Medium for culturing stem cells, cell-produced protein composition, and method for inducing differentiation into osteoprogenitor cells or osteoblasts

Abstract

The present invention provides a stem cell culture medium that induces differentiation of mesenchymal stem cells and osteoprogenitor cells into osteoblasts, comprising 60-90 vol% of a basal medium and 10-40 vol% of physiological saline, to which 1-100 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 are further added.

Description

[Technical field]

[0001] The present invention relates to a medium for stem cell culture, a cell-produced protein composition obtained by using the medium, and a method for inducing differentiation into osteoblasts and osteoblast precursor cells using the same. In particular, the present invention provides a method for inducing differentiation of mesenchymal stem cells or osteoblast precursor cells into osteoblasts by the cell-produced protein composition obtained by using the medium for stem cell culture. [Background technology]

[0002] Bone metabolism is maintained by a balance between bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts are thought to originate from mesenchymal stem cells, whereas osteoclasts are thought to originate from monocytes / macrophages in the modeling system.

[0003] Conventionally, a method for differentiating mesenchymal stem cells into osteoblasts is known in which mesenchymal stem cells (MSCs) are cultured in a dish or plate containing a cell proliferation medium, and when the cells reach a confluent state of 80% or more, the medium is replaced with an osteoblast differentiation-inducing medium and the cells are cultured for 2 weeks. As an index for evaluating differentiation induction into osteoblasts, a method is known in which calcification caused by the differentiation induction of mesenchymal stem cells into osteoblasts is evaluated by staining calcium with a dye such as alizarin red (for example, Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2005-124460 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the above-mentioned methods, calcification occurs as a result of the differentiation of mesenchymal stem cells into osteoblasts, making it difficult to detach the cells from the dish or plate, and the differentiated osteoblasts cannot be reseeded, administered, transplanted, etc., at the desired location.

[0006] On the other hand, the stage of osteoprogenitor cells, which is a differentiation state preceding osteoblasts, was judged by the formation of nodules (calcified spheres), which indicate the start of calcification, and the osteoprogenitor cells were collected at that stage and reseeded, administered, transplanted, etc. However, in this case, no calcification was observed in the reseeded, administered, or transplanted sites, and furthermore, the osteoprogenitor cells reverted to an undifferentiated state. This indicates that osteoblast differentiation does not proceed when osteoprogenitor cells are simply reseeded, administered, transplanted, etc. without the use of an osteoblast differentiation-inducing medium.

[0007] Furthermore, if an osteoblast differentiation-inducing medium is used at the site of reseeding, administration, or transplantation, it is possible to induce differentiation into osteoblasts in about 10 days. However, when the administration / transplantation site is in vivo, it is difficult to administer / supplement the osteoblast differentiation-inducing medium at the site of administration / transplantation for about 10 days.

[0008] Dexamethasone, ascorbic acid, and β-glycerophosphate are believed to be necessary to induce differentiation of mesenchymal stem cells into osteoblasts, and these are contained in the osteoblast differentiation induction medium. In addition, osteoblast differentiation induction medium generally contains fetal bovine serum (FBS), which has a strong effect of differentiating mesenchymal stem cells into osteoblasts and does not contain any cytotoxic components. FBS is a component derived from a different animal, and has the risk of antigenicity and zoonotic virus infection, making it difficult to use in the above-mentioned administration / transplantation destinations.

[0009] In view of the above circumstances, the present inventors have conducted further research and have newly discovered cell producing proteins (CPPs) containing osteoblast differentiation-inducing components that can be used in place of osteoblast differentiation-inducing medium and that can be prepared from mesenchymal stem cells, thereby completing the present invention.

[0010] The present invention has been made in view of the above-mentioned circumstances, and aims to provide a method for inducing differentiation of mesenchymal stem cells and osteoblast precursor cells into osteoblasts. More specifically, the present invention aims to provide a method for inducing differentiation into osteoblasts, which enables differentiation of mesenchymal stem cells and osteoblast precursor cells into osteoblasts using components obtained from mesenchymal stem cells without using an osteoblast differentiation-inducing medium. [Means for solving the problem]

[0011] The present invention has been made based on the above findings and has an object to advantageously solve the above problems. A first aspect of the present invention is 60-90 vol% basal medium, 10 to 40 vol% saline solution, and 1 to 100 ng / mL EGF, 0.2 to 20.0 ng / mL of FGF-2, 0.2 to 20.0 ng / mL PDGF, 0.5 to 8.0 mM magnesium ascorbyl phosphate; The present invention relates to a medium for culturing stem cells, the medium being composed of: Such a medium for stem cell culture can be used to prepare a cell-produced protein (CPPs) composition from mesenchymal stem cells, and the CPPs composition can be used to induce differentiation of mesenchymal stem cells and osteoprogenitor cells into osteoblasts. As the basal medium, IMDM, DMEM, and α-MEM can be used. In the composition of the above-mentioned medium for stem cell culture, the purpose of adding physiological saline is to lower the Ca concentration in the above-mentioned basal medium to 10-40%. Therefore, when the Ca concentration of the basal medium used is 40-50 μg / mL, there is no need to dilute it with physiological saline.

[0012] A second aspect of the present invention is Cultivating mesenchymal stem cells for 1 to 10 days in the stem cell culture medium according to the first aspect of the present invention; The protein components are then precipitated with an organic solvent. The precipitate is separated and dissolved in physiological saline or basal medium. The present invention relates to a cell-produced protein (CPP) composition for inducing differentiation of mesenchymal stem cells or osteoprogenitor cells into osteoblasts, the composition being obtained by the above steps. According to such a CPPs composition, it is possible to induce differentiation of mesenchymal stem cells and osteoblast precursor cells into osteoblasts using components obtained from mesenchymal stem cells, without using an osteoblast differentiation-inducing medium.

[0013] Moreover, in the above second aspect, the CPPs composition may contain type I procollagen and hyaluronic acid, and calcium may be bound to the type I procollagen. Type I procollagen forms a complex with calcium to serve as a scaffold for cells and to facilitate the supply of calcium to cells.

[0014] A third aspect of the present invention is 1) Mesenchymal stem cells are cultured in osteoblast differentiation-inducing medium for 2 to 6 days while attached to a culture vessel, and allowed to proliferate until confluent or greater; or 2) Mesenchymal stem cells are placed in a culture vessel in a suspended state and stored in a refrigerator for 2 to 4 days. Mesenchymal stem cells are differentiated into osteoblast precursor cells, and then The osteoprogenitor cells are collected and seeded in a culture vessel, and cultured in a basal medium containing the CPPs composition according to the second aspect of the present invention. The present invention relates to a method for inducing differentiation into osteoblasts, which comprises differentiating the osteoblast precursor cells into osteoblasts. According to such a method for inducing differentiation into osteoblasts, it is possible to induce differentiation of osteoblast precursor cells into osteoblasts using components obtained from mesenchymal stem cells without using an osteoblast differentiation-inducing medium. In the above aspect, the mesenchymal stem cells may be derived from bone marrow, fat, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

[0015] A fourth aspect of the present invention is 1) Mesenchymal stem cells are cultured in osteoblast differentiation-inducing medium for 2 to 6 days while attached to a culture vessel, and allowed to proliferate until confluent or greater; or 2) Mesenchymal stem cells are placed in a culture vessel in a suspended state and stored in a refrigerator for 2 to 4 days. Differentiating mesenchymal stem cells into osteoprogenitor cells, This is a method for inducing differentiation into osteoblast precursor cells. According to such a method for inducing differentiation into osteoblast precursor cells, osteoblast precursor cells for reseeding, administration or transplantation can be prepared without calcification occurring before reseeding, administration or transplantation. In the above aspect, the mesenchymal stem cells may be derived from bone marrow, fat, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

[0016] A fifth aspect of the present invention is The osteoprogenitor cells obtained by the differentiation induction method according to the fourth aspect are seeded and, at the same time, the CPPs composition according to the second aspect is added to differentiate the osteoprogenitor cells into osteoblasts; or seeding osteoprogenitor cells obtained by the differentiation induction method according to the fourth aspect above, and then adding the CPPs composition according to the second aspect above, thereby initiating differentiation of the osteoprogenitor cells into osteoblasts; This is a method for inducing differentiation into osteoblasts. According to such a method for inducing differentiation into osteoblasts, it is possible to induce differentiation of osteoblast precursor cells into osteoblasts at a desired timing in the reseeding, administration or transplantation destination. Effect of the Invention

[0017] According to the present invention, mesenchymal stem cells and osteoblast precursor cells can be differentiated into osteoblasts using components obtained from mesenchymal stem cells without using an osteoblast differentiation-inducing medium. [Brief description of the drawings]

[0018] [Figure 1] Figure 1 shows: A: a photograph of Alizarin Red staining, B: the results of measuring absorbance at OD450nm, and C: a magnified microscopic photograph (x40) of Alizarin Red staining. [Diagram 2] FIG. 2 shows the results of Example 2. [Diagram 3] FIG. 3 shows the results of Example 2. [Figure 4] FIG. 4 shows the results of Example 2. [Diagram 5] FIG. 5 shows the results of Example 2. [Figure 6] FIG. 6 shows the results of Example 3. [Figure 7] FIG. 7 shows the results of Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Hereinafter, an embodiment of the present invention will be described in detail. (Stem cell culture medium) The medium for stem cell culture according to the first aspect of the present invention comprises: 60-90 vol% basal medium, 10 to 40 vol% saline solution, and 1 to 100 ng / mL EGF, 0.2 to 20.0 ng / mL of FGF-2, 0.2 to 20.0 ng / mL PDGF, 0.5 to 8.0 mM magnesium ascorbyl phosphate; The present invention is characterized in that it is a medium for stem cell culture, comprising: The present inventors have newly discovered that a cell-produced protein (CPPs) composition, which will be described later, can be obtained from mesenchymal stem cells cultured using the above-mentioned stem cell culture medium. As the basal medium, IMDM, DMEM, and α-MEM can be used. In the composition of the above-mentioned medium for stem cell culture, the purpose of adding physiological saline is to lower the Ca concentration in the above-mentioned basal medium to 10-40%. Therefore, when the Ca concentration of the basal medium used is 40-50 μg / mL, there is no need to dilute it with physiological saline.

[0020] Regarding the supplemental factors added to the above-mentioned stem cell culture medium, EGF is epidermal growth factor, FGF-2 is fibroblast growth factor 2, and PDGF is platelet-derived growth factor. The above-mentioned supplemental factors are generally used as supplemental factors to basal media for the purpose of cell proliferation, etc. In the medium for stem cell culture according to the first aspect of the present invention, EGF 1-10ng / mL, FGF-2, 0.5 to 5.0 ng / mL; PDGF 0.5-5.0ng / mL, When the amount of the CPPs added is within this range, the CPPs composition can be produced more efficiently.

[0021] In addition, in the medium for stem cell culture according to the first aspect of the present invention, 0.1 to 10.0 μg / mL transferrin, and / or 0.2–20.0 μg / mL insulin, and / or 0.1-3.0ng / mL sodium selenite It is preferable to further add the above in terms of enhancing the proliferation ability of the cells.

[0022] Specific methods for preparing the stem cell culture medium include, for example, From the 500 mL of IMDM, remove 50 mL to 200 mL, add an equal amount of saline to make 500 mL, add 5 mL of antibiotics, moreover, EGF 1-10ng / mL, FGF-2, 0.5 to 5.0 ng / mL; PDGF 0.5-5.0ng / mL, Transferrin 0.1 to 10.0 μg / mL, Insulin 0.2-20.0μg / mL, Sodium selenite 0.1-3.0ng / mL, Magnesium ascorbyl phosphate, 0.5 to 8.0 mM; It can be prepared by adding in the range.

[0023] (Cell-Produced Proteins (CPPs) Composition) The CPPs composition according to the second aspect of the present invention comprises: Cultivating mesenchymal stem cells for 1 to 10 days in the stem cell culture medium according to the first aspect of the present invention; The protein components are then precipitated with an organic solvent. The precipitate is separated and dissolved in physiological saline or basal medium. The present invention is characterized by being a CPPs composition for inducing differentiation of mesenchymal stem cells or osteoprogenitor cells into osteoblasts, which is obtained by the above-mentioned method. According to such a CPPs composition, mesenchymal stem cells and osteoblast precursor cells can be induced to differentiate into osteoblasts. Furthermore, according to such a CPPs composition, mesenchymal stem cells and osteoblast precursor cells can be induced to differentiate into osteoblasts using components obtained from mesenchymal stem cells and even components derived from autologous cells, without using an osteoblast differentiation-inducing medium.

[0024] Moreover, in the above second aspect, the CPPs composition may contain type I procollagen and hyaluronic acid, and calcium may be bound to the type I procollagen. The CPPs composition contains, for example, 30 to 100 μg / mL of type I procollagen and 49 to 140 μg / mL of hyaluronic acid. The binding ratio of type I procollagen to calcium is, for example, 30 to 45%, and preferably 35 to 42%.

[0025] In the second embodiment, the mesenchymal stem cells may be bone marrow-derived mesenchymal stem cells, adipose-derived stem cells (ASC), peripheral blood-derived mesenchymal stem cells, umbilical cord Wharton's Jelly-derived mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, etc. The basal medium may be IMDM, DMEM, or α-MEM. The culture period is 1 to 10 days, preferably 3 to 4 days.

[0026] In the second embodiment, in the step of "collecting the precipitated portion and dissolving it in physiological saline or a basal medium" following the step of "precipitating the protein components with an organic solvent", the protein components precipitated with the organic solvent may be suspended in hydrochloric acid (e.g., 1N HCl) at pH 1, vortexed (e.g., for about 10 minutes), and then neutralized (e.g., with 1.2N NaOH) to simultaneously perform virus inactivation treatment. In this case, the neutralized solution is centrifuged, and the resulting supernatant is used as the cell-produced protein (CPPs) composition.

[0027] In this specification, the term "osteoblast precursor cells" is defined as "cells that have formed (partially calcified) nodules, which indicate the start of calcification, and that can be detached with trypsin-EDTA." The present inventors have newly discovered that mesenchymal stem cells can be induced to differentiate into osteoprogenitor cells by short-term culture in the step 1) or 2) described below.

[0028] (Method for inducing differentiation of mesenchymal stem cells into osteoblasts) A method for inducing differentiation into osteoblasts according to a third aspect of the present invention comprises: 1) Mesenchymal stem cells are cultured in osteoblast differentiation-inducing medium for 2 to 6 days while attached to a culture vessel, and allowed to proliferate until confluent or greater; or 2) Mesenchymal stem cells are placed in a culture vessel in a suspended state and stored in a refrigerator for 2 to 4 days. Mesenchymal stem cells are differentiated into osteoblast precursor cells, and then The osteoprogenitor cells are collected and seeded in a culture vessel, and cultured in a basal medium containing the CPPs composition according to the second aspect of the present invention. The method is characterized in that the osteoblast precursor cells are differentiated into osteoblasts.

[0029] According to such a method for inducing differentiation into osteoblasts, it is possible to induce differentiation of osteoblast precursor cells into osteoblasts using components obtained from mesenchymal stem cells without using an osteoblast differentiation-inducing medium. In the third aspect, the mesenchymal stem cells may be derived from bone marrow, fat, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

[0030] In the above third aspect, the concentration of the CPPs composition in the "basal medium supplemented with the CPPs composition" is preferably 25 to 75 vol%, more preferably 50 vol%. "Refrigerated storage" means storage at 4 to 10°C. Moreover, the term "confluent or more" means overgrowth of up to about 200%. In this specification, the term "confluent" refers to a state in which adherent cells completely cover the adhesive surface of the culture vessel, that is, a state of 100% confluence.

[0031] (Method for inducing differentiation of mesenchymal stem cells into osteoblast precursor cells) A fourth aspect of the present invention is 1) Mesenchymal stem cells are cultured in osteoblast differentiation-inducing medium for 2 to 6 days while attached to a culture vessel, and allowed to proliferate until confluent or greater; or 2) Mesenchymal stem cells are placed in a culture vessel in a suspended state and stored in a refrigerator for 2 to 4 days. It is characterized by differentiating mesenchymal stem cells into osteoprogenitor cells. This is a method for inducing differentiation into osteoblast precursor cells.

[0032] According to such a method for inducing differentiation into osteoblast precursor cells, osteoblast precursor cells for reseeding, administration or transplantation can be prepared without calcification occurring before reseeding, administration or transplantation. In the fourth aspect, the mesenchymal stem cells may be derived from bone marrow, fat, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

[0033] In the fourth embodiment, "refrigerated storage" means storage at 4 to 10°C. Moreover, the term "confluent or greater" means an overgrowth of up to 200%.

[0034] (Method for inducing differentiation of osteoblast precursor cells into osteoblasts) A fifth aspect of the present invention is The osteoprogenitor cells obtained by the differentiation induction method according to the fourth aspect are seeded and, at the same time, the CPPs composition according to the second aspect is added to differentiate the osteoprogenitor cells into osteoblasts; or seeding osteoprogenitor cells obtained by the differentiation induction method according to the fourth aspect above, and then adding the CPPs composition according to the second aspect above, thereby initiating differentiation of the osteoprogenitor cells into osteoblasts; This is a method for inducing differentiation into osteoblasts.

[0035] According to such a method for inducing differentiation into osteoblasts, it is possible to induce differentiation of osteoprogenitor cells into osteoblasts at a desired timing in the reseeding, administration, or transplantation destination. In particular, when a CPPs composition is added after seeding osteoprogenitor cells, the CPPs composition can be used as a booster for initiating differentiation into osteoblasts. Furthermore, in the above fifth aspect, the "CPPs composition" is added to the medium used for culturing osteoprogenitor cells so as to account for 25 to 75 vol%, preferably 50 vol%. EXAMPLES

[0036] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0037] Example 1 Preparation of stem cell culture medium and CPPs composition 5 mL of Antibiotic-Antimycotic Mixed Stock Solution (Nacalai Tesque, product number: 02892-54) was added as an antibiotic to 500 mL of BSCM-PL2 medium for stem cell proliferation (Bio Mirai Kobo) to prepare a stem cell culture medium for creating a CPPs composition. Next, 4 × 10 adipose-derived stem cells (ASCs) (manufactured by Lonza, product number: PT-2501) belonging to mesenchymal stem cells (MSCs) were 5 2 × 10 cells / mL were cultured in a T-75 flask (SARSTEDT, product number: 83.3911.002) using BMCM-PL1 medium (Bio Mirai Kobo) containing 2% human serum until the cells reached 90% confluence, and then detached with trypsin-EDTA solution (Nacalai Tesque, product number: 32777-44). Then, 2 × 10 cells / mL were cultured in a Hyper Flask (Corning, Inc.) until the cells reached 90% confluence. 6 The cells were seeded at 200 cells / mL and then cultured again in 560 mL of BMCM-PL1 medium (Bio Mirai Kobo) containing 1% human serum until the cells became confluent.

[0038] The medium was then replaced with BSCM-PL2 medium for stem cell proliferation (manufactured by Bio Mirai Kobo Co., Ltd.) and cultured for 5 days. The entire medium was collected and 560 mL of new medium for stem cell culture was added, and cultured for another 5 days. The entire medium was collected and added until the cells were detached, and the culture supernatant thus collected was centrifuged (3000 rpm, 5 minutes) to remove cell debris. The protein fraction was then separated by organic solvent precipitation using 80% ethanol or acetone. The protein fraction was dissolved in PBS(-) (manufactured by Nacalai Tesque, product number: 07269-84), the insoluble fraction was removed by centrifugation (8000 rpm, 10 minutes), and precipitated again with 80% ethanol or acetone.

[0039] The precipitate was dissolved in 10 mL of 1N hydrochloric acid (Nacalai Tesque, product number: 18320-15), stirred at room temperature at 2,500 rpm for 10 minutes on a vortex, neutralized with 1.2N caustic soda (Nacalai Tesque, product number: 31511-05) to inactivate the virus, and then centrifuged to remove insoluble matter. Finally, the solution was sterilized through a 0.45 μm filter (Cytiva, product number: 6900-2504) to obtain a CPPs composition.

[0040] (Comparative Example 1) Preparation of control culture supernatant (CM) Next, as a comparative control, a general culture supernatant, CM, was prepared as follows. ASC (Lonza, product number: PT-2501) 4 x 10 5The cells / mL were cultured in a T-75 flask (ARSTEDT, product number: 83.3911.002) in BMCM-PL1 medium (Bio Mirai Kobo) containing 2% human serum until 90% confluent, detached with trypsin-EDTA solution (Nacalai Tesque, product number: 32777-44), and cultured again in a Hyper Flask (Corning) in 560 mL of BMCM-PL1 medium (Bio Mirai Kobo) containing 1% human serum until confluent, and the culture supernatant was collected. The culture supernatant was then centrifuged to remove insoluble matter. Finally, the cells were sterilized through a 0.45 μm filter (Cytiva, product number: 6900-2504) to obtain CM.

[0041] Example 2 Preparation of osteoprogenitor cells by adhesion culture ASC (Lonza, product number: PT-2501) 3 x 10 5 cells / mL were seeded in a 10 cm dish (SARSTEDT, product number: 83.3902) using 10 mL of mesenchymal stem cell proliferation medium (Fukoku Co., Ltd., product number: FKCM 301T) to a confluence of 20 to 30%, and cultured. Next, the cells were cultured under the following culture conditions (1) to (4), and then the medium was replaced with osteoblast differentiation induction medium BMK-R008 (manufactured by Bio Mirai Kobo Co., Ltd.) and cultured for 2, 4, or 6 days, respectively. Culture conditions: (1) Culture until 80-90% confluent. Culture condition (2) When the cells were 80-90% confluent, the medium was replaced with IMDM (Nacalai Tesque, product number: 11506-05) and cultured until the cells were confluent. Culture conditions (3) Culture until confluent. Culture conditions (4) Culture for 2 days until the cells become confluent and then overgrow.

[0042] Next, ASCs cultured in a 10 cm dish were detached and collected, suspended in the above IMDM, and diluted to 1 × 10 5The cells were prepared at a concentration of 1.0 cells / mL / well and seeded onto a 24-well plate (SARSTEDT, product number: 83.3922). The next day, four media were cultured: Culture medium (A) IMDM, Medium (B) CM+IMDM (mixing ratio 1:1), Medium (C) CPPs composition + IMDM (mixing ratio 1:1), Medium (D) osteoblast differentiation induction medium (positive control); The medium was replaced with new one, and the cells were cultured for 1 to 8 days while the medium was replaced. The CM and CPPs compositions used were those prepared in (Example 1) and (Comparative Example 1) above. The above mixing ratios are volume ratios.

[0043] After 1 to 8 days of culture as described above, each of wells (1) to (4) was fixed using the Alizarin Red S staining kit BMK-R009 (manufactured by Bio Miraikobo Co., Ltd.), the calcified nodules (calcium phosphate) were stained, and the dye bound to the calcium phosphate was extracted with 5% formic acid and the absorbance was measured at OD450nm.

[0044] When stained red with Alizarin Red, the absorbance at OD450nm of the dye eluted with formic acid is 0.100 or more. The experiment that supports this is shown below. The above culture condition (4): "Cultured for 2 days to allow overgrowth after reaching confluence (100%)", then the medium was changed to osteoblast differentiation induction medium and cultured for 4 days. The cells were then cultured for an additional 3 to 8 days in each of the following media. (a)CM+IMDM (mixing ratio 1:1) (b) CPPs composition + IMDM (mixing ratio 1:1) (c) Osteoblast differentiation induction medium (positive control)

[0045] The results are shown in Figure 1. A: Photograph of Alizarin Red staining (culture day 6), B: Measurement results of absorbance at OD450nm (culture days 3, 6, and 8, respectively, from the left column), C: Microscopic magnified photograph (x40) of Alizarin Red staining (culture day 6). As for (a), A(a) and C(a) in Figure 1 show that it is not stained. The absorbance at this time is 0.009, as shown in B(a) in Figure 1. Regarding (b), A(b) and C(b) in Figure 1 show that the nodules (calcified spheres) are stained and calcification has begun. The absorbance at this time is 0.121, as shown in B(b) in Figure 1, which is about 10 times that of B(a). (c) shows a fully calcified state as a positive control. From the above results, an absorbance at OD450nm of 0.100 or more was judged to indicate the presence of calcification, and this was used as the criterion for judging that mesenchymal stem cells and osteoprogenitor cells had been induced to differentiate into osteoblasts.

[0046] The tables in Figures 2 to 5 show the results for the above culture conditions (1) to (4), respectively. Under culture conditions (1) and (2), almost no calcification was observed except in the positive control medium (D), and no induction of differentiation into osteoblasts was observed (Figures 2 and 3). On the other hand, under culture condition (3), when the cells were cultured in osteoblast differentiation medium at confluence, detached and seeded on a plate 2 and 4 days later, and then culture medium (C): CPPs composition + IMDM was used, calcification occurred 8 days later. Furthermore, when the cells were cultured in osteoblast differentiation induction medium at confluence, detached and seeded on a plate 6 days later, and then culture medium (C) was used, calcification occurred 3 and 8 days later, confirming that differentiation into osteoblasts had been induced (Figure 4). Furthermore, under culture condition (4), the cells were allowed to overgrow for 2 days from confluence, then exchanged for osteoblast differentiation-inducing medium, and detached 4 and 6 days later, seeded onto plates, and exchanged for medium (C). It was confirmed that calcification occurred 6 to 8 days and 1 to 8 days later, respectively, and that differentiation into osteoblasts had been induced (Figure 5).

[0047] As shown in the tables of Figures 2 to 5, when cultured in the positive control medium (D), calcification occurred 3 days after seeding on the plate, but when cultured in medium (A): IMDM and medium (B): CM + IMDM, calcification did not occur at all under any of culture conditions (1) to (4). In contrast, when cultured in medium (C): CPPs composition + IMDM, calcification was promoted according to the culture period under culture conditions (3) and (4) as described above, and differentiation into osteoblasts could be induced. From this, it is considered that the cells that were induced to differentiate into osteoprogenitor cells under culture conditions (3) and (4) were further induced to differentiate into osteoblasts by culture in (C) CPPs composition+IMDM.

[0048] (Example 3) Preparation of osteoprogenitor cells by suspension Next, we investigated a method for differentiating MSCs into osteoprogenitor cells in a suspended state without adhering them to a culture vessel. ASC (Lonza, product number: PT-2501) 1 x 10 6 The cells / mL were cultured in a T-175 flask (SARSTEDT, product number: 83.3912.002) using 40 mL of mesenchymal stem cell proliferation medium FKCM 301T (Fukoku), and 1.7 × 10 6 The cells were cultured in the four media used in Example 2: Culture medium (A) IMDM, Medium (B) CM+IMDM (mixing ratio 1:1), Medium (C) CPPs composition + IMDM (mixing ratio 1:1), Medium (D) osteoblast differentiation induction medium (positive control); 4 mL of each solution was added to four 15 mL tubes (SARSTEDT, product number: 62.554.5.2) and mixed, and the tubes were left to stand in a suspended state in a refrigerator (4-5°C) for 2 days, 4 days, and 6 days, respectively. The above mixing ratios are by volume.

[0049] After each refrigerated storage period, 10 μL was sampled from each tube and the number of viable cells was measured using a hemocytometer (Elma Sales, product number: 4025). Then, the medium in each of the four tubes was centrifuged to remove the cells, and the cells were suspended in IMDM (Nacalai Tesque, product number: 11506-05), and then seeded on a 48-well plate (SARSTEDT, product number: 83.3923).

[0050] The viability (survival) rate of ASC counted by the above-mentioned viable cell count measurement was 84-89% on the second day, 88-93% on the fourth day, and 79-91% on the sixth day. The cells were seeded on the 48-well plate, and the next day after adhesion, the medium was replaced with one of the four types of medium (A) to (D) above. In this way, the cells were cultured under 16 different conditions, and after 4 and 8 days, Alizarin Red staining was performed in the same manner as in Example 2.

[0051] The results are shown in the table of Figure 6. When the cells were stored in a suspended state for 2 and 4 days under refrigeration, calcification was observed after 8 days of culture in (C): CPPs composition + IMDM. However, when the cells were stored in a suspended state for 6 days under refrigeration, calcification was observed only in medium (D): osteoblast differentiation induction medium (positive control). In medium (A): IMDM and medium (B): CM + IMDM, no calcification was observed under any of the culture conditions. From the above results, it is believed that ASCs were induced to differentiate into osteoprogenitor cells after short-term storage in a suspended state under refrigeration, and then, by culturing in a medium containing the CPPs composition, the osteoprogenitor cells were induced to differentiate into osteoblasts. Furthermore, it was found that the medium used for storage in a suspended state under refrigeration did not affect the subsequent induction of differentiation into osteoblasts, even if it did not contain the CPPs composition.

[0052] (Example 4) Verification of differentiation induction into osteoprogenitor cells by suspension The four types of media used in Example 2 were used without the step of refrigerated storage in a suspended state: Culture medium (A) IMDM, Medium (B) CM+IMDM (mixing ratio 1:1), Medium (C) CPPs composition + IMDM (mixing ratio 1:1), Medium (D) osteoblast differentiation induction medium (positive control); ASC (Lonza, product number: PT-2501) was added to a 24-well plate (SARSTEDT, product number: 83.3922) containing 0.5 mL / well. 5 cells / mL were seeded and cultured for 7 to 11 days, after which Alizarin Red staining was performed in the same manner as in Example 2. The above mixing ratio is a volume ratio. The results are shown in Table 1. Only the positive control medium (D) was calcified, and no calcification was observed in the media (A) to (C). This shows that the process of refrigerated storage in a suspended state (a process of leaving the cells suspended in a culture vessel and storing them in a refrigerator for 2 to 4 days) before culturing in a basal medium containing the CPPs composition is one of the essential processes for inducing differentiation into osteoblasts.

[0053] [Table 1]

[0054] (Example 5) Analysis of CPPs composition 1 To characterize the CPPs composition, the concentrations of type I collagen, hyaluronic acid, and collagen and Ca were measured and compared with those of the control culture supernatant (CM). The concentration of type I collagen in the CPPs was measured by ELISA (a method equivalent to human collagen type 1 ELISA (Acel, product number: EC1-E105)) and SDS-PAGE (performed according to the Protein Experimental Notebook, Revised 4th Edition, Yodosha), and was found to be 60-100 μg / mL, with an average of 80 μg / mL. The hyaluronic acid concentration in the CPPs was measured using a hyaluronic acid quantification kit (Cosmo Bio Co., Ltd., product number: HA-96KIT) and was found to be 140 μg / mL. The TGF-β1 in the CPPs was measured using a human TGF-β1 ELISA kit (Bioss Inc., product number: BSKH1021) and found to be 384 to 810 pg / mL. The same measurements were also performed on the CM, and the results are shown in Table 2.

[0055] [Table 2]

[0056] When the CPPs composition was compared with CM, a common culture supernatant, the type I collagen concentration in the CPPs composition was approximately 42 times higher, the hyaluronic acid concentration was approximately 50 times higher, and the TGF-β1 concentration was up to 3.1 times higher, indicating that the CPPs composition is rich in these components.

[0057] In addition, from the results so far, it is clear that the CPPs composition has a calcification-promoting activity similar to the osteoblast differentiation-inducing medium, and therefore it is highly likely that it contains type I collagen and Ca, which are considered to be essential for calcification. In the above Example 5, since the concentration of type I collagen was confirmed to be high, the Ca concentration of the CPPs composition was 40 μg / mL when measured by metalloassay calcium measurement (OCPC) (manufactured by Metallogenics Co., Ltd., product number: CA30M). However, during the preparation of the CPPs composition, Ca was not precipitated with an organic solvent, and the hydrochloric acid and caustic soda used to inactivate the precipitated protein did not contain Ca, so the Ca concentration of the CPPs composition should be calculated to be approximately 0 μg / mL. From this, it was predicted that Ca was bound to type I collagen in the CPPs composition.

[0058] (Example 6) Analysis of CPPs composition 2 To confirm the above prediction, the following measurements were carried out. First, in order to eliminate the effect of non-specific Ca adsorption, 10 mL of the CPPs composition was concentrated to about 1.7 mL using a centrifugal concentrator (Corning, Spin-X UF20). The Ca concentration of the concentrated sample was measured by the metalloassay calcium measurement and found to be 174 μg. Then, 10.3 mL of physiological saline was added to the concentrated sample, and the sample was concentrated to 1.5 μL using the centrifugal concentrator. The Ca (calcium) concentrations of the filtrate and concentrated solution were measured, and the results were: Ca concentration of the filtrate (10.5 ml): 4.1 μg / mL; Ca concentration of concentrated solution (1.5 ml): 38.8 μg / mL, It was. On the other hand, when the concentration of type I collagen was measured by the above-mentioned ELISA, Type I collagen concentration of the filtrate: 0.1 μg / mL or less Concentration of collagen type I in the concentrate: 360 μg / mL, It was. These results show that there is a correlation between the type I collagen concentration and Ca concentration in the concentrate, and that the behavior of type I collagen and Ca coincide. In other words, most of the Ca remains in the concentrate without being filtered, indicating that Ca in the medium binds to the type I collagen in the CPPs composition.

[0059] (Example 7) Analysis of CPPs composition 3 The CPPs composition was electrophoresed on a 10% gel of SDS-PAGE together with type I atelocollagen (manufactured by Daiichi Fine Chemical Co., Ltd., product number: Y-1), then transferred to a nitrocellulose membrane and subjected to Western blotting (performed according to Protein Experimental Notebook, revised 4th edition, Yodosha). The results are shown in Figure 7. Figure 7A shows the results of CBB staining (manufactured by Nacalai Tesque, product number: 04543-51), and Figure 7B shows the electrophoretic pattern obtained by reacting with anti-Procollagen 1C-Terminal Propeptide (anti-PICP) antibody (manufactured by Cloud-Clone Crop, product number: PAA570Hu08). As shown in Figure 7A(b), type I atelocollagen was shown as a two-chain structure of approximately 120 kDa (mass) by CBB staining, but did not react with anti-PICP antibody that labels the C-terminus of the propeptide, and no band was detected, as shown in Figure 7B(b). On the other hand, at least five bands (five chains) were detected at approximately 120 to 200 kDa in the CPPs compositions shown in Figure 7 (c) and (d). This result indicated that the type I collagen in the CPPs composition contains procollagen (collagen precursor). The above-mentioned procollagen contained in the CPPs composition forms a complex with calcium, thereby serving as a scaffold for cells and facilitating the supply of calcium to cells.

[0060] According to the CPPs composition having such characteristics, differentiation into osteoblasts can be induced at a desired timing by locally administering the CPPs composition simultaneously with osteoprogenitor cells, or by subsequently administering the CPPs composition as a booster. Furthermore, it is possible to induce differentiation of mesenchymal stem cells and osteoprogenitor cells into osteoblasts using components obtained from mesenchymal stem cells, or even components derived from autologous cells, without using an osteoblast differentiation-inducing medium.

Claims

1. 60 to 90 vol% of a basal medium; 10 to 40 vol% physiological saline; and 1 to 100 ng / mL EGF; 0.2 to 20.0 ng / mL of FGF-2; 0.2 to 20.0 ng / mL of PDGF; 0.5 to 8.0 mM magnesium ascorbyl phosphate; A medium for stem cell culture comprising:

2. Cultivating mesenchymal stem cells for 1 to 10 days in the stem cell culture medium according to claim 1; The protein components are then precipitated with an organic solvent. The precipitate is separated and dissolved in physiological saline or basal medium. The present invention relates to a cell-produced protein (CPP) composition for inducing differentiation of mesenchymal stem cells or osteoprogenitor cells into osteoblasts, the composition being obtained by the above method.

3. Type I procollagen and Hyaluronic acid and Including, Calcium is bound to the type I procollagen. The CPPs composition according to claim 2.

4. 1) Cultivating mesenchymal stem cells in an osteoblast differentiation-inducing medium for 2 to 6 days in a state in which the mesenchymal stem cells are attached to a culture vessel and allowed to proliferate until the cells become confluent or greater; or 2) Mesenchymal stem cells are placed in a culture vessel in a suspended state and stored in a refrigerator for 2 to 4 days. Mesenchymal stem cells are differentiated into osteoblast precursor cells, and then The osteoprogenitor cells are collected and seeded in a culture vessel, and cultured in a basal medium containing the CPPs composition according to claim 2, A method for inducing differentiation into osteoblasts, which comprises differentiating the osteoblast precursor cells into osteoblasts.

5. The method for inducing differentiation into osteoblasts according to claim 4 , wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

6. 1) Cultivating mesenchymal stem cells in an osteoblast differentiation-inducing medium for 2 to 6 days in a state in which the mesenchymal stem cells are attached to a culture vessel and allowed to proliferate until the cells become confluent or greater; or 2) Mesenchymal stem cells are placed in a culture vessel in a suspended state and stored in a refrigerator for 2 to 4 days. Mesenchymal stem cells are differentiated into osteoprogenitor cells, a) seeding the obtained osteoprogenitor cells and simultaneously adding the CPPs composition according to claim 2 to differentiate the osteoprogenitor cells into osteoblasts; or b) seeding the obtained osteoprogenitor cells, and then adding the CPPs composition according to claim 2 to initiate differentiation of the osteoprogenitor cells into osteoblasts; A method for inducing differentiation into osteoblasts (excluding those performed on humans).

7. The method for inducing differentiation into osteoblasts according to claim 6 , wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.