Method for inducing differentiation into nerve cells

Abstract

Provided is a method for inducing differentiation into nerve cells, the method comprising: a) culturing mesenchymal stem cells for 1-10 days in a stem cell culture medium configured by including a basal medium at 60.0–90.0 vol% and physiological saline at 10.0–40.0 vol%, the culture medium being further supplemented with 1.0–100.0 ng / mL of EGF, 0.2–20.0 ng / mL of FGF-2, 0.2–20.0 ng / mL of PDGF, and 0.5–8.0 mM of magnesium ascorbyl phosphate; b) thereafter, precipitating protein components from the culture solution obtained in step a) using an organic solvent; and c) isolating the precipitate portion and dissolving the same in a physiological saline or a basal medium to obtain a medium comprising a cell-produced protein (CPPs) composition, and seeding, using the medium, the mesenchymal stem cells at a density of 3×103 to 2×104 cells / mL and culturing for 1-3 days, thereby inducing differentiation of the mesenchymal stem cells into nerve cells.

Description

Method for inducing differentiation into nerve cells

[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.

[0002] In recent years, somatic stem cells, which can be extracted from adult tissues, have been attracting attention. Somatic stem cells are present 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 establish the nervous system, but after that, they have little self-regenerative ability, and it is thought that once damaged, nerve tissue cannot be repaired. Therefore, there is a need for a technology to 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 of immature nerve 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] Furthermore, Patent Document 2 describes the differentiation of neural stem cells into neurons using TAT-VHL (157-171), a peptide synthesized by binding a fusion protein TAT (amino acid sequence of SEQ ID NO: 2: YGRKKRRQRRRD), which has the ability to easily penetrate cell membranes, to 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.

[0006] JP 2013-63088 A JP 2005-330206 A

[0007] As mentioned above, since nerve cells do not generally divide or proliferate, there is a need for a technology that can efficiently and safely obtain nerve cells from mesenchymal stem cells for treatments such as transplantation. Furthermore, nerve cell differentiation-inducing media generally contain fetal bovine serum (FBS), which has a strong effect of differentiating mesenchymal stem cells into nerve cells and does not contain any cytotoxic components. FBS is a component derived from a xenogeneic animal, and therefore 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 research and have discovered a new method for inducing differentiation into neurons using cell producing proteins (CPPs) containing neuron differentiation-inducing components, which can be used in place of a neuron differentiation-inducing medium and can be prepared from mesenchymal stem cells, thereby completing the present invention.

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

[0010] The present invention has been made based on the above findings and aims to advantageously solve the above problems. A first aspect of the present invention comprises the steps of: a) culturing mesenchymal stem cells for 1 to 10 days in a stem cell culture medium comprising 60.0 to 90.0 vol% of a basal medium and 10.0 to 40.0 vol% of physiological saline, to which 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; b) thereafter precipitating protein components of the culture medium obtained by the above culture with an organic solvent; and c) isolating and dissolving the precipitate in physiological saline or a basal medium, thereby producing a medium containing a cell-produced protein (CPP) composition obtained by: ３ ~2 x 10４ This method of inducing differentiation into neurons involves culturing mesenchymal stem cells seeded at 0.1% MSCs / mL for 1 to 3 days, thereby differentiating the mesenchymal stem cells into neurons. This method of inducing differentiation into neurons allows the differentiation of mesenchymal stem cells into neurons to be induced using components obtained from mesenchymal stem cells, without using a neuronal differentiation-inducing medium.

[0011] In the above-mentioned aspect, the CPP composition may contain collagen. The present inventors have also newly discovered that the collagen contained in the CPP composition has the property of contributing to the induction of differentiation of stem cells into nerve cells. Furthermore, the collagen may contain type I collagen and / or procollagen.

[0012] In the above aspect, the medium containing the CPP composition may further contain TAT-VHL, which allows for more efficient differentiation of mesenchymal stem cells into neurons.

[0013] According to the present invention, mesenchymal stem cells can be differentiated into neurons using components obtained from mesenchymal stem cells, without using a neuronal differentiation-inducing medium.

[0014] FIG. 1 is a diagram showing the results of Example 2. FIG. 2 is a diagram showing the results of Example 2. FIG. 3 is a diagram showing the results of Example 2. FIG. 4 is a diagram showing the results of Example 3. FIG. 5 is a diagram showing the results of Example 3. FIG. 6 is a diagram showing the results of Example 4. FIG. 7 is a diagram showing the results of Example 5. FIG. 8 is a diagram showing the results of Example 6. FIG. 9 is a diagram showing the results of Example 6. FIG. 10 is a diagram showing the results of Example 6. FIG. 11 is a diagram showing the results of Example 7.

[0015] The method for inducing differentiation into nerve cells according to a first aspect of the present invention comprises: a) culturing mesenchymal stem cells for 1 to 10 days in a stem cell culture medium comprising 60.0 to 90.0 vol% basal medium and 10.0 to 40.0 vol% physiological saline, to which 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; b) subsequently precipitating protein components of the culture medium obtained by the above culture with an organic solvent; and c) isolating and dissolving the precipitate in physiological saline or a basal medium, thereby producing 3 x 10 mesenchymal stem cells using a medium containing a cell-produced protein (CPP) composition obtained by: ３ ~2 x 10 ４ The method is characterized in that mesenchymal stem cells seeded at 1000 cells / mL are cultured for 1 to 3 days, thereby differentiating the mesenchymal stem cells into nerve cells.

[0016] The present inventors have newly discovered that the cell-produced protein (CPP) composition described below can be obtained from mesenchymal stem cells cultured using the above-mentioned stem cell culture medium. IMDM, DMEM, and α-MEM can be used as the basal medium. The purpose of adding physiological saline to the composition of 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, there is no need to dilute it with physiological saline.

[0017] Regarding the supplemental factors added to the stem cell culture medium, EGF is epidermal growth factor, FGF-2 is fibroblast growth factor 2, and PDGF is platelet-derived growth factor. The supplemental factors are generally used as supplemental factors to basal media for purposes such as cell proliferation. In the stem cell culture medium according to the first aspect of the present invention, adding EGF in the ranges 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 enables more efficient production of a CPP composition.

[0018] Furthermore, from the viewpoint of enhancing cell proliferation, it is preferable to further add 0.1 to 10.0 μg / mL transferrin, and / or 0.2 to 20.0 μg / mL insulin, and / or 0.1 to 3.0 ng / mL sodium selenite to the above-mentioned stem cell culture medium.

[0019] A specific method for preparing stem cell culture medium is, for example, to remove 50 mL to 200 mL from 500 mL of IMDM, add an equal volume of physiological saline to make 500 mL, add 5 mL of antibiotics to the mixture, and further add EGF at 1 to 10 ng / mL, FGF-2 at 0.5 to 5.0 ng / mL, PDGF at 0.5 to 5.0 ng / mL, transferrin at 0.1 to 10.0 μg / mL, insulin at 0.2 to 20.0 μg / mL, sodium selenite at 0.1 to 3.0 ng / mL, and magnesium ascorbyl phosphate at 0.5 to 8.0 mM.

[0020] (Cell-produced protein (CPP) composition) The CPP composition is characterized in that it is a CPP composition for inducing differentiation of mesenchymal stem cells into nerve cells, obtained by culturing mesenchymal stem cells in the stem cell culture medium for 1 to 10 days, then precipitating protein components of the culture solution obtained by the culture with an organic solvent, and separating and dissolving the precipitate in physiological saline or a basal medium.

[0021] The CPP composition may contain type I procollagen and hyaluronic acid, with calcium bound to the type I procollagen. The CPP 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 calcium to type I procollagen is, for example, 30 to 45%, preferably 35 to 42%.

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

[0023] Furthermore, in the first aspect, 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., 1 N HCl) at pH 1, vortexed (e.g., for about 10 minutes), and then neutralized (e.g., with 1.2 N 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 neurons) The present inventors have newly discovered that mesenchymal stem cells can be induced to differentiate into neurons by culturing them under specific conditions using the above-mentioned CPP composition. The differentiation induction method according to the present invention involves culturing mesenchymal stem cells at a concentration of 3 × 10 ３ ~2 x 10 ４ The method is characterized in that mesenchymal stem cells seeded at 5×10 cells / mL are cultured for 1 to 3 days, whereby the mesenchymal stem cells are differentiated into neural cells. ３ ~1 x 10 ４ Culturing mesenchymal stem cells seeded at 0.1 μg / mL for 1 to 3 days is preferred, as it results in higher differentiation induction efficiency. According to this method for inducing differentiation into neurons, mesenchymal stem cells can be induced to differentiate into neurons using components obtained from mesenchymal stem cells without using a neuronal differentiation-inducing medium. The CPP composition preferably has a protein concentration of 80 to 120 μg / mL, and is contained in a medium:CPP composition ratio of 1:1 / 8 to 1, i.e., 12.5 to 50.0 vol% of the medium. Furthermore, in the first aspect, the mesenchymal stem cells may be derived from bone marrow, adipose tissue, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

[0025] In the first aspect, the CPP composition may contain collagen. The collagen may also contain type I collagen and / or procollagen. Type I collagen is a type of collagen found in large amounts in skin and bones and is a heterotrimer consisting of two α1(I) chains (α1-chain collagen) and one α2(I) chain (α2-chain collagen). Procollagen (collagen precursor) is a precursor in the collagen synthesis process and contains procollagen peptides at the N- and C-termini of the peptide chain. While atelocollagen (collagen with telopeptides cleaved) has been used to coat culture vessels to enhance cell adhesion to the vessel, collagen has never been used as a differentiation inducer. Thus, it was previously unknown that collagen possesses the properties of a differentiation inducer that contribute 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 any treatment (e.g., pepsin treatment) that would cleave the telopeptides during 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 CPP composition may further contain TAT-VHL. As described in Japanese Patent Application Laid-Open No. 2005-330206, TAT-VHL is a peptide synthesized by conjugating 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, with the fusion protein TAT (amino acid sequence of SEQ ID NO: 2: YGRKKRRQRRRD), which has the ability to easily penetrate cell membranes. By combining the CPP composition with TAT-VHL, mesenchymal stem cells can be differentiated into neurons with higher efficiency. Preferably, TAT-VHL is contained in the medium containing the CPP composition at a concentration of 0.34 to 3.40 ng / mL.

[0027] The present invention will be specifically explained 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 Co., Ltd.) to prepare a stem cell culture medium for producing a CPPs composition. Next, 4 x 10 adipose-derived stem cells (ASCs) (Lonza, product number: PT-2501) belonging to the mesenchymal stem cells (MSCs) were added. 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). ６ The cells were seeded at 200 cells / mL and cultured again in 560 mL of BMCM-PL1 medium (Bio Mirai Kobo) containing 1% human serum until they became confluent.

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

[0030] The precipitate was dissolved in 10 mL of 1 N hydrochloric acid (Nacalai Tesque, product number: 18320-15), vortexed at room temperature for 10 minutes at 2,500 rpm, neutralized with 1.2 N 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 1 of Differentiation Induction into Neurons (CPP Composition) The ability to induce differentiation into neurons was analyzed using a medium containing a CPP composition. 4 x 10 adipose-derived stem cells (ASCs) (manufactured by Lonza, product number: PT-5006), which belong to the mesenchymal stem cells (MSCs), were used. ５ Cells / mL were cultured in a T-75 flask (SARSTEDT, product number: 83.3911.002) in BMCM-PL1 medium (Bio Mirai Kobo) containing 2% human serum until they reached 90% confluence, 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 x 10 in a 24-well plate (manufactured by SARSTEDT, product number: 83.3922). ４ The next day, the medium was replaced with 1 μM TAT-VHL and CPPs composition (protein concentration: 80-120 μg / mL solution): IMDM (1:1), and the cells were cultured for 3 days.

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

[0034] Next, staining with anti-βIII-tubulin antibody was performed as follows. Cells were fixed using the same procedure as for Nissl staining, blocked with 1% human albumin solution, and then washed with water. Subsequently, the cells were incubated with anti-βIII-tubulin antibody Alexa Fluor 488 (Merck Millipore, product number: AB15708A) for 1 hour in a refrigerator, washed with water, and then 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, nuclei were stained with 5 μg / mL Hoechst 33342 (ThermoFisher, product number: H3570) in water and incubated for 3–5 minutes in the dark. The solution was removed, washed with water, and photographed under a fluorescence microscope. The results are shown in Figure 2. No neurite outgrowth was observed in IMDM (Figure 2A). In contrast, cells cultured in a medium containing a CPP composition showed process extension and network formation, and were stained green with anti-βIII-tubulin antibody-Alexa Fluor 488 (Fig. 2B). This confirmed, from a morphological perspective, that mesenchymal stem cells were induced to differentiate into neurons by culturing in a medium containing a CPP composition.

[0035] Next, for genetic analysis, RNA extraction from the cells cultured as described above was performed using Maxwell RSC simplyRNA (Promega, product number: AS1390). qPCR was performed using StepOne PLUS (ThermoFisher) and the following TaqMan primers (ThermoFisher) to analyze the expression of the following genes, which are believed 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 medium containing TAT-VHL, and cells cultured in medium containing the CPPs composition. The results are shown in Figure 3. The vertical axis of the graph represents the mRNA expression ratio relative to IMDM. 3 shows that only cells induced to differentiate with the CPP composition exhibit significantly higher expression of nestin, which is one of the indicators for evaluating the induction of differentiation into neurons. This confirms from a genetic perspective that mesenchymal stem cells were induced to differentiate into neurons by culturing them in a medium containing the CPP composition. These results demonstrate that cells cultured in a medium containing the CPP composition were induced to differentiate into neurons.

[0036] (Example 3) Analysis 2 of Neuronal Differentiation Induction (CPP Composition + TAT-VHL) A medium containing a CPP composition and TAT-VHL was used to analyze the ability to induce differentiation into neurons. As in Example 2, a different lot of ASCs (manufactured by Lonza, product number: PT-5006) was used as mesenchymal stem cells, and the analysis was performed using the same procedure as in Example 2, except that TAT-VHL was added to the medium. Specifically, the ASCs were suspended in IMDM medium containing 5% FBS (manufactured by Cytiva, product number: SH30910.03), and 1 x 10 cells were placed in a 24-well plate (manufactured by SARSTEDT, product number: 83.3922). ４The next day, the medium was changed to either (A) 2 μM TAT-VHL:IMDM (1:1), or (B) 1 μM TAT-VHL and CPPs composition (protein concentration: 80-120 μg / mL solution):IMDM (1:1), and the cells were cultured for 3 days. ImDM was used as a control. The results are shown in Figure 4.

[0037] As in Example 2, cytoplasm was stained with anti-neuron-specific anti-βIII-tubulin antibody-Alexa Fluor 488, and nuclei were stained with Hoechst 33342. As shown in Figure 4, when comparing TAT-VHL alone (Figure 4A) with CPPs composition + TAT-VHL (Figure 4B), it is clear that cells induced with CPPs composition + TAT-VHL (B) showed more process extension and longer processes. This confirmed, from a morphological perspective, that mesenchymal stem cells were more efficiently induced to differentiate into neurons by culturing them 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, neurons, and neural stem cells: 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 relative to IMDM. Figure 5 shows that cells induced to differentiate with the CPP composition + TAT-VHL showed significantly higher expression of Nestin and MAP2 than cells cultured with TAT-VHL, the CPP composition, or either alone, compared to the control (IMDM), TAT-VHL alone, or the CPP composition alone. This confirmed from a genetic perspective that mesenchymal stem cells were more efficiently induced to differentiate into neurons by culturing them in a medium containing TAT-VHL in addition to a CPPs composition. These results demonstrate that cells induced in a medium containing TAT-VHL + CPPs composition were more efficiently induced to differentiate into neurons.

[0039] Example 4 Analysis of CPP Composition 1 The CPP 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 the Protein Experimental Notebook, revised 4th edition, Yodosha). The results are shown in Figure 6. Panel A of Figure 6 shows the results of CBB staining (manufactured by Nacalai Tesque, product number: 04543-51), and Panel B of Figure 6 shows the electrophoresis pattern obtained by reacting the CPP composition with an 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 stained by CBB staining as a two-chain structure of approximately 120 kDa (mass). However, it did not react with anti-PICP antibodies that label the C-terminus of the propeptide, and no band was detected, as shown in Figure 6B(b). On the other hand, in the CPP compositions shown in Figure 6C and Figure 6D, at least four bands (four-chain structure) were detected at approximately 120-200 kDa. These results indicated that the collagen in the CPP compositions contains type I procollagen in addition to type I collagen. The arrow in Figure 6B indicates the procollagen band.

[0040] Example 5: Analysis of CPP Composition 2 Collagen contained in small amounts in the CPP composition was purified separately for analysis. The ASCs used in Example 2 were grown in BSCM-PL1 medium in a T-175 flask until 100% confluent. They were then cultured for 4 days in 50 mL of medium supplemented with 5 mM magnesium ascorbyl phosphate, and the culture supernatant was collected. Culture was repeated several times until the cells detached. The collected culture supernatant was centrifuged to remove cell debris and subjected to salting out. This salting out process was repeated eight times to purify collagen. Figure 7 shows the results of SDS-PAGE stained with CBB (Nacalai Tesque, product number: 04543-51). The positions of the observed bands indicate that the purified collagen (c) contains α1-chain collagen and α2-chain collagen in a 2:1 ratio. This further confirmed that the CPP composition contains type I collagen.

[0041] (Example 6) Analysis 3 of Differentiation Induction into Neurons (Purified Collagen) The ability to induce differentiation into neurons was analyzed using a medium containing purified collagen (0.1 mg / mL) as shown in Figure 7(c). The same ASCs (manufactured by Lonza, product number: PT-5006) as in Example 3 were used as mesenchymal stem cells, and the analysis was carried out using the same procedures as in Example 3, except that purified collagen (0.1 mg / mL) was added to the medium. Specifically, the ASCs were suspended in IMDM medium containing 5% FBS (manufactured by Cytiva, product number: SH30910.03), and 1 x 10 cells were placed in a 24-well plate (manufactured by SARSTEDT, product number: 83.3922). ４ The next day, the medium was changed to (A) IMDM, (B) IMDM containing 50 μg / mL purified collagen, (C) 1 μM TAT-VHL, or (D) IMDM containing 50 μg / mL purified collagen: 2 μM TAT-VHL: IMDM (1:1), and the cells were cultured for 3 days. IMDM was used as a control. The results are shown in Figures 8 to 10.

[0042] In the cells cultured in IMDM containing 50 μg / mL of purified collagen (B) above, granular blue-stained areas (Nissl bodies) were observed in the cytoplasm, 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 differentiation-inducing effect of mesenchymal stem cells into neurons.

[0043] As in Example 2, the cytoplasm was stained with anti-neuron-specific anti-βIII-tubulin antibody-Alexa Fluor 488, and the nuclei were stained with Hoechst 33342. Figure 9 shows the results. Comparing the control cells (A) cultured in IMDM (Figure 9A) with the cells (D) cultured in IMDM containing 50 μg / mL purified collagen (Figure 9B), it is clear that the cells in (B) have more process extension. This confirms, from a morphological perspective, that the collagen component contained in the CPPs composition contributes to the differentiation-inducing effect of mesenchymal stem cells into neurons.

[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, neurons, and neural stem cells: 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 represents the mRNA expression ratio relative to IMDM. 10 shows that (B) cells cultured in IMDM containing 50 μg / mL purified collagen expressed neurofilament (NF) at a level comparable 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 confirms, from a genetic perspective, that the collagen component contained in the CPPs composition contributes to the ability to induce differentiation of mesenchymal stem cells into neurons. These results demonstrate that the collagen component contained in the CPPs composition contributes to the ability to induce differentiation of mesenchymal stem cells into neurons.

[0045] (Example 7) Analysis of differentiation induction into nerve cells 4 (bone marrow-derived stem cells) Bone marrow-derived stem cells (BM-MSC) (manufactured by Lonza, product number: PT-2501) 4 x 10 ５ BM-MSCs were cultured at 1 × 10 cells / mL in a T-75 flask (SARSTEDT, product number: 83.3911.002) in BMCM-PL1 medium (Bio Mirai Kobo) containing 2% human serum until they reached 90% confluence, and then detached with trypsin-EDTA solution (Nacalai Tesque, product number: 32777-44). BM-MSCs were suspended in IMDM medium containing 5% FBS (cytiva, catalog number SH30910.03) and plated at 1 × 10 cells / mL in a 24-well plate (SARSTEDT, product number: 83.3922). ４The cells were seeded at 1000 cells / mL / well, and the next day, the medium was replaced with one of the following media (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), and cultured for 3 days. Immedium-free medium was used as a control. As in Example 2, the cytoplasm was stained with anti-neuron-specific anti-βIII-tubulin antibody-Alexa Fluor 488, and the nuclei were stained with Hoechst 33342. Figure 11 shows the results. The vertical axis of the graph represents the mRNA expression ratio relative to IMDM. As shown in Figure 11, staining was faint in medium (A) (Figure 11A), and little process outgrowth was observed in medium (B) (Figure 11B). In contrast, neurite outgrowth was observed in medium (C) (Figure 11C), and more pronounced neurite outgrowth was observed in medium (D) (Figure 11D). These results demonstrate that bone marrow-derived stem cells can be induced to differentiate into neurons by culturing using the CPP composition. Furthermore, it was demonstrated that the use of the CPP composition in combination with TAT-VHL can more efficiently induce the differentiation of mesenchymal stem cells into neurons.

[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 even components derived from autologous cells, without using a neuronal differentiation induction medium.

Claims

1. a) culturing mesenchymal stem cells for 1 to 10 days in a stem cell culture medium comprising 60.0 to 90.0 vol% basal medium, 10.0 to 40.0 vol% physiological saline, and further comprising 1.0 to 100.0 ng / mL EGF, 0.2 to 20.0 ng / mL FGF-2, 0.2 to 20.0 ng / mL PDGF, and 0.5 to 8.0 mM magnesium ascorbyl phosphate; b) thereafter precipitating protein components of the culture medium obtained by the above culture with an organic solvent; and c) isolating and dissolving the precipitated portion in physiological saline or a basal medium, thereby culturing 3 x 10 mesenchymal stem cells using a medium containing a cell-produced protein (CPP) composition obtained by the above culture. 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 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 medium containing the CPPs composition further contains TAT-VHL.

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