Method for predicting osteoblast differentiation potential of mesenchymal stem cell populations - Patents.com

Abstract

To provide a novel method for predicting osteoblast differentiation potential of a mesenchymal stem cell which is different for each cell lot.SOLUTION: A method for predicting osteoblast differentiation potential of a mesenchymal stem cell population includes: a process of culturing an undifferentiated mesenchymal stem cell population on an elastic body having the elasticity modulus of less than 5kPa; and a process of measuring the ratio of cells, in which RUNX2 is held inside a nucleus, in the mesenchymal stem cell population cultured in the foregoing process.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for predicting cell differentiation potential in a stem cell population. [Background technology]

[0002] Biomaterial design methods based on the "mechanobiology mechanism" in which cells respond to the surrounding mechanical environment have been attracting attention in the fields of cell biology and regenerative medicine (Non-Patent Documents 1 and 2). In the field of regenerative medicine, the elastic modulus of the culture substrate is considered to be one of the mechanical stimuli that can stably and precisely control the lineage and fate determination of stem cells (Non-Patent Documents 3 to 6). On the other hand, mesenchymal stem cells, which have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes, are highly sensitive to the surrounding mechanical environment, and it is difficult to maintain their undifferentiated state, which is a problem. For example, it has been reported that mesenchymal stem cells are biased to differentiate into osteoblasts when cultured for more than 5 days on a polystyrene culture substrate, which has been used conventionally for proliferation. To solve this problem, it is considered necessary to have a new culture method for obtaining the required number of cells while maintaining the quality of mesenchymal stem cells, based on a mechanical-biochemical signal conversion mechanism that converts mechanical stimuli derived from the mechanical properties of the culture substrate into a differentiation response (Non-Patent Documents 7 and 8). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Li, L., Eyckmans, J., and Chen, CS, Designer biomaterials for mechanobiology, Nature Materials, Vol.16, No.12 (2017), pp.1164-1168. [Non-Patent Document 2] Miyoshi, H., Suzuki, K., Ju, J., Ko, J. S., Adachi, T., and Yamagata, Y., A Perturbation Analysis to understand the mechanism how migrating cells sense and respond to a topography in the extracellular environment, Analytical Sciences, Vol.32, No.11 (2016), pp.1207-1211. [Non-Patent Document 3] Engler, A. J., Sen, S., Sweeney, H. L., and Discher, D. E., Matrix elasticity directs stem cell lineage specification, Cell, Vol.126, No.4 (2006), pp.677-689. [Non-Patent Document 4] Lee, J., Abdeen, A. A., Huang, T. H., and Kilian, K. A., Controlling cell geometry on substrates of variable stiffness can tune the degree of osteogenesis in human mesenchymal stem cells, Journal of the Mechanical Behavior of Biomedical Materials, Vol.38 (2014), pp.209-218. [Non-Patent Document 5] Lee, J., Abdeen, A. A., and Kilian, K. A., Rewiring mesenchymal stem cell lineage specification by switching the biophysical microenvironment, Scientific Reports, Vol.4, No.1 (2014). [Non-Patent Document 6] Yang, C., Tibbitt, MW, Basta, L., and Anseth, KS, Mechanical memory and dosing influence stem cell fate, Nat Mater, Vol.13, No.6 (2014), pp.645-652. [Non-Patent Document 7] Pittenger, MF, Discher, DE, Peault, BM, Phinney, DG, Hare, JM, and Caplan, AI, Mesenchymal stem cell perspective: cell biology to clinical progress, npj Regenerative Medicine, Vol.4, No.1 (2019). [Non-Patent Document 8] Sipp, D., Robey, GP, and Turner, L., Clear up this stem-cell mess, Nature, Vol.561, No.455-457 (2018). [Non-Patent Document 9] Kuboki, T., and Kidoaki, S., Fabrication of Elasticity-Tunable Gelatinous Gel for Mesenchymal Stem Cell Culture, Methods in Molecular Biology, Vol.1416 (2016), pp.425-441. [Non-Patent Document 10] Chen, Q., Shou, P., Zheng, C., Jiang, M., Cao, G., Yang, Q., Cao, J., Xie, N., Velletri, T., Zhang, X., Xu, C., Zhang, L., Yang, H., Hou, J., Wang, Y., and Shi, Y., Fate decision of mesenchymal stem cells: adipocytes or osteoblasts?, Cell Death & Differentiation, Vol.23, No.7 (2016), pp.1128-1139.

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Summary of the Invention

Problems to be Solved by the Invention

[0004] An objective of the present invention is to provide a new method for predicting the osteoblast differentiation potential of mesenchymal stem cells, which differs depending on the cell lot. [Means for solving the problem]

[0005] Under such circumstances, the present inventors conducted intensive research and found that among the various factors present in mesenchymal stem cells, the behavior of Runt-related transcription factor 2 (RUNX2) differs depending on the osteoblast differentiation potential. After much trial and error, the present inventors found that when mesenchymal stem cells are cultured on a gel, no difference in the behavior of RUNX2 is observed on a gel with a high elastic modulus, while when a gel with an elastic modulus of less than 5 kPa is used, the behavior of RUNX2 that differs depending on the osteoblast differentiation potential can be measured. The present invention is based on such novel findings. Effect of the Invention

[0006] According to the present invention, a new method for predicting the osteoblast differentiation potential of mesenchymal stem cells, which differs depending on the cell lot, can be provided. [Brief description of the drawings]

[0007] [Figure 1] Differentiation ability of mesenchymal stem cells into osteoblasts and adipocytes. (a) Alizarin Red S staining after 14 days of osteoblast differentiation. (b) Oil Red O staining after 20 days of adipocyte differentiation. Scale bars, 5 mm. [Diagram 2] Changes in the localization of YAP and RUNX2 before and after adhesion to StG gel. (a) Fluorescent staining of DNA (cyan), YAP (green), and RUNX2 (red) before adhesion to StG gel and (b) after 4 days of culture on StG gel. (a) Scale bar, 10 μm. (b) Scale bar, 50 μm. [Diagram 3]Dependence of YAP and RUNX2 localization on elasticity. (a) Relationship between the percentage of cells with YAP localized to the nucleus and the elasticity of the substrate. (b) Relationship between the percentage of cells with RUNX2 localized to the nucleus and the elasticity of the substrate. The dashed lines are approximations of the linear increase region. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Method for predicting osteoblast differentiation potential The present invention relates to a method for producing a mesenchymal stem cell culture medium comprising the steps of: culturing an undifferentiated mesenchymal stem cell population on an elastic body having an elastic modulus of less than 5 kPa; A step of measuring the proportion of cells in which RUNX2 is retained in the nucleus in the mesenchymal stem cell population cultured in the above step. The present invention provides a method for predicting the osteoblast differentiation potential of a mesenchymal stem cell population, comprising: a. In the present invention, osteoblast differentiation potential refers to the ability of a mesenchymal stem cell population to be measured to differentiate into osteoblasts. In the present invention, osteoblast differentiation potential can be evaluated by the method described in "Evaluation of osteoblast differentiation potential" in the Examples. Since osteoblast differentiation potential can vary depending on the cell lot, the method of the present invention is useful because it allows the osteoblast differentiation potential of a target cell lot to be evaluated in a simple manner in a relatively short time.

[0009] Mesenchymal stem cells may be derived from mammals such as humans, mice, rats, rabbits, and pigs, and are preferably derived from humans. Mesenchymal stem cells can be collected from bone marrow, fat, umbilical cord, dental pulp, synovium, and the like, based on methods known in the technical field to which the present invention pertains.

[0010] In a preferred embodiment of the present invention, the mesenchymal stem cells used in the method of the present invention may be primary cultured cells or subcultured cells. When subcultured cells are used, a medium used for the subculture may be appropriately used that is used for the proliferation of mesenchymal stem cells in the technical field to which the present invention belongs. Examples of such a medium include DMEM and αMEM. In addition, a medium containing, for example, serum or the like may be used as the medium. As such a medium, a medium commercially available from Lonza or the like may be appropriately used. In addition, a pH adjuster, L-glutamine, antibiotics, or the like may be added to such a medium. The culture temperature is not particularly limited, but can be set, for example, in the range of 30 to 40°C, preferably 36 to 38°C. The pH of the medium is also not particularly limited, but can be set, for example, in the range of 6.8 to 8.0, preferably 7.2 to 7.6. The culture time is also not particularly limited, but can be set, for example, in the range of 1 to 80 days, preferably 1 to 25 days. In such culture, it is preferable to change the medium once every 1 to 4 days, preferably about once every 2 to 3 days. For example, the culture may be static culture or shaking culture, but static culture is preferred. In an embodiment using subcultured cells, cells that have been subcultured 1 to 20 times are preferred, and cells that have been subcultured 2 to 6 times are more preferred.

[0011] In the method of the present invention, first, a step of culturing an undifferentiated mesenchymal stem cell population on an elastic body having an elastic modulus of less than 5 kPa is performed. The elastic body is not particularly limited as long as it allows mesenchymal stem cells to be cultured on the elastic body, but examples include gel, rubber, etc., and gel is preferred. In a preferred embodiment of the present invention, it is preferable to use styrenated gelatin as the gel from the viewpoint of adjusting the elastic modulus. The surface shape of the elastic body is not particularly limited, and may be flat or have irregularities, but is preferably flat. The method of the present invention is characterized in that an elastic body having an elastic modulus of less than 5 kPa is used. The elastic modulus of the elastic body is less than 5 kPa, preferably 3 kPa or less. In the present invention, the elastic modulus can be measured at 20-26°C by an indentation test using an atomic force microscope. More specifically, the elastic modulus can be measured by the method described in the Examples of the present application. In a typical embodiment of the present invention, it is preferable to place a suspension of an undifferentiated mesenchymal stem cell population on the elastic body and culture it. In such an embodiment, as a medium for suspending cells, a liquid medium used for growing mesenchymal stem cells in the technical field to which the present invention belongs can be appropriately used. As such a medium, for example, the medium described above for the subculture of mesenchymal stem cells can be used. In this step, the amount of mesenchymal stem cells seeded in the elastic body at the start of the culture is, for example, 100 to 3000 cells / cm per unit area of ​​the elastic body. 2 , preferably 2400 to 2600 cells / cm 2The culture temperature is not particularly limited, but can be set within a range of, for example, 30 to 40°C, preferably 36 to 38°C. The pH of the medium is also not particularly limited, but can be set within a range of, for example, 6.8 to 8.0, preferably 7.2 to 7.6. The culture time can be set within a range of, for example, 24 to 168 hours, preferably 24 to 96 hours. The above culture time is preferable from the viewpoint of ensuring sufficient time for RUNX2 to migrate from the nucleus to the cytoplasm in a cell population with high osteoblast differentiation potential, and evaluating the osteoblast differentiation potential in a relatively short culture time. In such culture, it is preferable to replace the medium once every 1 to 4 days, preferably once every 2 to 3 days. The culture may be static culture or shaking culture, but static culture is preferable. In such a culture step, by culturing on an elastic body with the above elastic modulus, RUNX2 migrates from the nucleus to the cytoplasm in a relatively large number of cells in a cell population with high osteoblast differentiation potential. On the other hand, in cell populations with low osteoblast differentiation potential, RUNX2 was retained in the nucleus and was not translocated into the cytoplasm much in a relatively large number of cells, and this behavior is thought to be influenced by the physical stimuli that the mesenchymal stem cell population receives from the elastic material.

[0012] In the method of the present invention, the proportion of cells in which RUNX2 is retained in the nucleus is then measured in the mesenchymal stem cell population cultured in the above step. As a method for measuring cells in which RUNX2 is retained in the nucleus, methods used in the technical field to which the present invention belongs can be widely used. For example, RUNX2 is fluorescently labeled using an anti-RUNX2 antibody and a fluorescent secondary antibody, and at the same time, DNA in the nucleus is stained with a fluorescent label having a different wavelength from that of the fluorescent secondary antibody, such as DAPI (4',6-diamidino-2-phenylindole). A fluorescent image of the mesenchymal stem cell that has been subjected to the above treatment is taken, and cells in which RUNX2 is retained in the nucleus can be measured by image analysis. Specifically, the range of the cell nucleus is specified from the position of the stained DNA, and the average brightness value of RUNX2 in the cell nucleus and the average brightness value of RUNX2 in the cytoplasm are calculated using image analysis software, and the ratio of the brightness value of RUNX2 in the cell nucleus to the brightness value of RUNX2 in the cytoplasm from the obtained value (intranuclear-extranuclear brightness ratio) is calculated, whereby whether RUNX2 is localized in the nucleus of a certain cell can be evaluated. In this embodiment, in calculating the brightness value of RUNX2 in the cytoplasm, for example, the average brightness value of any region of the cytoplasm (for example, any three regions (each region 10 × 10 pixels at 1.24 μm pixel pitch)) may be used as the brightness value of RUNX2 in the cytoplasm of the cell. In this embodiment, for example, a cell having an intranuclear / extranuclear ratio of the brightness value equal to or greater than a threshold t can be evaluated as a cell in which RUNX2 is localized in the nucleus. In this embodiment, the threshold t can be set, for example, in the range of 1.4≦t≦1.8, preferably 1.5≦t≦1.7, and more preferably 1.55≦t≦1.65. For example, 50 to 10,000 cells, preferably 500 to 1,000 cells per mesenchymal stem cell population to be measured, are evaluated as to whether or not the above-mentioned RUNX2 is localized in the nucleus, and the proportion of cells in the mesenchymal stem cell population in which RUNX2 is retained in the nucleus can be calculated. A cell population with a low percentage of cells retaining RUNX2 in the nucleus has a higher osteoblast differentiation potential than a cell population with a high percentage. Therefore, the osteoblast differentiation potential of the mesenchymal stem cell population to be measured can be evaluated based on the percentage of cells retaining RUNX2 in the nucleus. For example, for a mesenchymal stem cell population cultured on an elastic body with a predetermined elastic modulus, if the percentage of cells retaining RUNX2 in the nucleus is equal to or lower than a preset value T1, the osteoblast differentiation potential can be evaluated to be high. In such an embodiment, the threshold value T1 can be set, for example, in the range of 0≦T1≦0.4, preferably 0≦T1≦0.2. In addition, for a mesenchymal stem cell population divided into three groups according to osteoblast differentiation potential and cultured on an elastic body with a predetermined elastic modulus, if the percentage of cells retaining RUNX2 in the nucleus is equal to or lower than a preset value T 1-1 The following cases are evaluated as belonging to a group with high osteoblast differentiation potential, and a preset value T 1-1 A larger and predefined value T 1-2 In the following cases, the system belongs to the intermediate group and the preset value T 1-2 In this embodiment, if the threshold T 1-1 For example, 0≦T 1-1 ≦0.4, preferably 0.15≦T 1-1 The threshold T can be set in the range of ≦0.25. 1-2 For example, 0.5≦T 1-2 ≦0.9, preferably 0.55≦T 1-2 It can be set in the range of ≦0.85.

[0013] In the present invention, a cell population with a low percentage of cells retaining RUNX2 in the nucleus has a higher osteoblast differentiation potential than a cell population with a high percentage. Therefore, the osteoblast differentiation potential of the mesenchymal stem cell population to be measured can be evaluated based on the percentage of cells retaining RUNX2 in the nucleus. In a preferred embodiment of the present invention, for example, a mesenchymal stem cell population cultured on an elastic body with a predetermined elastic modulus E (kPa) can be evaluated as having a high osteoblast differentiation potential when the percentage of cells retaining RUNX2 in the nucleus is equal to or lower than a preset value T1. In such an embodiment, the threshold T1 can be set, for example, in the range of 0.09E≦T1≦0.09E+0.3, preferably 0.09E≦T1≦0.09E+0.25. In addition, a mesenchymal stem cell population divided into three groups according to osteoblast differentiation potential and cultured on an elastic body with a predetermined elastic modulus can be evaluated as having a high osteoblast differentiation potential when the percentage of cells retaining RUNX2 in the nucleus is equal to or lower than a preset value T 1-1 The following cases are evaluated as belonging to a group with high osteoblast differentiation potential, and a preset value T 1-1 A larger and predefined value T 1-2 In the following cases, the system belongs to the intermediate group and the preset value T 1-2 In this embodiment, if the threshold T 1-1 For example, 0.09E+0.15≦T 1-1 ≦0.09E+0.35, preferably 0.09E+0.2≦T 1-1 The threshold T can be set in the range of ≦0.09E+0.3. 1-2 For example, 0.09E+0.4≦T 1-2 < 1, preferably 0.09E+0.45≦T 1-2 It can be set in the range ≦0.09E+0.55.

[0014] In another preferred embodiment of the present invention, the culture step may be performed using two or more types of elastic bodies (e.g., two, three, four, etc.) with different elastic moduli as the elastic body with an elastic modulus of less than 5 kPa. In the present invention, when the same lot of mesenchymal stem cell population is divided into a plurality of groups and cultured on two or more types of elastic bodies with different elastic moduli of less than 5 kPa, the proportion of cells with RUNX2 retained in the nucleus is lower when cultured on the elastic body with a lower elastic modulus. In this embodiment, the elastic modulus of the elastic body is plotted on a graph with the horizontal axis representing the elastic modulus of the elastic body and the vertical axis representing the proportion of cells with RUNX2 retained in the nucleus, and an approximation line in the graph can be obtained. In this embodiment, the gradient of the approximation line increases in a cell population with a higher osteoblast differentiation potential. Therefore, in this embodiment of the present invention, the osteoblast differentiation potential can be evaluated based on the gradient of the approximation line. For example, when the slope of the approximate straight line for a certain mesenchymal stem cell population is equal to or greater than a preset value T2, the osteoblast differentiation potential can be evaluated as high. In this embodiment, the threshold value T2 can be set, for example, in the range of 0.09≦T2≦0.11, preferably 0.095≦T2≦0.105. In addition, the osteoblast differentiation potential of a certain mesenchymal stem cell population can be divided into three groups according to the osteoblast differentiation potential, and the osteoblast differentiation potential of a certain mesenchymal stem cell population can be evaluated as high when the slope of the approximate straight line for a certain mesenchymal stem cell population is equal to or greater than a preset value T 2-1 In the above cases, the osteoblast differentiation potential is evaluated as being in a high group, and the predetermined value T 2-1 A smaller and predefined value T 2-2 If it is more than this, it belongs to the intermediate group and is set to the preset value T 2-2 If the threshold value T 2-1 For example, 0.09≦T 2-1 ≦0.11, preferably 0.095≦T 2-1 The threshold T can be set in the range of ≦0.105. 2-2 For example, 0.07≦T 2-2≦0.09, preferably 0.075≦T 2-2 It can be set in the range of ≦0.085.

[0015] Furthermore, in this embodiment, the intercept value (intercept at elastic modulus=0) extrapolated from the approximation line is smaller for cell populations with higher osteoblast differentiation potential. Therefore, in the present invention, osteoblast differentiation potential can also be evaluated based on the intercept of the approximation line. For example, for a certain mesenchymal stem cell population, if the intercept is equal to or less than a preset value T3, the osteoblast differentiation potential can be evaluated to be high. In this embodiment, the threshold value T3 can be set, for example, in the range of 0≦T3≦0.3, preferably 0≦T3≦0.25. Also, by dividing the cells into three groups according to their osteoblast differentiation potential, a certain mesenchymal stem cell population can be evaluated as having a high osteoblast differentiation potential when the intercept is equal to or less than a preset value T 3-1 The following cases are evaluated as belonging to a group with high osteoblast differentiation potential, and a preset value T 3-1 A larger and predefined value T 3-2 In the following cases, the system belongs to the intermediate group and the preset value T 3-2 In this embodiment, if the threshold T 3-1 For example, 0.1≦T 3-1 ≦0.4, preferably 0.2≦T 3-1 The threshold T can be set in the range of ≦0.3. 3-2 For example, 0.4≦T 3-2 ≦0.6, preferably 0.45≦T 3-2 It can be set in the range of ≦0.55.

[0016] In the present invention, RUNX2 is localized in the nucleus of undifferentiated mesenchymal stem cells before stimulation by an elastic body, and when the mesenchymal stem cells are cultured on an elastic body with an elastic modulus of less than 5 kPa, RUNX2 migrates from the nucleus to the cytoplasm in mesenchymal stem cells with high osteoblast differentiation potential, and tends to be retained in the nucleus in mesenchymal stem cells with high osteoblast differentiation potential. Therefore, after culturing mesenchymal stem cells on an elastic body with an elastic modulus of less than 5 kPa, the osteoblast differentiation potential can be evaluated using the percentage of cells in which RUNX2 has migrated from the nucleus to the cytoplasm as an index. Therefore, in another embodiment, the present invention provides a method for predicting the osteoblast differentiation potential of a mesenchymal stem cell population, comprising the steps of: culturing an undifferentiated mesenchymal stem cell population on an elastic body with an elastic modulus of less than 5 kPa; and measuring the percentage of cells in which RUNX2 has migrated to the cytoplasm in the mesenchymal stem cell population cultured in the above step. In this embodiment, the culture step is also as described above. When measuring the proportion of cells in which RUNX2 has been transferred to the cytoplasm in a mesenchymal stem cell population, first, in the same manner as in the step of measuring the proportion of cells in which RUNX2 is retained in the nucleus in the mesenchymal stem cell population described above, the number of RUNX2 present in the nucleus and the number of RUNX2 present in the cytoplasm outside the nucleus are measured as an index of brightness value for the cells in the mesenchymal stem cell population, and the ratio of brightness value between the outside and inside of the nucleus (brightness value of RUNX2 in the cytoplasm to the ratio of brightness value to RUNX2 in the cell nucleus) is calculated to determine whether the cell has RUNX2 transferred to the cytoplasm. For example, cells with a ratio of brightness value between the outside and inside of the nucleus of t' or more can be evaluated as cells in which RUNX2 has been transferred to the cytoplasm. In such an embodiment, the threshold value t' can be set, for example, in the range of 0.55≦t'≦0.72, preferably 0.58≦t'≦0.67, more preferably 0.60≦t'≦0.65. For example, 50 to 10,000 cells, preferably 500 to 1,000 cells per mesenchymal stem cell population to be measured, are evaluated to determine whether or not the above-mentioned RUNX2 has translocated into the cytoplasm, and the proportion of cells in the mesenchymal stem cell population in which RUNX2 has translocated into the cytoplasm can be calculated.

[0017] A cell population with a high percentage of cells in which RUNX2 has migrated into the cytoplasm has a higher osteoblast differentiation potential than a cell population with a low percentage. Therefore, the osteoblast differentiation potential of the mesenchymal stem cell population to be measured can be evaluated based on the percentage of cells in which RUNX2 has migrated into the cytoplasm. For example, for a mesenchymal stem cell population cultured on an elastic body with a predetermined elastic modulus, if the percentage of cells in which RUNX2 has migrated into the cytoplasm is equal to or greater than a preset value T1', the osteoblast differentiation potential can be evaluated to be high. In this embodiment, the threshold value T1' can be set, for example, in the range of 0.6≦T1'≦1, preferably 0.8≦T1'≦1. In addition, for a mesenchymal stem cell population cultured on an elastic body with a predetermined elastic modulus, which is divided into three groups according to the osteoblast differentiation potential, if the percentage of cells in which RUNX2 has migrated into the cytoplasm is equal to or greater than a preset value T 1-1 If the value is more than or equal to T 1-1 ' smaller than the preset value T 1-2 ' or more, it belongs to the intermediate group and is 1-2 In this embodiment, if the threshold value T 1-1 ', for example, 0.6 ≤ T 1-1 '≦1, preferably 0.75≦T 1-1 The threshold T can be set in the range of '≦0.85. 1-2 ' is, for example, 0.1 ≤ T 1-2 '≦0.5, preferably 0.15≦T 1-2 It can be set in the range '≦0.45.

[0018] As described above, in the present invention, a cell population with a high percentage of cells in which RUNX2 has migrated into the cytoplasm has a higher osteoblast differentiation potential than a cell population with a low percentage. Therefore, the osteoblast differentiation potential of the mesenchymal stem cell population to be measured can be evaluated based on the percentage of cells in which RUNX2 has migrated into the cytoplasm. For example, for a mesenchymal stem cell population cultured on an elastic body with a predetermined elastic modulus E, if the percentage of cells in which RUNX2 has migrated into the cytoplasm is equal to or greater than a preset value T1', the osteoblast differentiation potential can be evaluated to be high. In such an embodiment, the threshold value T1' can be set, for example, in the range of 0.7-0.09E≦T1'≦1-0.09E, preferably 0.75-0.09E≦T1'≦1-0.09E. In addition, for a mesenchymal stem cell population cultured on an elastic body with a predetermined elastic modulus, which is divided into three groups according to the osteoblast differentiation potential, if the percentage of cells in which RUNX2 has migrated into the cytoplasm is equal to or greater than a preset value T 1-1 If the value is more than or equal to T 1-1 ' smaller than the preset value T 1-2 ' or more, it belongs to the intermediate group and is 1-2 In this embodiment, if the threshold value T 1-1 ', for example, 0.65-0.09E≦T 1-1 '≦0.85-0.09E preferably 0.7-0.09E≦T 1-1 Threshold T can be set in the range of '≦0.8-0.09E. 1-2 ', for example, 0 <T 1-2 '≦0.6-0.09E, preferably 0.45-0.09E≦T 1-2 It can be set in the range of '≦0.55-0.09E.

[0019] In another preferred embodiment of the present invention, the culturing step may be performed using two or more types of elastic bodies (e.g., two, three, four, etc.) with different elastic moduli as the elastic body with an elastic modulus of less than 5 kPa. In the present invention, when the same lot of mesenchymal stem cell population is divided into a plurality of groups and cultured on two or more types of elastic bodies with different elastic moduli of less than 5 kPa, the proportion of cells in which RUNX2 has migrated into the cytoplasm is higher when cultured on the elastic body with a lower elastic modulus. In this embodiment, the elastic modulus of the elastic body is plotted on a graph with the horizontal axis representing the elastic modulus of the elastic body and the vertical axis representing the proportion of cells in which RUNX2 has migrated into the cytoplasm, and an approximation line in the graph can be obtained. In this embodiment, the gradient of the approximation line is lower in a cell population with a higher osteoblast differentiation potential (since the gradient is a negative value in this embodiment, the absolute value of the gradient increases). Therefore, in this embodiment of the present invention, the osteoblast differentiation potential can be evaluated based on the gradient of the approximation line. For example, when the slope of the approximate straight line for a certain mesenchymal stem cell population is equal to or less than a preset value T2', the osteoblast differentiation potential can be evaluated as high. In this embodiment, the threshold value T2' can be set, for example, in the range of -0.11≦T2'≦-0.09, preferably -0.105≦T2'≦-0.095. In addition, the osteoblast differentiation potential of a certain mesenchymal stem cell population can be divided into three groups according to the osteoblast differentiation potential, and when the slope of the approximate straight line for a certain mesenchymal stem cell population is equal to or less than a preset value T 2-1 'If the following conditions are met, the osteoblast differentiation potential is evaluated as being in the high group, and the pre-defined value T 2-1 'A larger value than the preset value T 2-2 'If it is below, it belongs to the intermediate group and the preset value T 2-2 In this embodiment, if the threshold T 2-1 ', for example, -0.11 ≤ T 2-1 '≦-0.09, preferably -0.105≦T 2-1The threshold T can be set in the range of '≦-0.095. 2-2 ', for example, -0.09 ≤ T 2-2 '≦-0.07, preferably -0.085≦T 2-2 It can be set in the range '≦-0.075.

[0020] Furthermore, in such an embodiment, the intercept value (intercept at elastic modulus=0) extrapolated from the approximation line is larger for cell populations with higher osteoblast differentiation potential. Therefore, in the present invention, osteoblast differentiation potential can also be evaluated based on the intercept of the approximation line. For example, for a certain mesenchymal stem cell population, if the intercept is equal to or greater than a preset value T3', the osteoblast differentiation potential can be evaluated as high. In addition, by dividing the cells into three groups according to their osteoblast differentiation potential, and for a certain mesenchymal stem cell population, if the intercept is equal to or greater than a preset value T 3-1 If the value is more than or equal to T 3-1 ' smaller than the preset value T 3-2 ' or more, it belongs to the intermediate group and is 3-2 In this embodiment, if the threshold value T 3-1 ', for example, 0.6 ≤ T 3-1 '≦0.9, preferably 0.7≦T 3-1 The threshold T can be set in the range of '≦0.8. 3-2 ', for example, 0.4 ≤ T 3-2 '≦0.6, preferably 0.45≦T 3-2 It can be set in the range '≦0.55.

[0021] A kit for predicting osteoblast differentiation potential In another embodiment, the present invention provides a method for culturing an undifferentiated mesenchymal stem cell population on an elastic body having an elastic modulus of less than 5 kPa; Provided is a kit for predicting the osteoblast differentiation potential of a mesenchymal stem cell population, comprising a means for measuring the proportion of cells in the mesenchymal stem cell population that retain RUNX2 in their nuclei.

[0022] In yet another embodiment, the present invention provides a method for culturing an undifferentiated mesenchymal stem cell population on an elastic body having an elastic modulus of less than 5 kPa; Also provided is a kit for predicting the osteoblast differentiation potential of a mesenchymal stem cell population, comprising a means for measuring the proportion of cells in which RUNX2 has translocated into the cytoplasm in the mesenchymal stem cell population.

[0023] In these embodiments, the definitions of terms such as elastic body and elastic modulus, the culture conditions, the method for measuring the percentage of cells in which RUNX2 is retained in the nucleus, the method for measuring the percentage of cells in which RUNX2 has translocated into the cytoplasm, etc. are as described above.

[0024] Examples of the means for culturing an undifferentiated mesenchymal stem cell population on an elastic body having an elastic modulus of less than 5 kPa include an elastic body having an elastic modulus of less than 5 kPa or a culture vessel having the elastic body, a medium for culturing a mesenchymal stem cell population, or a raw material thereof. Examples of the means for measuring the proportion of cells in which RUNX2 is retained in the nucleus include an anti-RUNX2 antibody, a fluorescent secondary antibody, and a dye for staining DNA in the nucleus. In addition, the kit of the present invention can contain other components as necessary. Examples of the other components include, but are not limited to, a tool for collecting undifferentiated mesenchymal stem cells (e.g., a syringe, etc.), a positive control sample, and a negative control sample. The kit of the present invention can also contain a written document on which the procedure for performing the above-mentioned test method is written. The kit of the present invention can be used according to the description of the "method for predicting osteoblast differentiation potential" described above, and the preferred embodiment is also as described above.

[0025] Specific embodiments of the present invention will be described in detail below using examples, but the present invention is not limited to these examples. EXAMPLES

[0026] 1 method 1.1 Preparation of culture substrate Styrenated gelatin (StG) was used as the elastic modulus adjustment gel (Non-Patent Document 9). A PBS(-) solution containing 2,2'-azobis [2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044, FUJIFILM Wako Pure Chemical Corporation) was added to a 33.3 wt. % styrenated gelatin solution to prepare 30 wt. % StG gel solutions with final concentrations of VA-044 of 0.032, 0.06, and 0.1%. 30 μL of the StG gel solution was added to a 18 mm diameter cover glass coated with Poly(N-isopropylacrylamide) (PNIPAAm) (KOHJIN), and the cover glass was sandwiched between vinylized 18 mm diameter coverslips. The gel was allowed to stand at 45°C for 270 or 300 minutes to gel. The gel was then left to stand overnight in PBS(-) at 4°C, and the StG gel was peeled off from the PNIPAAm-coated glass. The elastic modulus was adjusted by changing the combination of the final concentration of VA-044 and the heat curing time.

[0027] The elastic modulus of the StG gel was measured by indentation testing using an atomic force microscopy (AFM) (JPK NanoWizard 4, JPK Instruments, Bruker Nano GmbH). Silicon nitride cantilevers (qp-BioAC-CI CB3, Nanosensors, Neuchatel) with a nominal spring constant of 0.03-0.09 N / m were used for the indentation testing. The test was performed at a temperature of 20-26°C. The force curve was fitted to the Hertz model by the nonlinear least squares method to calculate the elastic modulus. The elastic modulus was measured at three points on the gel substrate, at the center, 2.5 mm from the center, and 5.5 mm from the center, and the average value was used as the elastic modulus of the gel. The StG gel after elastic modulus measurement was used as the culture substrate.

[0028] 1.2 Cell culture Three lots of human bone marrow-derived mesenchymal stem cells were used in the experiment (Lot 1: #70011720 (American Type Culture Collection), Lot 2: #471980 (Lonza), Lot 3: #00003525 (Lonza)). After thawing, the cells were seeded in a T25 cell culture flask (Corning) containing mesenchymal stem cell growth medium (basal medium with supplements and growth factors: MSCGM, Lonza) and cultured for proliferation at 5% CO2 and 37°C. The medium was changed every two days during the proliferation period. Mesenchymal stem cells that had been passaged four times were used for the subsequent experiments (evaluation of osteoblast differentiation potential, adipocyte differentiation potential, and fluorescent staining of YAP and RUNX2).

[0029] 1.3 Evaluation of osteoblast differentiation potential Each lot of mesenchymal stem cells subcultured in the above "1.2 Cell culture" was plated on a collagen-coated 12-well plate (Corning) at 3100 cells / cm. 2 The cells were seeded at 1000 x g / ml and cultured in MSCGM at 5% CO2 and 37°C. After 24 hours, the culture medium was replaced with osteoblast differentiation induction medium (Lonza) and cultured for 14 days. The medium was replaced once every three days during osteoblast differentiation induction. To evaluate the amount of bone matrix produced in each sample, the samples were left to stand in 95% ethanol at -20°C for 10 minutes to fix the proteins. The samples were immersed in 40 mM Alizarin Red S staining solution (Sigma-aldrich) and left to stand at room temperature for 10 minutes. The stained samples were photographed using a single-lens reflex camera (D7000, Nikon).

[0030] 1.4 Adipocyte differentiation potential evaluation Each lot of mesenchymal stem cells subcultured in "1.2 Cell culture" was cultured on a 24-well plate (AGC Techno Glass) at 21,000 cells / cm 2The cells were seeded in 100% CO2 and placed in MSCGM at 37°C in a 5% CO2 environment. The medium was changed every 2-3 days and cultured for 5 days until confluence was reached. A cycle of culturing for 3 days in adipocyte differentiation induction medium (Lonza) and 1 day in adipocyte differentiation maintenance medium (Lonza) was repeated 3 times. After that, the cells were cultured in adipocyte differentiation maintenance medium for 7 days. The samples were immersed in 4% paraformaldehyde for 60 minutes to fix the proteins. Each sample was immersed in 1.8 mg / mL Oil Red O staining solution for 20 minutes to stain the lipid droplets. The stained samples were photographed using a single-lens reflex camera (D7000, Nikon).

[0031] 1.5 Fluorescent staining images of YAP and RUNX2 Each lot of mesenchymal stem cells subcultured in the above "1.2 Cell culture" was cultured on StG gel at 2500 cells / cm 2The cells were seeded at 1:100 and cultured in MSCGM for 4 days. The MSCGM medium was replaced every 2-3 days. The cells were immersed in 4% paraformaldehyde for 20 minutes to fix proteins. The cells were then placed in PBS(-) containing 10% donkey serum (Sigma-Aldrich), 1% bovine serum albumin (BSA, FUJIFILM Wako Pure Chemical Corporation), and 0.5% Triton X-100 (Sigma-Aldrich) for 45 minutes to puncture the cell membrane and block the cells. The cells were then immersed overnight at 4°C in PBS(-) containing 10% donkey serum and 1% BSA, containing rabbit anti-YAP (1:100, Cell Signaling Technology Japan) and goat anti-RUNX2 (1:15, R&D Systems) as primary antibodies. Then, the sections were immersed in a 1% BSA solution containing anti-rabbit Alexa Fluor 488-labeled secondary antibody (1:1000; Cell Signaling Technology Japan) and anti-goat Alexa Fluor 568-labeled secondary antibody (1:1000; Cell Signaling Technology Japan) at 37°C for 1 hour. After washing with PBS(-) containing 0.05% Tween, the sections were immersed in PBS(-) containing 5 μg / mL DAPI for 10 minutes at room temperature to stain DNA. Fluorescence images were taken using a confocal laser microscope (FV3000, Olympus) equipped with a 10x objective lens (UPLFLN, numerical aperture (NA) = 0.3, Olympus).

[0032] To observe the localization of YAP and RUNX2 in cells just before they attached to the StG gel, i.e., before they received stimuli derived from the elasticity of the StG gel, fluorescent staining was performed on mesenchymal stem cells in suspension. Mesenchymal stem cells were plated at 5000 cells / cm on a glass bottom dish coated with poly-L-lysine (Sigma Aldrich). 2Cells were seeded at 100 nm and incubated in MSCGM. Proteins were fixed with 4% paraformaldehyde within 10 min, and stained for YAP, RUNX2, and DNA using the same procedure as above. Fluorescent images were captured using a confocal laser microscope (FV3000, Olympus) equipped with a 60x oil immersion objective (UPlanSApo, NA = 1.42, Olympus).

[0033] 1.6 Quantitative evaluation method for YAP and RUNX2 localization Image analysis was performed using ImageJ. Projection images were created from the z-slice images of YAP and RUNX2 using the maximum intensity projection method. The DAPI images were binarized using the automatic thresholding algorithm Otsu to determine the cell nucleus region. The average intensity value in the cell nucleus region of YAP and RUNX2 was divided by the average intensity value of three arbitrary regions of the cytoplasm (10 × 10 pixels at 1.24 μm pixel pitch) to calculate the nuclear-to-outernuclear ratio. Cells with an intensity value of 1.6 or higher were considered to be nuclear-localized cells. To evaluate the change in the localization of YAP and RUNX2 with respect to the substrate elasticity, a line was approximated by the least squares method for the region where the ratio of nuclear-localized cells increased linearly in the scatter plot showing the relationship between the ratio of nuclear-localized cells of YAP and RUNX2 and the substrate elasticity.

[0034] 2 Results 2.1 Elastic modulus of StG gel The results of measuring the elastic modulus of StG gels prepared by varying the VA-044 concentration and heat curing time are shown in Table 1. The elastic modulus increased by increasing the VA-044 concentration and heat curing time. The prepared StG gels were found to have an elastic modulus of 0.8-30 kPa. After measuring the elastic modulus, the StG gels were used as culture substrates.

[0035] [Table 1]

[0036] 2.2 Differentiation potential of mesenchymal stem cells The osteoblast and adipocyte differentiation capacities of mesenchymal stem cells were evaluated. All cell lots had the ability to differentiate into osteoblasts and adipocytes, but the efficiency differed between lots. Figure 1(a) shows the results of Alizarin Red S staining after 14 days of osteoblast differentiation induction. Bone matrix formation was confirmed in all cell lots. The amount of bone matrix observed was the highest in Lot 3 and the lowest in Lot 1. Figure 1(b) shows the results of Oil Red O staining after 20 days of adipocyte differentiation induction. Lipid droplets were observed in all lots, but Lot 3 had the least of the three lots.

[0037] 2.3 Changes in YAP and RUNX2 localization in response to substrate elasticity To evaluate the response of YAP and RUNX2 to substrate elasticity stimulation, we first confirmed the localization of YAP and RUNX2 in mesenchymal stem cells immediately before they adhered to the StG gel and immediately before they received stimulation from the substrate elasticity. As a representative example, cells of Lot2 in a cell suspension are shown in Figure 2(a). YAP was not localized in the cell nucleus, but was diffused between the cell nucleus and cytoplasm. On the other hand, RUNX2 was localized in the cell nucleus even in the non-adherent state. A similar tendency was observed in all cells in Lot1-3.

[0038] Next, we show the localization of YAP and RUNX2 in mesenchymal stem cells cultured for 4 days on StG gels with elastic modulus of 0.8-30 kPa. As a representative example, cells from Lot 2 cultured on StG gels with elastic modulus of 1.0 kPa and 13.4 kPa are shown in Figure 2(b). In mesenchymal stem cells cultured on the 1.0 kPa substrate, neither YAP nor RUNX2 was observed to be localized in the cell nucleus, but was diffused between the cell nucleus and cytoplasm. On the other hand, in mesenchymal stem cells cultured on the 13.4 kPa substrate, both YAP and RUNX2 were localized in the cell nucleus. A similar tendency was confirmed in all cells from Lots 1-3.

[0039] 2.4 Dependence of YAP and RUNX2 localization on substrate elasticity Finally, we show the relationship between the nuclear localization of YAP and RUNX2 and the elastic modulus of the StG gel. The relationship between the elastic modulus of the StG gel and the ratio of cells with nuclear YAP localization is shown in Figure 3(a). The ratio of cells with nuclear YAP localization increased linearly up to 10 kPa and saturated at 0.91, 0.79, and 0.87 in Lots 1, 2, and 3, respectively. In all cell lots, the slope of the approximation line in the linear increase region up to 10 kPa was approximately 0.1, and the intercept value was between 0 and 0.1.

[0040] The relationship between the elastic modulus of the substrate and the ratio of cells with RUNX2 localized in the nucleus is shown in Figure 3(b). The ratio of cells with RUNX2 localized in the nucleus increased linearly up to approximately 5 kPa and saturated at 0.88, 0.83, and 0.80 in Lots 1, 2, and 3, respectively. The slopes of the approximation lines in the linear increase region up to 5 kPa were 0.08, 0.09, and 0.10, respectively, and the intercept values ​​were 0.49, 0.32, and 0.20, respectively, and distributed over a wide range from 0.2 to 0.5. As with YAP, the number of cells with RUNX2 localized in the nucleus increased with increasing elastic modulus, but the lot difference in the slope of the approximation line in the linear increase region was about 10%, and the lot difference in the intercept was about 50%, which was significant.

[0041] 3 Discussion 3.1 Differentiation potential of mesenchymal stem cells The three lots used in this experiment had the ability to differentiate into osteoblasts and adipocytes, but it became clear that their differentiation efficiencies differed (Figure 1). It has been reported that in mesenchymal stem cells, differentiation into osteoblasts and adipocytes is in a competitive relationship in which the promotion of one inhibits the other (Non-Patent Document 10). It is presumed that, among the mesenchymal stem cells used in this experiment, Lot 3 was the cell with the highest osteoblast differentiation ability and the lowest adipocyte differentiation ability, and Lot 1 was the mesenchymal stem cell with the lowest osteoblast differentiation ability and the highest adipocyte differentiation ability.

[0042] 3.2 Localized change in YAP depending on substrate elastic modulus It was found that the intracellular localization of YAP in mesenchymal stem cells changes in response to the elastic modulus of the culture substrate (Figure 2). It has been reported that the development of the actin cytoskeleton is necessary for the nuclear localization of YAP (Non-Patent Documents 11, 12). In this experiment, it is thought that the development of the actin cytoskeleton in response to the culture environment plays an important role. In mesenchymal stem cells immediately before receiving stimuli from the StG gel, YAP was diffused between the cell nucleus and cytoplasm (Figure 2(a)). In cells suspended in a cell suspension, actin fibers are depolymerized, which may result in the suppression of nuclear localization of YAP and lead to diffusion.

[0043] It is known that the elastic modulus of the culture substrate affects the development of the actin cytoskeleton, and that the actin cytoskeleton weakens on a low elastic modulus substrate, while the actin cytoskeleton develops on a high elastic modulus substrate (Non-Patent Document 13). In this experiment, YAP was localized to the nucleus by culturing on a high elastic modulus substrate. It is believed that the development of actin fibers required for YAP nuclear localization was induced by culturing on a high elastic modulus substrate, resulting in YAP localization in the nucleus. Furthermore, the ratio of cells with YAP nuclear localization increased up to 10 kPa and then saturated (Figure 2(a)). These results suggest that the actin cytoskeleton functions as a regulator of YAP nuclear localization, and that an elastic modulus of 10 kPa or more is necessary for the development of the actin cytoskeleton sufficient to promote YAP nuclear localization.

[0044] 3.3 Changes in RUNX2 localization depending on substrate elasticity The intracellular localization of RUNX2 in mesenchymal stem cells also changed in response to the elastic modulus of the culture substrate. Like YAP, RUNX2 is known as a mechanomediator whose localization changes in response to the elastic modulus of the substrate, and it has been suggested that the localization of the two between the nucleus and cytoplasm is correlated (Non-Patent Document 6). In suspended cells before sensing the elastic modulus of the substrate, YAP diffuses between the cell nucleus and cytoplasm, so it was predicted that RUNX2 would also diffuse. However, RUNX2 was localized in the cell nucleus (Figure 2(a)). The mesenchymal stem cells used in this experiment were cultured in polystyrene tissue culture vessels for up to four passages. Previous studies have reported that culturing on a polystyrene substrate for more than five days biases the differentiation into osteoblasts (Non-Patent Document 6). In mesenchymal stem cells biased to differentiate into osteoblasts, RUNX2 is retained in the cell nucleus by condensed chromatin and the cell nucleoskeletal structure, not by depolymerization of the actin cytoskeleton (Non-Patent Document 14). It is possible that in this experiment, mesenchymal stem cells were biased towards differentiation into osteoblasts during the proliferation culture period, and RUNX2 was retained in the cell nuclei.

[0045] When cells with RUNX2 nuclear localization after proliferation culture were cultured on StG gels with an elastic modulus lower than 5 kPa, the nuclear localization of RUNX2 was suppressed (Figures 2(b) and 3(b)). The efficiency of RUNX2 nuclear export was higher with lower elastic modulus stimulation. The elastic modulus of the substrate affects the chromatin structure in the cell nucleus (Non-Patent Document 15). Since the nuclear retention of RUNX2 is involved in the nuclear skeleton and chromatin aggregation, it is presumed that the low elastic modulus stimulation of 5 kPa or less promoted structural changes in the nuclear skeleton in the cell nucleus and chromatin disaggregation, resulting in a decrease in the nuclear retention of RUNX2.

[0046] 3.4 Heterogeneity of localization and response of YAP and RUNX2 The relationship between the ratio of YAP-localized cells in the nucleus and the elastic modulus of the substrate (Fig. 3(b)) did not show any significant differences between cell lots, and the slope and intercept of the approximation line in the linear increase region up to 10 kPa were approximately the same between cell lots. Previous studies have reported that YAP localizes to the nucleus at substrate elastic moduli of 10 kPa or more and diffuses at substrate elastic moduli of 5 kPa or less (Non-Patent Documents 6, 11). The results obtained in this study were not inconsistent with previous studies, and the ratio of YAP-localized cells in the nucleus reached 80% or more at 10 kPa and saturated, regardless of cell lot, and showed a linear positive correlation with the elastic modulus below 10 kPa (Fig. 3(a)).

[0047] Reports on the localization of RUNX2 depending on the substrate elastic modulus vary from paper to paper (Non-Patent Documents 4-6). One possible reason for this is the difference in the ability of RUNX2 to be retained in the nucleus between cell lots. The ratio of cells with RUNX2 nuclear localization on low-elasticity substrates of 5 kPa or less varied depending on the lot (Figure 3(b)). As for Lot 1, as a result of culturing on StG gel of 2 kPa, RUNX2 was localized in the nucleus in many cells (ratio 0.78). This result was consistent with the previous study by Yang et al. that RUNX2 is localized in the nucleus at a substrate elastic modulus of about 2 kPa (Non-Patent Documents 6, 11). On the other hand, for Lots 2 and 3, the ratio of cells with RUNX2 nuclear localization was 0.5 or less at a substrate elastic modulus of around 2 kPa, and RUNX2 nuclear localization was suppressed in many cells. It has been reported that mesenchymal stem cells stimulated by long-term culture on a high-elasticity substrate are less likely to undergo chromatin structural changes in response to external mechanical stimuli (Non-Patent Document 15). These findings suggest that differences in the ability of RUNX2 to be retained in the nucleus due to chromatin condensation may explain the discrepancies in reports of changes in RUNX2 localization in response to substrate modulus among research groups.

[0048] 3.5 Differentiation potential of mesenchymal stem cells and RUNX2 localization response It is known that YAP and RUNX2 affect the progression of differentiation by regulating gene expression related to the differentiation of mesenchymal stem cells into osteoblasts (Non-Patent Documents 11, 16-18). The change in localization of YAP and RUNX2 in response to extracellular mechanical stimuli may differ depending on the differentiation ability of mesenchymal stem cells. Although the three lots of mesenchymal stem cells used in this experiment had different osteoblast differentiation abilities (Figure 1(a)), the proportion of cells with YAP localized in the nucleus showed a uniform change with respect to the elastic modulus. From this result, it is considered that the response of YAP to the elastic modulus of the substrate is similar regardless of the difference in the osteoblast differentiation ability of mesenchymal stem cells.

[0049] In contrast to YAP, the results showed that the elastic modulus dependency of the ratio of RUNX2 nuclear-localized cells differed between lots. In particular, the ratio of nuclear-localized cells in the intercept of the substrate elastic modulus of 0 kPa, obtained by extrapolating the increasing line of the RUNX2 nuclear-localized cell ratio against the substrate elastic modulus (Figure 3(b)), differed greatly between lots. This value is presumed to be the ratio of cells that continue to retain RUNX2 in the cell nucleus even under low elastic modulus stimulation, and is considered to indicate the RUNX2 nuclear retention ability of the cell population. Furthermore, it was found that there was a negative correlation between the intercept value and the osteoblast differentiation efficiency. RUNX2 is known as a protein essential for the transition from mesenchymal stem cells to the early stage of osteoblast differentiation, while it has been reported that it acts inhibiting the differentiation progression into mature osteoblast-differentiated cells (Non-Patent Document 16). The nuclear retention ability of RUNX2 in mesenchymal stem cell populations may be involved in the efficiency of RUNX2 discharge from the middle to late stages of osteoblast differentiation.

[0050] 3.6 Elastic substrates for controlling the localization of YAP and RUNX2 in mesenchymal stem cells Mesenchymal stem cells are highly sensitive to extracellular mechanical stimuli, and when cultured on a polystyrene substrate such as a conventional tissue culture dish, they are biased toward differentiation into osteoblasts, resulting in the loss of pluripotency and self-renewal ability (Non-Patent Documents 6, 19). Since the nuclear localization of YAP and RUNX2 promotes the transition from mesenchymal stem cells to the initial state of osteoblast differentiation, it is necessary to inhibit the nuclear localization of YAP and RUNX2 in order to suppress unintended osteoblast differentiation of mesenchymal stem cells.

[0051] The responsiveness of YAP localization to substrate elasticity was consistent regardless of cell lot. From the relationship between the ratio of YAP nuclear-localized cells and the ratio of substrate elasticity, it can be seen that culturing on a substrate with an elastic modulus of 10 kPa or higher promotes YAP nuclear localization. Furthermore, it is thought that the substrate elasticity required to inhibit YAP nuclear localization can be predicted based on the linear approximation of the relationship between the ratio of YAP nuclear-localized cells and substrate elasticity (Figure 3(a)).

[0052] The responsiveness of RUNX2 localization to substrate elasticity was consistent in the high elasticity range, and it was found that RUNX2 could be localized in the cell nucleus by culturing on substrates with an elastic modulus of 5 kPa or more. On the other hand, the ratio of cells with RUNX2 nuclear localization at elastic moduli of 5 kPa or less varied significantly from lot to lot. However, it was found that there was a tendency for the ratio to increase linearly up to 5 kPa in all cell lots. The lot differences in the increase rate were relatively small, at 0.08, 0.09, and 0.10, but the lot differences in the intercept were significant, at 0.2 to 0.5. From this, it was possible to estimate the efficiency of RUNX2 nuclear localization inhibition at any elastic modulus by measuring the ratio of cells with RUNX2 nuclear localization at substrate elastic moduli of less than 5 kPa. In addition, by measuring the ratio of cells with RUNX2 nuclear localization at two substrate elastic moduli, one near 5 kPa and one below 5 kPa, it is possible to create a predicted line between the substrate elastic modulus and the ratio of cells with RUNX2 nuclear localization, and precisely determine the efficiency of RUNX2 nuclear localization inhibition at any elastic modulus.

[0053] 4 Conclusion As described above, we resolved the instability of the localization response of YAP and RUNX2 to elastic modulus stimulation in mesenchymal stem cells and obtained insight into a method for designing the substrate elastic modulus to reproducibly control differentiation across multiple cell lots.

Claims

1. Culturing a plurality of populations of undifferentiated mesenchymal stem cells on an elastic body having an elastic modulus of less than 5 kPa; A step of measuring the proportion of cells in which RUNX2 is retained in the nucleus in the multiple populations of mesenchymal stem cells cultured in the above step; and evaluating that, among the plurality of populations of undifferentiated mesenchymal stem cells, a cell population having a low ratio of cells retaining RUNX2 in the nucleus has a higher osteoblast differentiation potential than a cell population having a high ratio. a method for predicting the osteoblast differentiation potential of said mesenchymal stem cell population, comprising:

2. Culturing a plurality of populations of undifferentiated mesenchymal stem cells on an elastic body having an elastic modulus of less than 5 kPa; A step of measuring the ratio of cells in which RUNX2 has been translocated into the cytoplasm in the multiple populations of mesenchymal stem cells cultured in the above step; and A step of evaluating, among the plurality of populations of undifferentiated mesenchymal stem cells, a cell population having a higher proportion of cells in which RUNX2 has been translocated into the cytoplasm as having a higher osteoblast differentiation potential than a cell population having a lower proportion of the cells. a method for predicting the osteoblast differentiation potential of said mesenchymal stem cell population, comprising:

3. A process for culturing an undifferentiated mesenchymal stem cell population on two or more types of elastomers having elastic moduli of less than 5 kPa and different elastic moduli, A step of measuring the proportion of cells in which RUNX2 is retained in the nucleus in the mesenchymal stem cell population cultured in the above step; and a step of plotting (elastic modulus of elastic body, proportion of cells retaining RUNX2 in the nucleus) of a mesenchymal stem cell population cultured on two or more types of elastic bodies on a graph with the elastic modulus of elastic body on the horizontal axis and the proportion of cells retaining RUNX2 in the nucleus on the vertical axis, obtaining an approximation line on the graph, and evaluating that the osteoblast differentiation potential is high when the intercept extrapolated from the approximation line (intercept when elastic modulus = 0) is equal to or less than a set value T3-1 (where T3-1 is set in the range of 0.1≦T3-1≦0.4); a method for predicting the osteoblast differentiation potential of said mesenchymal stem cell population, comprising:

4. A process for culturing an undifferentiated mesenchymal stem cell population on two or more types of elastomers having elastic moduli of less than 5 kPa and different elastic moduli, A step of measuring the proportion of cells in which RUNX2 has been transferred into the cytoplasm in the mesenchymal stem cell population cultured in the above step; and a process of plotting (elastic modulus of elastic body, proportion of cells in which RUNX2 has migrated into the cytoplasm) of a mesenchymal stem cell population cultured on two or more types of elastic bodies on a graph with the elastic modulus of elastic body on the horizontal axis and the proportion of cells in which RUNX2 has migrated into the cytoplasm on the vertical axis, obtaining an approximate straight line in the graph, and evaluating that the population belongs to a group with high osteoblast differentiation potential when the intercept extrapolated from the approximate straight line (the intercept at elastic modulus = 0) is equal to or greater than a set value T3-1' (wherein T3-1' is set within the range of 0.6≦T3-1'≦0.9); a method for predicting the osteoblast differentiation potential of said mesenchymal stem cell population, comprising:

5. The method according to any one of claims 1 to 4, wherein the culturing step is carried out using an elastic body with an elastic modulus of 3 kPa or less.

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