Method for inducing differentiation into nerve cells

Abstract

Provided is a method for inducing differentiation into nerve cells, wherein: a) mesenchymal stem cells are cultured for 1-10 days in a stem cell culture medium that is constituted by containing 60.0-90.0 vol% of a basal medium and 10.0-40.0 vol% of physiological saline and 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, a protein component in a culture solution that has been obtained by the culturing are precipitated with an organic solvent; and c) using a medium that contains a cell-produced protein (CPPs) composition obtained by isolating a precipitate portion and dissolving the precipitate portion in physiological saline or a basal medium, the mesenchymal stem cells seeded at a density of 3×104 to 2×105 cells / mL are cultured at 4-10°C for 8 hours to 2 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] Japanese Patent Application Laid-Open No. 2013-63088

[0006] 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.

[0007] 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.

[0008] 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 with extremely high efficiency, using components obtained from a culture medium of mesenchymal stem cells.

[0009] 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: ４ ~2 x 10 ５ This method of inducing differentiation into neurons involves culturing mesenchymal stem cells seeded at 1000 cells / mL for 8 hours to 2 days at 4 to 10°C, thereby differentiating mesenchymal stem cells into neurons. This method of inducing differentiation into neurons allows for extremely efficient differentiation of mesenchymal stem cells using components obtained from mesenchymal stem cells, without using a neuronal differentiation-inducing medium.

[0010] In the above aspect, the medium containing the CPP composition may further contain Ham's F12 medium, which allows for even more efficient differentiation of mesenchymal stem cells into neurons.

[0011] According to the present invention, mesenchymal stem cells can be differentiated into neurons with extremely high efficiency using components obtained from mesenchymal stem cells, without using a neuronal differentiation-inducing medium.

[0012] Fig. 1 is a diagram showing the results of Example 3. Fig. 2 is a diagram showing the results of Example 3. Fig. 3 is a diagram showing the results of Example 3. Fig. 4 is a diagram showing the results of Example 4. Fig. 5 is a diagram showing the results of Example 4. 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 5. Fig. 9 is a diagram showing the results of Example 5.

[0013] 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: ４ ~2 x 10 ５ The method is characterized in that mesenchymal stem cells seeded at 1000 cells / mL are cultured at 4 to 10°C for 8 hours to 2 days, thereby differentiating the mesenchymal stem cells into nerve cells.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] (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.

[0019] 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%.

[0020] 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.

[0021] 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.

[0022] (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 ４ ~2 x 10 ５ The method is characterized in that mesenchymal stem cells seeded at 1×10 cells / mL are cultured at 4 to 10°C for 8 hours to 2 days, thereby differentiating the mesenchymal stem cells into neurons. ５ Culturing mesenchymal stem cells seeded at 1000kJ / mL for 8 hours to 2 days at 4-10°C is preferred, as this results in higher differentiation induction efficiency. More preferably, the incubation period at 4-10°C is 12 hours to 2 days, even more preferably 1-2 days, and most preferably 1 day. According to this method for inducing differentiation into neurons, mesenchymal stem cells can be induced to differentiate into neurons using components derived from mesenchymal stem cells without using a neuronal differentiation-inducing medium. The CPP composition preferably has a protein concentration of 80-120 μg / mL, and is contained in a medium:CPP composition ratio of 1:1 / 8 to 1, i.e., 12.5-50.0 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.

[0023] In the first aspect, the medium containing the CPP composition may further contain Ham's F12 medium. By using the CPP composition in combination with Ham's F12 medium, it is possible to induce differentiation of mesenchymal stem cells into neurons with even higher efficiency. It is preferable that Ham's F12 medium is contained in an equal volume (1:1) to the medium (basal medium or physiological saline) containing the CPP composition at the above concentration (vol %).

[0024] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0025] 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 (manufactured by Bio Mirai Kobo Co., Ltd.) (medium composition: 75 vol% IMDM as a basal medium, 25 vol% physiological saline, and further composed of 10 ng / mL EGF, 10 ng / mL FGF-2, 2 ng / mL PDGF, and 1 mM magnesium ascorbyl phosphate) to prepare a stem cell culture medium for producing a CPPs composition. Next, 4 x 10 adipose-derived stem cells (ASCs) (manufactured by 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 (manufactured by Bio Mirai Kobo Co., Ltd.) supplemented with 1% human serum (a serum-free medium for mesenchymal stem cells, manufactured by Fukoku Co., Ltd., product number: FKCM301T, can also be used as a substitute) until the cells became confluent.

[0026] 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.

[0027] 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.

[0028] Example 2: Preparation of Adipose-Derived Stem Cells (ASCs), Gingival-Derived Stem Cells (GMSCs), and Bone Marrow-Derived Stem Cells (BM-MSCs) Three types of human mesenchymal stem cells (ASCs), gingival-derived stem cells (GMSCs), and bone marrow-derived stem cells (BM-MSCs) were prepared for use in Examples 3 and beyond. These three types of stem cells were isolated at Bio Mirai Kobo from tissue collected from volunteers at an affiliated clinic, according to the procedures described in the "Mesenchymal Stem Cell Handbook" (Information System, Inc., 2023, pp. 17-22). The isolated ASCs, GMSCs, and BM-MSCs were cultured for 2-3 passages in stem cell proliferation medium BSCM-PL1 (Bio Mirai Kobo, product number: BMK-S001) supplemented with 2% FBS (Cytiva, product number: SH30910.03) before being used in the following examples.

[0029] (Example 3) Analysis of differentiation induction into neural cells (ASC) ５ The cells were suspended in IMDM or F12 to a concentration of 1000 cells / mL, and subjected to low-temperature treatment (standing in a refrigerator (4-10°C) for 1-2 days), and then diluted 10-fold with either 1) CPPs composition (solution with a protein concentration of 80-120 μg / mL) + IMDM (volume ratio 1:1) [F12 / CPPs], or 2) CPPs composition + Ham's F12 (hereinafter referred to as "F12") (volume ratio 1:1) [IMDM / CPPs], and plated onto a 24-well plate. After 3 hours, the medium was replaced as in 1) or 2).

[0030] As a control without low-temperature treatment, ASCs were plated in a 24-well plate (SARSTADT, product number: 83.3922) at 1 × 10 ４ The cells were seeded at 100 cells / well, and the next day, the medium was replaced with either 1) CPPs composition + IMDM (volume ratio 1:1) [IMDM] or 2) CPPs composition + F12 (volume ratio 1:1) [F12], and cultured for 2 days.

[0031] After the incubation, one well was fixed with 10% neutral formalin (Nacalai Tesque, product number: 37152-51), washed, and then incubated overnight in the dark with anti-human βIII-Tubulin antibody-AlexaFluor488 (Merck Millipore, product number: AB15708A) diluted 200-fold with 1% FBS saline. The wells were then washed with distilled water and stained with 5 μg / mL Heochst 33342 (ThermoFisher, product number: H3570) in aqueous solution for 3-5 minutes in the dark. After rinsing with water, the wells were photographed (×100) under a fluorescence microscope (Keyence, product number: BZ-X800). The differentiation state of ASCs, such as the extension of processes, was visually observed after 0 days (no treatment), 1 day, and 2 days of low-temperature treatment. The results are shown in Figures 1 to 3 as F12 / CPPs, IMDM / CPPs, F12 (control), and IMDM (control).

[0032] After the above incubation, mRNA was extracted from another well using the Maxwell® RSC simplyRNA Cells Kit (Promega, product number AS1390), and qPCR was performed using Step One PLUS (ThermoFisher) with TaqMan Assays (GAPDH: Hs99999905_m1, Nestin: Hs04187831_g1, MAP2: Hs00258900_m1, Neurofilament: Hs00196245_m1) to measure the expression of the neuron-specific genes Nestin, MAP2, and Neurofilament (Table 1).

[0033]

[0034] The results in Figures 1 to 3 show that low-temperature treatment increased the number of extended processes in both IMDM / CPPs and F12 / CPPs compared to their respective controls, IMDM and F12. Furthermore, in this example, the number of extended processes was particularly high after one day of low-temperature treatment, and F12 extended a greater number of processes than IMDM. From this morphological standpoint, it was confirmed that low-temperature culture for a certain period in a medium containing a CPP composition induced the differentiation of ASCs, which are mesenchymal stem cells, into neurons with extremely high efficiency.

[0035] Table 1 shows the results of calculating the relative expression levels of IMDM / CPPs and F12 / CPPs, with the expression levels of the respective controls, IMDM and F12, set to 1.00 (reference value). The results in Table 1 indicate that the expression of Nestin, MAP2, and Neurofilament was increased compared to the respective controls, IMDM and F12. This indicates that low-temperature treatment significantly increased the expression of neuron-specific genes, guiding ASCs to differentiate into neurons. Furthermore, in this example, the expression levels of the above three genes were particularly high after one day of low-temperature treatment, with Neurofilament expression particularly high. Furthermore, the expression levels of the above three genes were higher in F12 than in IMDM. This confirms, from a genetic perspective, that low-temperature culture for a certain period in a medium containing a CPP composition induced the differentiation of ASCs, which are mesenchymal stem cells, into neurons with extremely high efficiency.

[0036] Example 4: Analysis 2 of Neuronal Differentiation (GMSCs) Neuronal differentiation was analyzed using the same procedure as in Example 3, except that GMSCs were used as stem cells. GMSCs were subjected to low-temperature treatment for 0 days (none), 1 day, and 2 days, respectively, using the same method as in Example 3. The differentiation-induced state of the cells, such as the extension of processes, was observed morphologically using a fluorescent microscope. The results are shown in Figures 4 to 6. Furthermore, using the same method as in Example 3, GMSCs were subjected to low-temperature treatment for 0 hours (none), 8 hours (8h), 12 hours (12h), and 1 day (1day), and the expression of MAP2 and Neurofilament was measured in the cells induced to differentiate into neurons (Table 2).

[0037]

[0038] The results in Figures 4 to 6 show that low-temperature treatment increased the number of extended processes in both IMDM / CPPs and F12 / CPPs compared to their respective controls, IMDM and F12. Furthermore, F12 extended a greater number of processes than IMDM. From this, it was confirmed from a morphological standpoint that low-temperature culture for a certain period in a medium containing a CPP composition induced the differentiation of mesenchymal stem cells (GMSCs) into neurons with extremely high efficiency.

[0039] Table 2 shows the results of calculating the relative expression levels of IMDM / CPPs and F12 / CPPs, with the expression levels of the respective controls, IMDM and F12, set to 1.00 (reference value). The results in Table 2 indicate that the expression of MAP2 and Neurofilament was increased compared to the respective controls, IMDM and F12. This indicates that low-temperature treatment significantly increased the expression of neuron-specific genes, guiding GMSCs to differentiate into neurons. In this example, the expression levels of all genes were higher in F12 than in IMDM, and in particular, the expression level of Neurofilament in cells subjected to low-temperature treatment for 1 day was nearly 100-fold higher than the reference value. This confirms, from a genetic perspective, that low-temperature culture in a medium containing a CPP composition for a certain period of time induced the differentiation of mesenchymal stem cells, GMSCs, into neurons with extremely high efficiency.

[0040] Example 5: Analysis 3 of Neuronal Differentiation Induction (BM-MSC) Analysis of neuronal differentiation induction was performed using the same procedure as in Example 3, except that BM-MSCs were used as stem cells. BM-MSCs were subjected to low-temperature treatment for two days using the same method as in Example 3, and then the differentiation induction state of the cells, such as extended processes, induced into neurons was observed morphologically using a fluorescent microscope. The results are shown in Figures 7 to 9. Furthermore, using the same method as in Example 3, the expression of MAP2 and Neurofilament was measured for BM-MSCs induced to differentiate into neurons on the second day of culture after two days of low-temperature treatment (Table 3).

[0041]

[0042] The results in Figures 7 to 9 show that low-temperature treatment increased the number of extended processes in both IMDM / CPPs and F12 / CPPs compared to their respective controls, IMDM and F12. Furthermore, in this example, F12 / CPPs had the greatest number of extended processes on day 2 of culture. This morphologically confirmed that low-temperature culture for a certain period in a medium containing a CPP composition induced the differentiation of mesenchymal stem cells (BM-MSCs) into neurons with extremely high efficiency.

[0043] Furthermore, the results in Table 3 show that low-temperature treatment significantly increased the expression of genes specific to nerve cells, indicating that BM-MSCs were induced to differentiate into nerve cells. In this example, the expression levels of all genes were higher in F12 than in IMDM, and in particular, the expression level of Neurofilament was more than 100 times the reference value. This confirms from a genetic perspective that low-temperature culture for a certain period in a medium containing a CPP composition induced the differentiation of BM-MSCs, which are mesenchymal stem cells, into nerve cells with extremely high efficiency.

[0044] These results demonstrate that mesenchymal stem cells can be induced to differentiate into neurons with extremely high efficiency by culturing them at low temperatures (low-temperature treatment) for a certain period of time in a medium containing a CPP composition before inducing differentiation. Furthermore, it is also demonstrated that the inclusion of Ham's F12 medium in the medium containing the CPP composition can further enhance the efficiency of differentiation into neurons.

[0045] According to the culture method of the present invention, it is possible to induce differentiation of mesenchymal stem cells into neurons with extremely high efficiency by 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 and 10.0 to 40.0 vol% physiological saline, to which 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 precipitate in physiological saline or a basal medium, ４ ~2 x 10 ５ A method for inducing differentiation into neurons, in which mesenchymal stem cells are differentiated into neurons by culturing mesenchymal stem cells seeded at 1000kJ / mL at 4-10°C for 8 hours to 2 days.

2. The method for inducing differentiation into nerve cells according to claim 1, wherein the medium containing the CPP composition further contains Ham's F12 medium.

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