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

Abstract

The present invention induces differentiation of mesenchymal stem cells and osteoblast precursor cells into osteoblasts. Provided is a medium for culturing stem cells, the medium including 60-90 vol% basal medium and 10-40 vol% 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

Stem cell culture medium, cell-produced protein composition, and method for inducing differentiation into osteoprogenitor cells or osteoblasts

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

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

[0003] Conventionally, a known method for differentiating mesenchymal stem cells into osteoblasts involves culturing mesenchymal stem cells (MSCs) in a dish or plate containing a cell growth medium, and when the cells reach a confluent state of 80% or more, replacing the medium with an osteoblast differentiation-inducing medium and culturing for 2 weeks. Furthermore, a known indicator for evaluating differentiation induction into osteoblasts is a method for evaluating calcification, which occurs when mesenchymal stem cells are induced to differentiate into osteoblasts, by staining calcium with a dye such as alizarin red (see, for example, Patent Document 1).

[0004] Japanese Patent Application Laid-Open No. 2005-124460

[0005] However, with 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 onset of calcification, and the osteoprogenitor cells were recovered and reseeded, administered, transplanted, etc. However, in this case, no calcification was observed in the reseeded, administered, or transplanted cells, and further, the osteoprogenitor cells reverted to an undifferentiated state. This demonstrated that simply reseeding, administering, transplanting, etc. of osteoprogenitor cells without an osteoblast differentiation-inducing medium does not promote differentiation into osteoblasts.

[0007] Furthermore, if an osteoblast differentiation-inducing medium is used at the site of reseeding, administration, or transplantation, differentiation into osteoblasts can be induced in about 10 days. However, when the administration / transplantation site is particularly 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 osteoblast differentiation-inducing media contain these ingredients. Furthermore, osteoblast differentiation-inducing media generally contain 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 xenogeneic animal-derived component, which carries 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 media 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 osteoprogenitor cells into osteoblasts. More specifically, the present invention aims to provide a method for inducing differentiation into osteoblasts, which enables mesenchymal stem cells and osteoprogenitor cells to differentiate into osteoblasts using components obtained from mesenchymal stem cells without using an osteoblast differentiation-inducing medium.

[0011] The present invention was made based on the above findings and aims to advantageously solve the above problems. A first aspect of the present invention is a stem cell culture medium comprising: 60-90 vol% basal medium; 10-40 vol% physiological saline; and further comprising: 1-100 ng / mL EGF; 0.2-20.0 ng / mL FGF-2; 0.2-20.0 ng / mL PDGF; and 0.5-8.0 mM magnesium ascorbyl phosphate. This stem cell culture medium allows for the preparation of a cell-produced protein (CPP) composition from mesenchymal stem cells, which can induce differentiation of mesenchymal stem cells and osteoprogenitor cells into osteoblasts. IMDM, DMEM, and α-MEM can be used as the basal medium. The purpose of adding physiological saline to the stem cell culture medium is to reduce the Ca concentration in the basal medium to 10-40%. Therefore, when the Ca concentration of the basal medium used is 40 to 50 μg / mL, there is no need to dilute it with physiological saline.

[0012] A second aspect of the present invention is a cell-produced protein (CPP) composition for inducing the differentiation of mesenchymal stem cells or osteoprogenitor cells into osteoblasts, which is obtained by culturing mesenchymal stem cells for 1 to 10 days in the stem cell culture medium described in the first aspect, then precipitating protein components with an organic solvent, and separating and dissolving the precipitate in physiological saline or a basal medium. With this CPP composition, mesenchymal stem cells and osteoprogenitor cells can be induced to differentiate into osteoblasts using components obtained from mesenchymal stem cells, without using an osteoblast differentiation-inducing medium.

[0013] In the second aspect, the CPP composition may comprise type I procollagen and hyaluronic acid, with calcium bound to the type I procollagen. By forming a complex with calcium, type I procollagen serves as a scaffold for cells and can facilitate the supply of calcium to cells.

[0014] A third aspect of the present invention is a method for inducing differentiation into osteoblasts, comprising: 1) culturing mesenchymal stem cells in an osteoblast differentiation-inducing medium for 2 to 6 days in a state where the mesenchymal stem cells are adhered to a culture vessel and allowed to proliferate until confluent or greater; or 2) differentiating the mesenchymal stem cells into osteoblastic progenitor cells by placing the mesenchymal stem cells in a suspended state in a culture vessel and storing them in a refrigerator for 2 to 4 days; and then recovering the osteoblastic progenitor cells, seeding them in a culture vessel, and culturing them in a basal medium supplemented with the CPP composition described in the second aspect, thereby differentiating the osteoblastic progenitor cells into osteoblasts. According to this method, osteoblastic differentiation can be induced by using components derived from mesenchymal stem cells without using an osteoblast differentiation-inducing medium. In the above-mentioned aspect, the mesenchymal stem cells may be derived from bone marrow, adipose tissue, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

[0015] A fourth aspect of the present invention is a method for inducing differentiation into osteoprogenitor cells, which involves differentiating mesenchymal stem cells into osteoprogenitor cells by: 1) culturing mesenchymal stem cells in an osteoblast differentiation-inducing medium for 2 to 6 days in a state where the stem cells are adhered to a culture vessel and allowed to proliferate until confluent or greater, or 2) allowing the stem cells to stand in a suspended state in a culture vessel and storing them in a refrigerator for 2 to 4 days. This method for inducing differentiation into osteoprogenitor cells allows osteoprogenitor cells for reseeding, administration, or transplantation to be prepared without calcification before reseeding, administration, or transplantation. Furthermore, in the above-mentioned aspect, the mesenchymal stem cells may be derived from bone marrow, adipose tissue, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

[0016] A fifth aspect of the present invention is a method for inducing differentiation into osteoblasts, comprising seeding osteoprogenitor cells obtained by the differentiation-inducing method described in the fourth aspect and simultaneously adding the CPP composition described in the second aspect to differentiate the osteoprogenitor cells into osteoblasts, or seeding osteoprogenitor cells obtained by the differentiation-inducing method described in the fourth aspect and then adding the CPP composition described in the second aspect to initiate differentiation of the osteoprogenitor cells into osteoblasts. According to such a method for inducing differentiation into osteoblasts, osteoprogenitor cells can be induced to differentiate into osteoblasts at a desired timing when reseeding, administering, or transplanting.

[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.

[0018] Figure 1 shows A: a photograph of Alizarin Red staining, B: the results of measuring absorbance at OD 450 nm, and C: a magnified microscope photograph (x40) of Alizarin Red staining. Figure 2 shows the results of Example 2. Figure 3 shows the results of Example 2. Figure 4 shows the results of Example 2. Figure 5 shows the results of Example 2. Figure 6 shows the results of Example 3. Figure 7 shows the results of Example 7.

[0019] Embodiments of the present invention are described in detail below. (Stem Cell Culture Medium) A stem cell culture medium according to a first aspect of the present invention is characterized by being a stem cell culture medium comprising: 60-90 vol% basal medium; 10-40 vol% physiological saline; and further comprising: 1-100 ng / mL EGF; 0.2-20.0 ng / mL FGF-2; 0.2-20.0 ng / mL PDGF; and 0.5-8.0 mM magnesium ascorbyl phosphate. The inventors have newly discovered that a cell-produced protein (CPP) composition, described below, can be obtained from mesenchymal stem cells cultured using the stem cell culture medium. IMDM, DMEM, and α-MEM can be used as the basal medium. The purpose of adding physiological saline to the stem cell culture medium is to reduce the Ca concentration in the basal medium to 10-40%. Therefore, when the Ca concentration of the basal medium used is 40 to 50 μg / mL, there is no need to dilute it with physiological saline.

[0020] 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.

[0021] Furthermore, in the stem cell culture medium according to the first aspect of the present invention, 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, from the viewpoint of enhancing the proliferation ability of cells.

[0022] 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.

[0023] (Cell-Produced Protein (CPP) Composition) The CPP composition according to a second aspect of the present invention is characterized in that it is a CPP composition for inducing the differentiation of mesenchymal stem cells or osteoprogenitor cells into osteoblasts, obtained by culturing mesenchymal stem cells for 1 to 10 days in the stem cell culture medium described in the first aspect above, then precipitating protein components with an organic solvent, and separating and dissolving the precipitate in physiological saline or a basal medium. Such a CPP composition can induce the differentiation of mesenchymal stem cells and osteoprogenitor cells into osteoblasts. Furthermore, such a CPP composition can induce the 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.

[0024] In the second aspect, 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%.

[0025] In the second aspect, examples of mesenchymal stem cells that can be used 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.

[0026] Furthermore, in the second 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 cell-produced protein (CPP) composition.

[0027] Here, in this specification, "osteoprogenitor cells" are defined as "cells in which nodules (calcified spheres) indicating the start of calcification have been formed (partially calcified), and which 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 culturing them for a short period of time in step 1) or 2) described below.

[0028] (Method for inducing mesenchymal stem cells to differentiate into osteoblasts) A method for inducing differentiation into osteoblasts according to a third aspect of the present invention is characterized in that the mesenchymal stem cells are differentiated into osteoprogenitor cells by: 1) culturing mesenchymal stem cells in a state where they are adhered to a culture vessel in an osteoblast differentiation-inducing medium for 2 to 6 days and growing them until they become confluent or more, or 2) allowing the mesenchymal stem cells to stand in a suspended state in a culture vessel and storing them in a refrigerator for 2 to 4 days, and thereafter recovering the osteoprogenitor cells and seeding them in a culture vessel, and culturing them in a basal medium supplemented with the CPPs composition according to the second aspect above, thereby differentiating the osteoprogenitor cells into osteoblasts.

[0029] According to this 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 addition, in the third aspect, the mesenchymal stem cells may be derived from bone marrow, adipose tissue, peripheral blood, umbilical cord, umbilical cord blood, or dental pulp.

[0030] In the third aspect, the concentration of the CPP composition in the "basal medium supplemented with the CPP composition" is preferably 25 to 75 vol%, more preferably 50 vol%. "Refrigerated storage" means storage at 4 to 10°C. "Confluent or more" means overgrowth of up to about 200%. In this specification, "confluent" refers to a state in which adherent cells completely cover the adhesive surface of the culture vessel, i.e., 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 a method for inducing differentiation of mesenchymal stem cells into osteoblast precursor cells, characterized by: 1) culturing mesenchymal stem cells in a state where they are attached to a culture vessel in an osteoblast differentiation-inducing medium for 2 to 6 days and growing them until they become confluent or more, or 2) placing the mesenchymal stem cells in a suspended state in a culture vessel and storing them in a refrigerator for 2 to 4 days, thereby differentiating mesenchymal stem cells into osteoblast precursor cells.

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

[0033] In the fourth aspect, "refrigerated storage" means storage at 4 to 10° C. Furthermore, "more than" in "confluent or more" means overgrowth of up to 200%.

[0034] (Method for inducing differentiation of osteoblast precursor cells into osteoblasts) A fifth aspect of the present invention is a method for inducing differentiation into osteoblasts, comprising: seeding osteoblast precursor cells obtained by the differentiation induction method described in the fourth aspect and simultaneously adding the CPP composition described in the second aspect to differentiate osteoblast precursor cells into osteoblasts; or seeding osteoblast precursor cells obtained by the differentiation induction method described in the fourth aspect, and then adding the CPP composition described in the second aspect to initiate differentiation of osteoblast precursor cells into osteoblasts.

[0035] According to this method for inducing differentiation into osteoblasts, osteoprogenitor cells can be induced to differentiate into osteoblasts at the desired timing when reseeding, administering, or transplanting. In particular, when the CPP composition is added after seeding osteoprogenitor cells, the CPP composition can be used as a booster to initiate differentiation into osteoblasts. Furthermore, in the fifth aspect, the "CPP composition" is preferably added to the medium used for culturing osteoprogenitor cells so as to account for 25 to 75 vol%, more preferably 50 vol%, of the medium.

[0036] The present invention will be specifically explained 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 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. ５2 × 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.

[0038] 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.

[0039] 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.

[0040] Comparative Example 1: Preparation of control culture supernatant (CM) Next, as a comparative control, CM, a general culture supernatant, was prepared as follows: 4 x 10 ASC (manufactured by Lonza, product number: PT-2501) ５Cells / 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. Cells were then detached with trypsin-EDTA solution (Nacalai Tesque, product number: 32777-44). Then, cells were cultured again in a HyperFlask (Corning) in 560 mL of BMCM-PL1 medium (Bio Mirai Kobo) containing 1% human serum until confluent. The culture supernatant was then collected by centrifugation 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 (manufactured by Lonza, product number: PT-2501) 3 x 10 ５ Cells / mL were seeded in 10 cm dishes (SARSTEDT, product number: 83.3902) using 10 mL of mesenchymal stem cell proliferation medium (Fukoku Co., Ltd., product number: FKCM 301T) to 20-30% confluence and cultured. Next, after culturing under the following culture conditions (1) to (4), the medium was replaced with osteoblast differentiation induction medium BMK-R008 (Bio Mirai Kobo Co., Ltd.) and cultured for 2, 4, or 6 days, respectively. Culture condition (1): Culture until 80-90% confluent. Culture condition (2): At 80-90% confluence, the medium was replaced with IMDM (Nacalai Tesque, product number: 11506-05) and cultured until confluent. Culture condition (3): Culture until confluent. Culture condition (4): Culture for 2 days from confluence to allow overgrowth.

[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 1000 cells / mL / well and seeded into a 24-well plate (SARSTEDT, product number: 83.3922). The following day, the medium was replaced with four types of medium: Medium (A) IMDM, Medium (B) CM + IMDM (mixing ratio 1:1), Medium (C) CPPs composition + IMDM (mixing ratio 1:1), and Medium (D) Osteoblast differentiation induction medium (positive control). The medium was replaced with medium and cultured for 1 to 8 days. The CM and CPPs composition prepared in Example 1 and Comparative Example 1 above were used. The above mixing ratios are by volume.

[0043] After culturing for 1 to 8 days, each well (1) to (4) was immobilized using Alizarin Red S staining kit BMK-R009 (manufactured by Bio Mirai Kobo Co., Ltd.), and the calcified nodules (calcium phosphate) were stained. The dye bound to the calcium phosphate was extracted with 5% formic acid, and the absorbance was measured at OD 450 nm.

[0044] When stained red with Alizarin Red, the absorbance at OD 450 nm of the dye eluted with formic acid is 0.100 or higher. The experiment that supports this is shown below. The above culture condition (4): "Cultivation for two days to allow overgrowth from a confluent (100%) state," then switched to osteoblast differentiation induction medium and cultured for four days. Cells were then cultured for an additional 3 to 8 days in 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 (day 6 of culture), B: Absorbance measurement results at OD 450 nm (days 3, 6, and 8 of culture, respectively, from the left column), C: Microscopic magnification (×40) of Alizarin Red staining (day 6 of culture). Regarding (a), A(a) and C(a) in Figure 1 show no staining. 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 stained nodules (calcified spheres), indicating the onset of calcification. The absorbance at this time is 0.121, as shown in B(b) in Figure 1, approximately 10 times that of B(a). (c) shows the positive control, showing a fully calcified state. From the above results, an absorbance at OD 450 nm of 0.100 or more was judged to indicate 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 results for the above culture conditions (1) to (4) are shown in the tables of Figures 2 to 5, respectively. Under culture conditions (1) and (2), almost no calcification was observed, and no induction of osteoblast differentiation was observed, except in the positive control medium (D) (Figures 2 and 3). On the other hand, under culture condition (3), when cells were switched to osteoblast differentiation medium at confluence, detached and plated 2 and 4 days later, and then switched to medium (C): CPP composition + IMDM, calcification occurred 8 days later. Furthermore, when cells were switched to osteoblast differentiation-inducing medium at confluence, detached and plated 6 days later, and then switched to medium (C), calcification occurred 3 and 8 days later, confirming induction of osteoblast differentiation (Figure 4). Furthermore, under culture condition (4), the cells were allowed to overgrow for 2 days after confluence, then switched to osteoblast differentiation-inducing medium, and detached 4 and 6 days later, seeded on plates, and switched to medium (C). Calcification occurred 6 to 8 days later and 1 to 8 days later, respectively, confirming that differentiation into osteoblasts had been induced (Figure 5).

[0047] As shown in the tables of Figures 2 to 5, when cells were cultured in the positive control medium (D), calcification occurred three days after seeding on the plate. However, when cells were cultured in medium (A): IMDM and medium (B): CM + IMDM, no calcification occurred under any of the culture conditions (1) to (4). In contrast, when cells were cultured in medium (C): CPPs composition + IMDM, as described above, calcification was promoted depending on the culture period under culture conditions (3) and (4), and differentiation into osteoblasts was induced. This suggests that cells 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 Static Floating Culture Next, we investigated a method for differentiating MSCs into osteoprogenitor cells in a static floating state without adhering them to a culture vessel. 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). ６ The cells were mixed with the four types of media used in Example 2: medium (A) IMDM, medium (B) CM + IMDM (mixing ratio 1:1), medium (C) CPPs composition + IMDM (mixing ratio 1:1), and medium (D) osteoblast differentiation induction medium (positive control) in four 15 mL tubes (SARSTEDT, product number: 62.554.5.2) at 4 mL each, and allowed to stand in a suspended state in a refrigerator (4-5°C) for 2, 4, and 6 days, respectively. Note that the above mixing ratios are by volume.

[0049] After each refrigerated storage period, 10 μL of each tube was sampled and the viable cell count was measured using a hemocytometer (Elma Sales, product number: 4025). The medium in each of the four tubes was then removed by centrifugation, suspended in IMDM (Nacalai Tesque, product number: 11506-05), and then seeded onto a 48-well plate (SARSTEDT, product number: 83.3923).

[0050] The viability (survival) rates of ASCs counted by the viable cell count measurement were 84-89% on day 2, 88-93% on day 4, and 79-91% on day 6. The cells were seeded onto the 48-well plate, and the day after adhesion, the media was replaced with one of the four media (A) to (D). In this way, the cells were cultured under 16 different conditions, and after 4 and 8 days, Alizarin Red staining was performed as in Example 2.

[0051] The results are shown in the table in Figure 6. When the suspension was stored in a refrigerator for 2 and 4 days, calcification was observed after 8 days of culture in (C): CPP composition + IMDM. However, when the suspension was stored in a refrigerator for 6 days, calcification was observed only in medium (D): osteoblast differentiation induction medium (positive control). No calcification was observed under any of the culture conditions in medium (A): IMDM or medium (B): CM + IMDM. These results suggest that ASCs were induced to differentiate into osteoprogenitor cells after short-term storage in a suspension, and that subsequent culture in a medium containing the CPP composition induced the osteoprogenitor cells to differentiate into osteoblasts. Furthermore, it was found that the medium used for storage in a suspension in a refrigerator, even without the addition of the CPP composition, did not affect the subsequent induction of differentiation into osteoblasts.

[0052] (Example 4) Verification of induction of differentiation into osteoblast precursor cells by suspension. Without carrying out the step of refrigerated storage in a suspension state, 2 x 10 ASCs (manufactured by Lonza, product number: PT-2501) were placed in a 24-well plate (manufactured by SARSTEDT, product number: 83.3922) containing 0.5 mL / well of the four types of media used in Example 2: medium (A) IMDM, medium (B) CM + IMDM (mixing ratio 1:1), medium (C) CPPs composition + IMDM (mixing ratio 1:1), and medium (D) osteoblast differentiation induction medium (positive control). ５ After culturing for 7 to 11 days, Alizarin Red staining was performed in the same manner as in Example 2. The above mixing ratios are volume ratios. The results are shown in Table 1. Only the positive control medium (D) calcified, while no calcification was observed in the media (A) to (C). This indicates that the step of refrigerated storage in a suspended state (a step 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 supplemented with a CPP composition is one of the essential steps for inducing differentiation into osteoblasts.

[0053]

[0054] Example 5 Analysis of CPP Composition 1 To confirm the characteristics of the CPP composition, the type I collagen concentration, hyaluronic acid concentration, and collagen and Ca were measured and compared with a control culture supernatant (CM). The type I collagen concentration in the CPPs was measured by ELISA (prepared equivalent to a human collagen type 1 ELISA (manufactured by E-Cell, product number: EC1-E105)) and SDS-PAGE (performed according to the Protein Experiment 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 (manufactured by Cosmo Bio Co., Ltd., product number: HA-96KIT), and was found to be 140 μg / mL. TGF-β1 in CPPs was measured using a human TGF-β1 ELISA kit (Bioss Inc., product number: BSKH1021) and found to be 384-810 pg / mL. Similar measurements were also performed on CM for comparison. The results are shown in Table 2.

[0055]

[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 was rich in these components.

[0057] Furthermore, based on the results obtained so far, it is clear that the CPP composition, like the osteoblast differentiation-inducing medium, has a calcification-promoting activity, suggesting a high likelihood of containing type I collagen and calcium, which are considered essential for calcification. In Example 5 above, the high concentration of type I collagen was confirmed, and the calcium concentration of the CPP composition was measured using a metalloassay calcium analyzer (OCPC) (Metalogenics, Inc., product number: CA30M) and found to be 40 μg / mL. However, since calcium is not precipitated with organic solvents during the preparation of the CPP composition, and the hydrochloric acid and caustic soda used to inactivate the precipitated proteins do not contain calcium, the calcium concentration of the CPP composition should theoretically be approximately 0 μg / mL. This suggests that calcium is bound to the type I collagen in the CPP composition.

[0058] Example 6 Analysis of CPP Composition 2 To confirm the above prediction, the following measurements were performed. First, to eliminate the influence of non-specific Ca adsorption, 10 mL of the CPP composition was concentrated to approximately 1.7 mL using a centrifugal concentrator (Corning, Spin-X UF20). The Ca concentration of the concentrated sample was measured using the metalloassay calcium measurement described above, and was found to be 174 μg. Subsequently, 10.3 mL of physiological saline was added to the concentrated sample, and the mixture was concentrated to 1.5 μL using the centrifugal concentrator. The Ca (calcium) concentrations of the filtrate and concentrate were measured, and were found to be: Ca concentration in the filtrate (10.5 mL): 4.1 μg / mL; Ca concentration in the concentrate (1.5 mL): 38.8 μg / mL, respectively. On the other hand, when the type I collagen concentrations of these samples were measured by the above-mentioned ELISA, the type I collagen concentration in the filtrate was 0.1 μg / mL or less, and the type I collagen concentration in the concentrate was 360 μg / mL. These results show that there is a correlation between the type I collagen concentration and the Ca concentration in the concentrate, and that the behavior of type I collagen and Ca is consistent. In other words, the majority of Ca remained in the concentrate without being filtered, indicating that Ca in the medium was bound to the type I collagen in the CPPs composition.

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

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

Claims

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

2. A cell-produced protein (CPP) composition for inducing differentiation of mesenchymal stem cells or osteoprogenitor cells into osteoblasts, obtained by culturing mesenchymal stem cells for 1 to 10 days in the stem cell culture medium according to claim 1, thereafter precipitating protein components with an organic solvent, and then isolating and dissolving the precipitate in physiological saline or a basal medium.

3. The CPPs composition according to claim 2, comprising type I procollagen and hyaluronic acid, wherein calcium is bound to the type I procollagen.

4. A method for inducing differentiation into osteoblasts, comprising: 1) culturing 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) differentiating the mesenchymal stem cells into osteoblast precursor cells by placing the mesenchymal stem cells in a suspended state in a culture vessel and storing them in a refrigerator for 2 to 4 days; and then recovering the osteoblast precursor cells and seeding them in a culture vessel and culturing them in a basal medium containing the CPPs composition according to claim 2, thereby differentiating the osteoblast precursor cells into osteoblasts.

5. The method for inducing differentiation into osteoblasts described in 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. A method for inducing differentiation into osteoblast precursor cells, comprising: 1) culturing mesenchymal stem cells attached to a culture vessel in an osteoblast differentiation inducing medium for 2 to 6 days and growing the cells until confluent or greater; or 2) placing the mesenchymal stem cells in a suspended state in a culture vessel and storing them in a refrigerator for 2 to 4 days, thereby differentiating mesenchymal stem cells into osteoblast precursor cells.

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

8. A method for inducing differentiation into osteoblasts, comprising: seeding osteoblast precursor cells obtained by the differentiation induction method according to claim 6 and simultaneously adding the CPPs composition according to claim 2 to differentiate the osteoblast precursor cells into osteoblasts; or seeding osteoblast precursor cells obtained by the differentiation induction method according to claim 6 and then adding the CPPs composition according to claim 2 to initiate differentiation of the osteoblast precursor cells into osteoblasts.

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