. Then, protein fractions were separated using an organic solvent precipitation method with 80% ethanol or acetone. These protein fractions were dissolved in PBS(-) (manufactured by Hanjing Reagent Co., Ltd., product number: 07269-84), and insoluble fractions were removed by centrifugation (8000 rpm, 10 minutes). Precipitation was then performed again with 80% ethanol or acetone.

[0081] The precipitate was dissolved in 10 mL of 1N hydrochloric acid (manufactured by Hani Reagent Co., Ltd., product number: 18320-15), and stirred at room temperature and 2,500 rpm for 10 minutes using a vortex mixer. The mixture was then neutralized with 1.2N sodium hydroxide (manufactured by Hani Reagent Co., Ltd., product number: 31511-05) to inactivate the virus, followed by centrifugation to remove insoluble matter. Finally, the mixture was sterilized through a 0.45 μm filter (manufactured by Cytiva Co., Ltd., product number: 6900-2504) to obtain the CPPs composition.

[0082] (Example 2) Analysis of differentiation induction into nerve cells 1 (CPPs composition)

[0083] The differentiation-inducing properties to neural cells were analyzed using a culture medium containing a CPPs composition.

[0084] Using BMCM-PL1 medium (manufactured by BioMirai Kobo) containing 2% human serum, in T-75 culture flasks (manufactured by SAST, product number: 83.3911.002), 4 × 10⁴ 5 Adipose-derived stem cells (ASCs) belonging to mesenchymal stem cells (MSCs) (manufactured by Lonza, product number: PT-5006) were cultured at 90% confluence and then dissected using trypsin-EDTA solution (manufactured by Hanjing Reagent Co., Ltd., product number: 32777-44).

[0085] The above-mentioned ASCs were suspended in IMDM medium containing 5% FBS (manufactured by Stofavente, product number: SH30910.03) at a concentration of 1×10⁻⁶. 4 Cells were seeded at 1 / mL in 24-well plates (manufactured by SAST, product number: 83.3922). The next day, the solution was replaced with 1 μM TAT-VHL and CPPs combination (a solution with a protein concentration of 80–120 μg / mL): IMDM (1:1) and cultured for 3 days.

[0086] First, Nissl staining was performed as follows: Cells cultured for 3 days with the CPPs composition added as described above were fixed with 10% neutral formaldehyde solution (Hanjing Reagent Co., Ltd., product number: 37152-51) for 10 minutes, then washed with water. Next, cresol violet solution (Mutoh Chemical Co., Ltd., product number: 41022) was added, and after 30 minutes, the cells were washed with water and photographed. The results are shown below. Figure 1 .

[0087] No staining sites were observed in cells cultured with IMDM as a control. Figure 1 A). In contrast, in cells cultured in a medium containing CPPs, granular portions (Nissl bodies) stained blue were observed in the cytoplasm, as indicated by the arrow. Figure 1 B). Thus, it was morphologically confirmed that mesenchymal stem cells were induced to differentiate into nerve cells by culturing in a medium containing CPPs compositions.

[0088] Next, staining based on anti-βIII-tubulin antibody was performed as follows. Cells fixed using the same steps as in Nissl staining were blocked with 1% human albumin aqueous solution and washed with water. Afterward, the cells were reacted with anti-βIII-tubulin antibody Alexa Fluor 488 (Merck Millipore, product number: AB15708A) at refrigeration for 1 hour, washed with water, and photographed using a fluorescence microscope (Keyence, BZ-X800).

[0089] After staining the cytoplasm with the neuron-specific anti-βIII-tubulin antibody Alexa Fluor 488 using the method described above, 5 μg / mL of Hoechst 33342 (Thermo Fisher Scientific, product number: H3570) aqueous solution was added, and the mixture was incubated in the dark for 3–5 minutes to stain the nuclei. The aqueous solution was then removed, the cells were washed with water, and photographed using a fluorescence microscope. The results are shown below. Figure 2 .

[0090] No protrusion extension was observed in IMDM. Figure 2 A). In contrast, in cells cultured with a medium containing CPPs compositions, the extension and networking of protrusions were observed, and they stained green by staining with the anti-βIII-tubulin antibody-Alexa Fluor 488. Figure 2 B). Thus, it was morphologically confirmed that mesenchymal stem cells were induced to differentiate into nerve cells by culturing in a medium containing CPPs compositions.

[0091] Next, as part of gene 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 (Thermo Fisher Scientific) and the following TaqMan primers (Thermo Fisher Scientific) to analyze the expression of the following genes believed to be specifically expressed in neural progenitor cells, neural cells, and neural stem cells:

[0092] Oct3 / 4: Hs00999632_g1

[0093] Nestin: Hs04187831_g1

[0094] MAP2: Hs00258900_m1.

[0095] The expression of neural-specific genes was compared between cells cultured with IMDM (control), cells cultured in medium containing TAT-VHL, and cells cultured in medium containing a CPPs composition. The results are presented in… Figure 3 The vertical axis in the chart represents the mRNA expression ratio based on IMDM. Figure 3 It was found that nestin, considered an indicator of induction into neural cell differentiation, was significantly elevated only in cells induced by the CPPs composition. This genetically confirms that mesenchymal stem cells can be induced to differentiate into neural cells by culturing in a medium containing the CPPs composition.

[0096] The results above show that cells cultured in a medium containing CPPs were induced to differentiate into nerve cells.

[0097] (Example 3) Analysis of differentiation induction into nerve cells 2 (CPPs composition + TAT-VHL)

[0098] The differentiation-inducing properties to neural cells were analyzed using a culture medium containing a CPPs composition and TAT-VHL.

[0099] As mesenchymal stem cells, similar to Example 2, different batches of ASC (manufactured by Lonza, product number: PT-5006) were used, and TAT-VHL was added to the culture medium. Otherwise, the analysis was performed according to the same steps as in Example 2.

[0100] Specifically, the above-mentioned ASCs were suspended in IMDM medium containing 5% FBS (manufactured by Stofavente, product number: SH30910.03) at a concentration of 1×10⁻⁶.4 Cells were seeded at 1 / mL in 24-well plates (Sarstar, product number: 83.3922). The next day, the solution was replaced with (A) 2 μM TAT-VHL:IMDM (1:1) or (B) a 1 μM TAT-VHL and CPPs combination (80–120 μg / mL protein concentration):IMDM (1:1), and cultured for 3 days. IMDM was used as a control. Results are presented below. Figure 4 .

[0101] Similar to Example 2, the cytoplasm was stained with an anti-neuron-specific anti-βIII-tubulin antibody—Alexa Fluor 488—and the nucleus was stained with Hoechst 33342. Figure 4 As shown, only TAT-VHL ( Figure 4 A) with CPPs composition + TAT-VHL ( Figure 4 B) Comparison shows that cells induced by the CPPs composition + TAT-VHL (B) exhibit significantly more and longer process extension. This morphologically confirms that mesenchymal stem cells are more efficiently induced to differentiate into neural cells when cultured in a medium containing TAT-VHL in addition to the CPPs composition.

[0102] Next, similar to Example 2, as a gene analysis, RNA was extracted from the cells cultured as described above, and qPCR was performed to analyze the expression of the following genes that are believed to be specifically expressed in neural progenitor cells, neural cells, and neural stem cells:

[0103] Oct3 / 4: Hs00999632_g1

[0104] Nestin: Hs04187831_g1

[0105] MAP2: Hs00258900_m1.

[0106] The results are shown in Figure 5 The vertical axis in the chart represents the mRNA expression ratio based on IMDM.

[0107] Depend on Figure 5 It was found that, compared with the control (IMDM), TAT-VHL alone, and CPPs combination alone, the expression of nestin and MAP2 in cells induced by differentiation using CPPs combination + TAT-VHL was significantly increased compared with the use of TAT-VHL or CPPs combination alone. Therefore, from a genetic perspective, it was confirmed that mesenchymal stem cells were more efficiently induced to differentiate into neural cells by culturing in a medium containing TAT-VHL in addition to CPPs combination.

[0108] These results indicate that cells induced in a culture medium containing the TAT-VHL+CPPs composition are more efficiently induced to differentiate into nerve cells.

[0109] (Example 4) Analysis of CPPs Composition 1

[0110] Regarding the CPPs composition, after electrophoresis with type I determinate collagen (manufactured by Daiichi Fine Chemicals Co., Ltd., product number: Y-1) on a 10% gel by SDS-PAGE, it was transferred to a nitrocellulose membrane for Western blotting (performed according to the revised 4th edition of the Protein Experiment Notes, Volume 2, published by Yodosha). The results are shown below. Figure 6 . Figure 6 A is the result of CBB staining (manufactured by Hanjing Reagent Co., Ltd., product number: 04543-51). Figure 6 Image B is an electrophoretic pattern obtained after reacting with an antibody against type I procollagen C-terminal propeptide (anti-PICP (Procollagen 1 C-Terminal Propeptide)) (manufactured by Cloud-Clone Corp., product number: PAA570Hu08).

[0111] like Figure 6 As shown in A(b), type I determinate collagen appears as two chains of approximately 120 kDa (mass) upon CBB staining, but does not react with anti-PICP antibody labeled at the C-terminus of the propeptide, as... Figure 6 No bands were detected as shown in B(b).

[0112] on the other hand, Figure 6 The CPPs compositions shown in (c) and (d) exhibited at least four bands (four chains) detected at approximately 120–200 kDa. This result indicates that the collagen in the CPPs compositions contains type I procollagen in addition to type I collagen. It should be noted that... Figure 6 The arrow in B represents a band of precollagen.

[0113] (Example 5) Analysis of CPPs Composition 2

[0114] Further purification was performed to analyze the trace amounts of collagen contained in the CPPs composition.

[0115] Using BSCM-PL1 medium, the ASCs used in Example 2 were proliferated in T-175 culture flasks until 100% confluence was achieved. They were then cultured for 4 days in 50 mL of medium supplemented with 5 mM magnesium ascorbate phosphate, and the culture supernatant was collected. This culture was repeated several times until cell exfoliation. Cell debris was removed from the collected culture supernatant by centrifugation, followed by salting out. This salting out was repeated 8 times to purify the collagen. Figure 7 The results of SDS-PAGE based on CBB staining (manufactured by Hani Reagents Co., Ltd., product number: 04543-51) are shown. From the observed band positions, it can be seen that the refined collagen in (c) contains α1-chain collagen and α2-chain collagen in a 2:1 ratio. This further confirms the presence of type I collagen in the CPPs composition.

[0116] (Example 6) Analysis of differentiation induction into nerve cells 3 (refined collagen)

[0117] Use containing Figure 7 (c) The culture medium containing purified collagen (0.1 mg / mL) was analyzed to assess its ability to induce differentiation into nerve cells.

[0118] As mesenchymal stem cells, the same ASC (manufactured by Lonza, product number: PT-5006) as in Example 3 was used, with purified collagen (0.1 mg / mL) added to the culture medium, and the analysis was performed according to the same procedure as in Example 3.

[0119] Specifically, the above-mentioned ASCs were suspended in IMDM medium containing 5% FBS (manufactured by Stofavente, product number: SH30910.03) at a concentration of 1×10⁻⁶. 4 Cells were seeded per mL / well in 24-well plates (SAST, product number 83.3922). The next day, the cells were replaced with (A) IMDM, (B) IMDM containing 50 μg / mL refined collagen, (C) 1 μM TAT-VHL, and (D) IMDM containing 50 μg / mL refined collagen: 2 μM TAT-VHL:IMDM (1:1), and cultured for 3 days. IMDM was used as a control. Results are presented below. Figures 8-10 .

[0120] In cells cultured with IMDM containing 50 μg / mL refined collagen as described in (B), such as Figure 8 The middle arrow indicates the blue-stained granular portions (Nissl bodies) observed in the cytoplasm. This morphologically confirms that the collagen component in the CPPs composition contributes to the induction of mesenchymal stem cells into neural cells.

[0121] Similar to Example 2, the cytoplasm was stained with an anti-neuron-specific anti-βIII-tubulin antibody—Alexa Fluor 488—and the nucleus was stained with Hoechst 33342, as shown in the figure. Figure 9 Cells cultured with IMDM were used as a control (A). Figure 9 Cells cultured with IMDM containing 50 μg / mL refined collagen in (A) and (D) Figure 9 A comparison with B) clearly shows that cells in (B) have extended protrusions. Therefore, from a morphological perspective, this confirms that the collagen component in the CPPs composition helps induce mesenchymal stem cells to differentiate into nerve cells.

[0122] Next, similar to Example 2, as a gene analysis, RNA was extracted from the cells cultured as described above, and qPCR was performed to analyze the expression of the following genes that are believed to be specifically expressed in neural progenitor cells, neural cells, and neural stem cells:

[0123] Sox2: Hs00415716_m1

[0124] Oct3 / 4: Hs00999632_g1

[0125] Nestin: Hs04187831_g1

[0126] MAP2: Hs00258900_m1

[0127] NF (Neurofilament): Hs00196245_m1.

[0128] The results are shown in Figure 10 The vertical axis represents the mRNA expression ratio based on IMDM. Figure 10 It was found that cells cultured with IMDM containing 50 μg / mL refined collagen (B) expressed neurofilaments (NF) as readily as cells cultured with 1 μM TAT-VHL (C). However, in cells cultured with IMDM containing 50 μg / mL refined collagen and 2 μM TAT-VHL containing IMDM (1:1), nestin expression was significantly higher than NF expression. Therefore, from a genetic perspective, it was confirmed that the collagen component in the CPPs composition contributes to the induction of mesenchymal stem cells into neural cells.

[0129] These results indicate that the collagen component in the CPPs composition helps induce mesenchymal stem cells to differentiate into nerve cells.

[0130] (Example 7) Analysis of differentiation induction into nerve cells 4 (bone marrow-derived stem cells)

[0131] Using BMCM-PL1 medium (manufactured by BioMirai Kobo) containing 2% human serum, in T-75 culture flasks (manufactured by SAST, product number: 83.3911.002), 4 × 10⁴ 5 Bone marrow-derived stem cells (BM-MSCs) (manufactured by Lonza, product number: PT-2501) were cultured at 90% confluence and then dissected using trypsin-EDTA solution (manufactured by Hanjing Reagent Co., Ltd., product number: 32777-44).

[0132] Using these cells, BM-MSCs were suspended in IMDM medium containing 5% FBS (Stonevan, catalog number SH30910.03) at a concentration of 1×10⁻⁶. 4 Cells were seeded per mL per well in 24-well plates (Sarstar, product number: 83.3922). The culture medium was replaced with (A) to (D) the following day, and the plates were cultured for 3 days. An original IMDM sample was used as a control.

[0133] (A)IMDM

[0134] (B) 1μM TAT-VHL:IMDM(1:1)

[0135] (C)CPPs composition: IMDM (1:1),

[0136] (D) 2 μM TAT-VHL:CPPs composition (1:1).

[0137] Similar to Example 2, the cytoplasm was stained with an anti-neuron-specific anti-βIII-tubulin antibody—Alexa Fluor 488—and the nucleus was stained with Hoechst 33342, as shown in the figure. Figure 11 The vertical axis in the chart represents the mRNA expression ratio based on IMDM.

[0138] like Figure 11 As shown, the staining is lighter in culture medium (A). Figure 11 In culture medium (B), almost no protrusion extension was observed (A). Figure 11 B). In contrast, protrusion extension was observed in culture medium (C). Figure 11 C), and more significant protrusion extension was observed in culture medium (D). Figure 11 D).

[0139] These results confirm that bone marrow-derived stem cells can be induced to differentiate into neural cells through culture using a CPPs composition. Furthermore, it was confirmed that combining TAT-VHL with a CPPs composition can induce mesenchymal stem cells to differentiate into neural cells with even greater efficiency.

[0140] According to the culture method of this invention, mesenchymal stem cells can be induced to differentiate into nerve cells without using a neural cell differentiation induction culture medium, but using components obtained from mesenchymal stem cells and components derived from autologous cells.

Claims

1. A method for inducing differentiation into nerve cells, comprising using a culture medium containing a composition of cell-producing proteins (CPPs) at a concentration of 3 × 10⁻⁶ 3 ~2×10 4 Mesenchymal stem cells seeded at a concentration of cells / mL were cultured for 1–3 days to differentiate into nerve cells. The culture medium containing a cell-derived protein (CPP) composition was obtained through the following steps: a) Mesenchymal stem cells were cultured for 1 to 10 days in a stem cell culture medium consisting of 60.0–90.0 vol% basal medium and 10.0–40.0 vol% physiological saline, with the addition of 1.0–100.0 ng / mL EGF, 0.2–20.0 ng / mL FGF-2, 0.2–20.0 ng / mL PDGF and 0.5–8.0 mM magnesium ascorbate phosphate. b) Subsequently, an organic solvent is used to precipitate the protein components in the culture medium obtained through the culture. c) Separate and collect the precipitate and dissolve it in physiological saline or basal culture medium.

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

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

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

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