Method for maintaining undifferentiation potency of mesenchymal stem cell, employing shaking suspension culture

Abstract

To culture mesenchymal stem cells for a long time while maintaining their undifferentiation potency.SOLUTION: Provided is a method of culturing mesenchymal stem cells as a cell construct while maintaining their undifferentiation potency, the method including subjecting the mesenchymal stem cells to shaking culture.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to mesenchymal stem cells and a method for culturing the same. [Background technology]

[0002] In the medical and dental fields, bone tissue defects are treated with artificial materials such as β-TCP, titanium, and autologous bone grafts. In treatments using artificial materials, including autologous bone grafts, the absorption of regenerated bone after treatment is an issue (Non-Patent Documents 1 and 2). In the field of bone regeneration, scaffolds and membranes that prevent the intrusion of granulation tissue are often required to make space during surgery, and their effects on the regeneration site must also be considered.

[0003] In recent years, research and treatment using autologous cells have also been conducted, the most representative of which are mesenchymal stem cells (MSCs) (hereafter, in this specification, mesenchymal stem cells will be abbreviated to MSCs).

[0004] MSCs were originally defined as cells that attach to and proliferate in plastic culture dishes when bone marrow cells are seeded onto them, and have the ability to differentiate into mesodermal tissues and cells such as fat, cartilage, and bone (Non-Patent Document 3).

[0005] A search for markers for mouse bone marrow MSCs was carried out, and in 2009, Morikawa et al. - / CD45 - / Ter119 - / Sca-1 + / PDGFRα + During fractionation It has been reported that mouse MSCs are enriched in mouse bone marrow cells (Non-Patent Documents 4 and 5). Details of this purification technique have been published by Houlihan et al. (Non-Patent Document 6). Human bone marrow MSCs have been enriched in mouse bone marrow cells by Mabuchi et al. + / CD90 (Thy-1) + It has been reported that it is present at high concentrations in fractions (Non-Patent Document 7).

[0006] MSCs exist in various tissues, but bone marrow is the tissue from which many MSCs can generally be obtained stably. MSCs are defined as cells that can adhere to and proliferate in a plastic culture dish when seeded on the bone marrow and differentiate into osteoblasts, chondrocytes, and adipocytes (Non-Patent Document 3), and are attracting attention as a cell source for regenerative medicine. However, repeated long-term adhesion culture reduces proliferation and differentiation abilities, which causes clinical problems such as differences in results between facilities and patients.

[0007] Specifically, it is known that conventional MSCs have limited proliferation capacity in an adherent culture environment, and accordingly lose their differentiation capacity (Non-Patent Documents 8 and 9). It is also known that purified MSCs gradually lose proliferation capacity in an adherent culture environment (see Non-Patent Document 5, Fig 1G).

[0008] In recent years, it has been reported that when MSCs are cultured on a special culture dish, they can form three-dimensional cell masses (spheres or spheroids) in a floating state without adhering to the culture dish (Non-Patent Document 10). However, this method does not allow for long-term culture, There have been no reports on what changes occur to cells when cultured in a suspension environment. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Hatano et al. Clin Oral Impl Res, vol.15, p339~345, 2004 [Non-Patent Document 2] Verhoeven et al. Clin Oral Impl Res, vol.11, p583~594, 2000 [Non-Patent Document 3] Pittenger et al. Science, vol 284 2 April, 1999 [Non-Patent Document 4] Morikawa et al. BBRC, vol.379, p1114~1119, 2009. [Non-Patent Document 5] Morikawa et al. JEM, vol.206, p2483~2496, 2009. [Non-Patent Document 6] Houlihan et al. Nature Protocol, vol.7(12), p2103~2111, 2012. [Non-Patent Document 7] Mabuchi et al. Stem cell reports, vol.1(2), p152~165, 2013 [Non-Patent Document 8] Bonab et al. BMC Cell Biol, vol.7, p14, 2006. [Non-Patent Document 9] Bork et al. Aging Cell, vol.9(1), p54~63, 2010. [Non-Patent Document 10] Baraniak et al. Cell Tissue Res, vol.347(3), p701~711, 2012 [Non-Patent Document 11] Doetsch et al. Cell, 97 (6), p703~716, 1999. [Non-Patent Document 12] Laura et al. Protoc Exch, Doi:10.1038 / nprot.2006.215, 2006 Summary of the Invention [Problem to be solved by the invention]

[0010] The problem to be solved by the present invention is to culture MSCs for a long period of time while maintaining their undifferentiated state. [Means for solving the problem]

[0011] Under these circumstances, the present inventors conducted intensive research and found that it is possible to induce the formation of cell masses by shaking culture of MSCs, and that MSCs can be cultured for a long period of time while maintaining their undifferentiated state. The present invention is based on this novel finding.

[0012] Thus, the present invention provides the methods and cell masses described in the following sections.

[0013] Item 1. A method for culturing MSCs while maintaining their undifferentiated state, comprising culturing the MSCs with shaking.

[0014] Item 2. The method according to Item 1, wherein the shaking culture is carried out at a rotation speed of 20 to 200 rpm, preferably 60 to 120 rpm, and more preferably 85 to 95 rpm.

[0015] Item 3. The method according to Item 1 or 2, wherein the shaking culture is carried out with an amplitude of 10 to 40 mm, preferably 25 to 40 mm, and more preferably 30 to 40 mm.

[0016] Item 4. The method according to any one of Items 1 to 3, wherein the subculture is performed two or more times.

[0017] Item 5. The MSCs to be subjected to shaking culture are cells that have lost the ability to differentiate into a specific cell. and the ability to differentiate into the predetermined cells is restored by shaking culture.

[0018] Item 6. A cell mass obtained by the method according to any one of items 1 to 5.

[0019] Item 7. A cell differentiation method comprising a step of culturing the cell mass according to Item 6 in a differentiation-inducing medium.

[0020] Item 8. A cell mass differentiated into a predetermined cell by the method according to Item 7. Effect of the Invention

[0021] According to the present invention, shaking culture can induce MSCs to form cell masses and enable long-term culture while maintaining their undifferentiated state. In addition, according to the present invention, when MSCs that have lost their differentiation potential due to long-term conventional adhesion culture or the like are used, shaking culture can In the field of stem cells to which the present invention pertains, physical stimuli have been thought to induce differentiation. Therefore, the effect of the present invention, that undifferentiated state can be maintained for a long period of time by shaking culture of MSCs, is unexpected and could not be predicted from the prior art. [Brief description of the drawings]

[0022] [Figure 1] This shows cell mass formation of mouse and human MSCs by shaking culture (for 2 months). Figure 1, left: Mouse MSCs. Used after 12 adherent subcultures. Figure 1, right: Human MSCs. Used after 6 adherent subcultures. [Diagram 2] The effect of shaking culture on maintaining the differentiation potential of mouse MSCs is shown. Figure 2a, top: 11 adherent subcultures (Fig. 2a, top left: osteoblasts, top center: chondrocytes, top right: adipocytes). Figure 2a, bottom: Shaking culture after 36 adherent subcultures (Fig. 2a, bottom left: osteoblasts, bottom right: adipocytes). Figure 2b, left: 8 adherent subcultures. Figure 2b, right: Shaking culture after 44 adherent subcultures. Figure 2c: RT-PCR analysis of PDGFRα expression in cell aggregates subjected to shaking culture for 2 months after 9, 25, and 41 adherent subcultures and 11 and 37 adherent subcultures. [Diagram 3] The mesodermal differentiation potential of OricellTM mouse MSCs cultured with shaking is shown. Left column in Fig. 3: Osteoblasts. Center column in Fig. 3: Chondrocytes. Right column in Fig. 3: Adipocytes. Top row in Fig. 3: One month of shaking culture after nine adherent subcultures. Bottom row in Fig. 3: One month of shaking culture after 30 adherent subcultures. [Figure 4] Gene expression analysis (RT-PCR) after induction of differentiation into mesodermal cells is shown in Fig. 4a: After induction of bone differentiation. Fig. 4b: After induction of cartilage differentiation. Fig. 4c: After induction of adipocyte differentiation. The numbers above Fig. 4a, Fig. 4b, and Fig. 4c indicate the number of adherent subcultures (times). [Diagram 5]The effect of shaking culture on maintaining the differentiation potential of human MSCs is shown. Left column in Fig. 5a: Osteoblasts. Right column in Fig. 5a: Adipocytes. Top row in Fig. 5a: 20 adherent subcultures. Middle row in Fig. 5a: Shaking culture after 6 adherent subcultures. Bottom row in Fig. 5a: 20 adherent subcultures without osteogenic differentiation induction. Top row in Fig. 5b: 21 adherent subcultures. Bottom row in Fig. 5b: Shaking culture after 7 adherent subcultures. Fig. 5c: The numbers on the top of Fig. 5c indicate the number of adherent subcultures (times). [Figure 6] The chondrocyte differentiation ability of human MSCs is shown. Figure 6a, upper: Cartilage (stained with toluidine blue). 9 cycles of adherent subculture. Figure 6a, lower: Cartilage (stained with toluidine blue). Shaking culture after 9 cycles of adherent subculture. Figure 6b, upper: Cartilage (stained with toluidine blue). 19 cycles of adherent subculture. No cartilage pellet formed. Figure 6b, lower: Cartilage (stained with toluidine blue). Shaking culture after 19 cycles of adherent subculture. [Figure 7] The results of adhesion culture of MSC cell aggregates using a three-dimensional suspension culture vessel, which is a prior art, are shown. Figure 7a: Human MSCs (used after 9 adhesion subcultures). Cultured for 7 days. Figure 7b: Mouse MSCs. Figure 7b, upper row: Used after 25 adhesion subcultures (second from the left: three-dimensional suspension culture vessel, immediately after seeding. Third from the left: three-dimensional suspension culture vessel, cultured for 7 days. Fourth from the left: re-adhesion to culture dish, re-adhesion for 7 days. Fifth from the left: re-adhesion to culture dish, induction of adipogenesis). Figure 7b, lower row: Used after 41 adhesion subcultures (second from the left: three-dimensional suspension culture vessel, immediately after seeding. Third from the left: three-dimensional suspension culture vessel, cultured for 7 days. Fourth from the left: re-adhesion to culture dish, re-adhesion for 7 days. Fifth from the left: re-adhesion to culture dish, induction of adipogenesis). [Figure 8] Differentiation potential analysis of OricellTM mouse MSC cell aggregates using a three-dimensional suspension culture vessel (prior art). Figure 8a: Used after 25 adherent subcultures (Figure 8a left: bone, Figure 8a right: fat). Figure 8b: Used after 41 adherent subcultures (Figure 8b left: osteoblasts, Figure 8b right: fat cells). [Figure 9]The morphology of human MSC cell clusters cultured with shaking is shown, as well as their cell supplying ability. Figure 9a, top row, first from the left: cell clusters seeded onto an adhesion culture dish, 0 days. Figure 9a, top row, second from the left: 1 day. Figure 9a, top row, third from the left: 7 days. Figure 9a, top row, fourth and fifth from the left: re-adhesion of cell clusters (first time). Figure 9a, middle row, first from the left: 12 days. Figure 9a, middle row, second from the left: 15 days. Figure 9a, middle row, third from the left: 27 days. Figure 9a, middle row, fourth from the left: 44 days. Figure 9a, middle row, fifth from the left: 54 days. Figure 9a, bottom row, left: 57 days. Figure 9a, bottom row, right: 67 days. Figure 9b: Differentiation of migratory cells after the third re-adhesion (Figure 9b, left: osteoblasts; Figure 9b, right: adipocytes). [Figure 10] This shows cell clusters formed by shaking culture of purified mouse MSCs. Figure 10a right: Purified mouse MSCs. Figures 10b and 10c: After two adhesion subcultures, shaking culture was performed, and after cell clusters were formed, the cell clusters were allowed to re-adhere to the culture dish. Figure 10b shows the first day of adhesion. Adipocytes (fat droplets) after differentiation induction are shown in Figure 10c. [Figure 11] This shows cell aggregate formation using OricellTM mouse MSCs in neural stem cell medium. Figure 11a: Shaking culture for one month after 9 adherent subcultures. Figure 11b: Shaking culture for one month after 9 adherent subcultures (First from the left in Figure 11b: Osteogenesis. Second from the left in Figure 11b: Fatogenesis. Third from the left in Figure 11b: Cartilage differentiation.) Figure 11c Neuronal differentiation. Figure 11d: Shaking culture for one month after 39 adherent subcultures. Figure 11e: RT-PCR after one month of shaking culture (numbers on top of Figure 11d are passage numbers) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] How to culture MSCs while maintaining their undifferentiated state The present invention provides a method for culturing MSCs in cell clusters while maintaining their undifferentiated state, which method is characterized by culturing MSCs with shaking.

[0024] The type of MSC used in the present invention is not particularly limited, and examples thereof include bone marrow-derived cells, dental pulp-derived cells, adipose tissue-derived cells, etc. Furthermore, the animal species of MSC is also not particularly limited, and examples thereof include those derived from mammals such as primates (e.g., humans, monkeys, etc., preferably humans) and rodents (e.g., mice, rats, rabbits, etc.).

[0025] The present invention is characterized in that such MSCs are cultured with shaking. The culture vessel is not particularly limited and can be appropriately set according to the amount of culture solution required, the type of shaking method, etc. Examples of the shape of the culture vessel include Erlenmeyer flasks and seesaw-type bioreactor flasks (e.g., flasks with a polygonal (4-8) bottom) as shown in the figures of WO2015 / 064705. Examples of the capacity of the culture vessel include those of 125 to 3000 ml, preferably 125 to 500 ml. As the culture vessel, a non-adhesive culture vessel (e.g., a non-adhesive culture dish, a non-adhesive well, a non-adhesive flask, etc.) that has been treated to suppress adhesion of cells to the bottom of the inner wall of the vessel may be used, or a normal culture vessel that has not been particularly treated may be used.

[0026] The type of shaking method is not particularly limited, and examples thereof include rotation, figure-of-eight, reciprocation, and seesaw type. In the present invention, rotation is preferred, but other shaking modes can be adopted. In the case of shaking culture by rotation, the rotation speed is not particularly limited, but can typically be set within the range of, for example, 20 to 200 rpm, preferably 60 to 120 rpm, and more preferably 85 to 95 rpm. Shaking culture within the above rotation speed range is preferable because it makes it difficult for the spheres to stick to the side and bottom (floor) inside the culture vessel.

[0027] In this specification, the shaking conditions such as the rotation speed and amplitude have been described using the rotation type as an example of a preferred embodiment, but as long as it can give the stem cells a similar physical stimulus, the figure-of-eight, reciprocating, seesaw, etc. can be adopted. For example, in the case of the figure-of-eight shaking culture, the culture vessel is shaken through two approximately circular tracks that are in contact at one point, so that the conditions can be set so that the rotation speed and amplitude are as exemplified for the rotation type for each circle forming the figure-of-eight. In addition, for example, in the case of the reciprocating type, the number of reciprocations per minute can be set to 20 to 250 reciprocations / min, and the amplitude can be appropriately set in the range of 10 to 40 mm. For example, in the case of the seesaw type, the angle of the shaking culture can be set to 2° to 12°, and the shaking cycle can be appropriately set in the range of 5 to 60 rpm.

[0028] The medium used is not particularly limited as long as it is a liquid medium suitable for culturing stem cells. Examples of such media include Minimum Essential Medium (MEM) media. The media may contain additives that are typically added when culturing stem cells, as necessary. Specific examples of additives include fetal bovine serum (FBS), Examples of such antibacterial agents include L-glutamine, L-alanyl-L-glutamine, and the like; and antibiotics (eg, penicillin, streptomycin, and the like).

[0029] A preferred embodiment of the culture of the present invention is subculture. In subculture, stem cells are collected before reaching a confluent state, and for example, 1 × 10 in the case of MSCs are collected. 3 ~ 1×10 7 Approximately 1 x 10 cells / ml 5 ~1×10 6 cells / ml The cells are seeded in a new medium so that the number of times of passage is equal to the number of times of passage. In the culture of the present invention, it is preferable to replace the medium appropriately (for example, every 1 to 5 days, preferably every 3 to 4 days). According to the method of the present invention, MSCs can be passage-cultured in an undifferentiated state without losing their differentiation ability. In the present invention, the cells can be passage-cultured, for example, by shaking culture, one or more times, preferably three or more times, preferably five or more times. In the present invention, the upper limit of the number of passages by shaking culture is not particularly limited, but the cells can be passage-cultured, for example, three or less times, preferably two or less times, preferably one or less times.

[0030] The shaking culture time is, for example, preferably about 1 to 90 days (about 24 to 2160 hours), more preferably 10 to 75 days (about 240 to 1800 hours), and more preferably 14 to 60 days (about 336 to 1440 hours). The shaking culture temperature in this step is not particularly limited, and is, for example, preferably 30 to 42°C, and more preferably 35 to 39°C. Such culture is preferably carried out in an atmosphere of 3 to 10% CO2.

[0031] According to the method of the present invention, MSCs can be cultured while still being in an undifferentiated state and including those in a differentiation potential state. Moreover, the shaking culture causes MSCs to proliferate and aggregate to form cell clusters. Thus, the present invention also provides a method for forming (undifferentiated) cell clusters, characterized in that MSCs are cultured with shaking; a method for inhibiting MSC differentiation, characterized in that MSCs are cultured with shaking, and the like, for culturing MSCs. The cell clusters obtained by the method of the present invention have the differentiation potential into various cells belonging to the mesoderm system, but in a preferred embodiment, the cell clusters have the differentiation potential into osteoblasts, adipocytes, etc., chondrocytes, and more preferably into adipocytes, etc. As described in the Examples, in conventional adhesion culture, the differentiation potential into adipocytes is lost, and therefore such an embodiment is preferred.

[0032] In the present invention, the term "undifferentiated" means that the cells are in an undifferentiated state and have the differentiation potential. In the present invention, the undifferentiated state of MSCs can typically be confirmed by, for example, whether or not they exhibit the differentiation potential into both osteoblasts and adipocytes, as shown in the Examples of the present application. In addition, in the present invention, "maintenance of undifferentiated state" includes not only a state in which the differentiation potential of MSCs that they originally had is maintained without losing, but also a state in which the differentiation potential of MSCs into at least one of the cells into which they can originally differentiate (as described above) is once reduced or lost during the culture process before the shaking culture, but the differentiation potential is restored by the shaking culture. Therefore, in the present invention, for example, as shown in the Examples of the present application, the state in which MSCs that have lost the differentiation potential into a specific cell (e.g., adipocytes, etc.) as a result of repeated subculture by a method other than shaking culture (e.g., adhesion culture, etc.) are restored to the differentiation potential into the cell by shaking culture is also included in the scope of "maintenance of undifferentiated state". Therefore, in this embodiment, the present invention also provides a method for recovering the ability to differentiate into a predetermined cell, which comprises subjecting MSCs that have lost the ability to differentiate into the predetermined cell to shaking culture.

[0033] In addition, the method of the present invention is characterized by the shaking culture of MSCs, but as long as the effects of the present invention can be obtained, static culture may be combined. For example, as shown in the examples of the present application, after forming cell clusters that retain undifferentiated state by shaking culture for a certain period of time, such The cell mass may be subjected to static culture. Such static culture may be performed as an adherent culture or a non-adherent culture. The medium, culture temperature, CO2 concentration, etc. in such static culture may be the same as those in the shaking The same conditions as those for culture can be appropriately adopted. As the culture vessel, a non-adhesive culture vessel (e.g., a non-adhesive culture dish, a non-adhesive well, a non-adhesive flask, etc.) that has been treated to suppress adhesion of cells to the bottom surface of the inner wall of the vessel may be used, or a normal culture vessel that has not been particularly treated may be used. The number of times of subculture in static culture is not particularly limited as long as the effects of the present invention can be obtained, but for example, in the present invention, subculture can be performed two or more times, preferably three or more times, and preferably five or more times. In addition, when static culture is performed, the upper limit of the number of subcultures in static culture is not particularly limited, but subculture can be performed, for example, five or less times, preferably three or less times, and preferably one or less times. In such an embodiment, the time for static culture is, for example, preferably about 0.1 to 60 days, and more preferably 1 to 30 days. In this embodiment of the present invention, the static culture time is, for example, preferably about 2.4 to 1440 hours, more preferably about 24 to 720 hours, and more preferably about 72 to 336 hours.

[0034] Cell mass that retains undifferentiated properties The present invention provides a cell mass obtained by the above-mentioned culture method of the present invention. In this specification, the cell mass means a mass of cells having a three-dimensional spread, not a group of cells that grow planarly along the bottom and / or wall of the inner wall of a culture vessel by adhesion culture or the like. The cell mass can also be called a sphere, a spheroid, or the like. The cell mass of the present invention exhibits the effect of remaining in an undifferentiated state and not losing its differentiation ability even after long-term culture. Here, in the present invention, all the cells constituting the cell mass may maintain their undifferentiated state, but it is sufficient that the cell mass contains cells that maintain their undifferentiated state to the extent that it can be used as a stem cell pool that continues to supply stem cells that maintain their undifferentiated state; for application in regenerative medicine, and the like.

[0035] It is clear from the examples of the present application that the effect of the cell clusters of the present invention that they maintain their undifferentiated state even after long-term culture is achieved by performing the shaking culture described above. However, it is very difficult to specify the characteristics of such cell clusters in words based on the structure or properties of the cell clusters themselves without using the description of the above steps.

[0036] In the above explanation of the method of the present invention, rotary shaking culture was mentioned as a preferred type of shaking method in the shaking culture for obtaining cell clumps. However, for example, the scope of "cell clumps obtained by rotary shaking culture" in the present invention naturally includes not only cell clumps actually obtained by rotary shaking culture, but also cell clumps that have been subjected to shaking culture by other methods, so long as they are similarly in an undifferentiated state and have not lost their differentiation ability.

[0037] Furthermore, in the case of mouse MSCs, the cell mass of the present invention may be positive for markers such as PDGFRα, Sca-1, or both of these, preferably PDGFRα. In the case of human MSCs, the cell mass of the present invention may be positive for markers such as CD90 and CD106. Positive for such a marker means that, for example, when measured according to the method described in the Examples of the present application, the proportion of the positive fraction in the cell mass is 50% or more.

[0038] Differentiation induction method The present invention provides a cell differentiation method comprising the step of culturing the above-mentioned cell mass of the present invention in a differentiation-inducing medium.

[0039] The method of the present invention can induce differentiation of MSCs into cells belonging to the mesodermal lineage. Examples of cells belonging to the mesodermal lineage or mesenchymal lineage include adipocytes, osteoblasts, chondrocytes, Examples of such cells include bone cells, cardiac muscle cells, tendon cells, dental pulp cells, odontoblasts, etc., and preferably include osteoblasts, adipocytes, etc. The cell mass of the present invention can also be applied to the culture of purified MSCs (Non-Patent Document 4) containing neural crest-derived cells, and examples of such cells include cells belonging to the neural crest, such as nerve cells, glial cells, smooth muscle cells, dental pulp cells, odontoblasts, cementoblasts, periodontal ligament cells, etc. (preferably, nerve cells, odontoblasts, more preferably nerve cells).

[0040] As the medium used in this step, a medium suitable for inducing differentiation into the desired type of cells can be appropriately used. Examples of such a medium include DMEM medium (e.g., sodium pyruvate-free DMEM medium, manufactured by Nacalai Tesque, Inc.); αMEM medium (e.g., MEM Alpha (1×) medium, manufactured by Gibco, Inc.); Mesenchymal stem cell growth medium, manufactured by Takara Bio Inc.; and MSCGM, manufactured by LONZA. TM Mesenchymal stem cell growth medium, hMSC-Human Mesenchymal Stem cell Osteogenic Differentiation Medium BullkKit TM , hMSC-Human Mesenchymal Stem cell Chondrogenic Differentiation Medium BullkKit TM , hMSC-Human Mesenchymal Stem cell Adipogenic Differentiation Medium BullkKit TM These media may be used alone or in combination of two or more.

[0041] In this step, the medium may contain an agent for promoting differentiation into the desired type of cells.

[0042] Examples of the differentiation promoter into adipocytes include insulin, dexamethasone, 3- isobutyl-1-methylxanthine, etc. These adipocyte differentiation promoters may be used alone or in combination of two or more.

[0043] Examples of agents for promoting differentiation into osteoblasts include ascorbic acid, ascorbic acid-2-phosphate, β-glycerophosphate, dexamethasone, hydrocortisone hemisuccinate, statins, isoflavone derivatives, and 3-benzothiepin derivatives TAK-778 ((2R,4S)-(-)-N-[4-(diethoxyphosphorylmethyl)phenyl]-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin). These include compounds such as epine-2-carboxamide, heloxanthin derivative TH (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide), phenamil (3,5-diamino-6-chloro-N-[imino(phenylamino)methyl]pyrazine-2-carboxamide, harmine and its analogues, acerogenin and its analogues, and resveratrol; and proteins involved in bone formation [BMP (Bone morphogenic Protein)-2, BMP-4, IGF (Insulin-like growth factor)-1, βFGF (basic fibroblast growth factor), TGF (Transforming Growth Factor)-β1, PTH (parathyroid hormone), Wnts, etc.]. These osteoblast differentiation promoters may be used alone or in combination of two or more.

[0044] Examples of the promoter for inducing differentiation into chondrocytes include BMP-6, TGF-β3, dexamethasone, ascorbic acid, etc. These promoters for inducing differentiation into chondrocytes may be used alone or in combination of two or more.

[0045] Examples of the promoter for inducing differentiation into cardiomyocytes include KY03I, KY02111, βFGF, etc. These promoters for inducing differentiation into cardiomyocytes may be used alone or in combination of two or more.

[0046] Examples of the agent for promoting differentiation into tendon cells include PDGF, VEGF, etc. These agents for promoting differentiation into tendon cells may be used alone or in combination of two or more.

[0047] Differentiation-induced cell mass The present invention provides a cell mass differentiated into a predetermined cell by the above-mentioned differentiation induction method of the present invention.

[0048] The differentiation-induced cell clusters according to the present invention use as a raw material the undifferentiated cell clusters obtained by the method of the present invention described above. As described above, the undifferentiated cell clusters according to the present invention, which serve as a raw material, are different from conventional MSC cell clusters, and have the unexpected effect of remaining undifferentiated and not losing their differentiation potential even after long-term subculture. Therefore, it is clear that the cell clusters induced to differentiate into desired cells using such undifferentiated cell clusters as a raw material are also different from conventional cell clusters.

[0049] The following examples are provided to more specifically explain the embodiments of the present invention and to illustrate the effects of the present invention. These examples are provided for illustrative and specific purposes, and the present invention is not limited to these examples. EXAMPLES

[0050] Experimental Method The experiment was carried out according to the following method.

[0051] Purification and sorting of mouse MSCs (see Non-Patent Document 6) The femurs and tibiae of five 4-week-old C57 / BL6 males (CLEA JAPAN) were crushed with a mortar and pestle, and then soaked in HBSS supplemented with 2% FBS (Cat.#SH30910.03:Hyclone), 10 mM HEPES (Cat.#346-01373:Dojindo), and 1% Penicillin / Streptomycin (P / S:Cat.#168-23191,Wako). + (Cat.#14025134:Gibco) (HBSS+ The bones were suspended in a 40 μm diameter cell suspension (prepared as a 40 μm diameter cell suspension) and red blood cells were removed. The crushed bones were enzymatically treated for 1 hour at 37°C with a solution of 0.2% collagenase (Cat.#034-10533: Wako), 10 mM HEPES (Cat.#346-01373: Dojindo), and 1% P / S (Cat.#168-23191: Wako) in DMEM (Cat.#08459-64: Nacalai tesque). The cells were passed through a strainer (Cat.#352340: Falcon) and centrifuged at 280G for 7 minutes at 4℃, the supernatant was removed, and the cells were collected. 7 ~ 3×10 7 The number of cells that can be collected is 1 x 10 7 1 ml of HBSS + adjustment solution The cells were suspended in 0.2 mg / ml PE-conjugated CD45 (30-F11: Cat. #12-0451-83, 0.2 mg / ml, eBioscience), TER119 (TER-119: Cat. #12-5921-83, 0.2 mg / ml, eBioscience), APC-conjugated PDGFRα (APA5: Cat. #17-14 01-81, 0.2mg / ml, eBioscience) and FITC-conjugated Sca-1 (Ly6A / E: Cat.#11-5981-85, 0.5mg / ml, eBioscience) antibodies were added at 2μl each, and the plate was left to stand for 30 minutes in a darkened environment at 4℃. Then, the mixture was centrifuged at 280 G for 7 minutes at 4°C to obtain 1 × 10 7 100 cells / 1 ml cell suspension HBSS + Adjust the solution and add propidium iodide (PI) to a final concentration of 1 μg / ml. After labeling dead cells with PI stain (Cat.#169-26281, WAKO), the cells were analyzed using an Aria III flow-cytometer (BD Bioscience). - / C D45 - / Ter119- / PDGFRα + / Sca-1 + The cells were sorted (Figure 6 -a).

[0052] FACS analysis of mouse MSCs Cell samples subcultured by adhesion culture: Cells cultured on a 10 cm culture dish (Cat.#664160-013: CELLSTAR) were washed twice with PBS, detached using 1 ml of Cell Dissociation Buffer (Cat.#13151014: Gibco), and then resuspended in HBSS. + Add the adjusted solution and centrifuge at 280G for 5 minutes at 4℃. The cells were centrifuged, the supernatant was removed, and the precipitated cells were collected.

[0053] Cell cluster samples formed by shaking culture: Considering the difficulty of separating and recovering single cells without damaging each cell or cell surface antigen of the cell cluster, An average of 10 cell clumps were placed on a 10 cm culture dish (Cat.#664160-013: CELLSTAR) for 7 days, and the cells that migrated from the cell clumps attached to the culture dish were washed twice with PBS. After removing the PBS, 1 ml of Cell Dissociation Buffer (Cat.#13151014: Gibco) was added to the culture dish and left to act for 2 to 3 minutes to detach the cells. Next, 9 ml of HBSS was added to the culture dish. + Add the adjusted solution, and rotate at 280G for 5 minutes, then The cells were centrifuged at 37 °C, the supernatant was discarded, and the precipitated cells were collected. The collected cells were 1 × 10 7 Pieces 1 ml HBSS + Suspended in the adjusted solution, APC-conjugated Sca-1(L y6A / E: Cat.#11-5981-85, 0.5mg / ml, eBioscience) antibody was added at 2μl each, and the plate was left to stand for 30 minutes in a darkened environment at 4℃. Centrifuge for 1 x 10 min at 4 °C. 7 HBSS to give a cell suspension of 1 cell per 1 ml +The cells were adjusted with an adjustment solution and analyzed using Aria III (BD Bioscience). Note that in the analysis of the cells derived from the cell clusters, subculture was not performed even once, and the cells that migrated from the cell clusters were directly used for the analysis.

[0054] Purification and sorting of donated human MSCs (see Non-Patent Document 7) Human MSCs provided by Tokyo Medical and Dental University were sorted from the femoral bone marrow of a 19-year-old male using PE-conjugated CD271 (LNGFR: Cat. #130-091-885, Miltenyi Biotec) and FITC-conjugated CD90 (Thy-1: Cat. #328110, Biolegend) antibodies with a MoFlo (BECKMAN COULTER) (Non-Patent Document 7). PI was used to label dead cells, and the negative fraction was sorted as live cells, after which the cells were passaged twice.

[0055] FACS analysis of human MSCs As with the analysis of mouse cells, adherent cells were cultured on a 10 cm culture dish (Cat. #664160-013: CELLSTAR) and washed twice with PBS. The cells were detached using 1 ml of Cell Dissociation Buffer (Cat. #13151014: Gibco) and then resuspended in 9 ml of HBSS. + Add the adjusted solution and centrifuge at 280G for 5 minutes at 4℃. The supernatant was removed and the cells were collected. For cell cluster analysis, an average of 10 cell clusters were placed on a 10 cm culture dish (Cat.#664160-013: CELLSTAR) for 7 days, and the cells that had migrated from the cell clusters attached to the culture dish were washed twice with PBS, detached using 1 ml of Cell Dissociation Buffer (Cat.#13151014: Gibco), and then washed with 9 ml of HBSS. + Add the adjusted solution and centrifuge at 280G for 5 minutes at 4°C. The supernatant was removed, and the cells were collected. In the analysis of the cell cluster-derived cells, subculture was not performed even once, and the cells that migrated from the cell cluster were directly used for the analysis. In both the case of adhesion culture and the case of cell cluster-derived adhesion culture, 7 × 10 6 ~1×107 The number of cells recovered was 1 × 10 7 1 ml of HBSS + The cells were suspended in the adjusted solution (PE-conjugated LNGFR: Cat. #130-091-885, 5.5 μg / ml, Miltenyi Biotec), FITC-conjugated Thy-1 Add 2 μl of each of CD106 (VCAM-1): Cat. #328110, 0.2 mg / ml, Biolegend, and APC-conjugated CD106 (VCAM-1): Cat. #305810, 0.5 mg / ml, Biolegend, and leave at room temperature for 30 minutes in the dark at 4°C. Then, the mixture was centrifuged at 280 G for 5 minutes at 4°C to obtain 1 × 10 7 100 cells / 1 ml cell suspension HBSS + The solution was adjusted and then incubated with Aria III (BD Bioscience ) was used for the analysis.

[0056] Adherent culture of mouse and human MSCs OriCell TM Strain C57BL / 6 mouse MSCs (MUBMX-0 1001: CYAGEN) and mouse purified MSCs were maintained in MEM-α+GlutaMAX-I (Cat.#32561-1001: CYAGEN) containing 10% FBS (Cat.#SH30910.03: Hyclone), 1% P / S (Cat.#168-23191: Wako) and 10 mM HEPES (Cat.#346-01373: Dojindo) as a growth maintenance medium. 2: Gibco).

[0057] Human MSCs were maintained in a growth medium containing 20% ​​FBS (Cat.#SH30910.03:Hyclone), 1% P / S (Cat.#168-23191:Wako), 10 mM HEPES (Cat.#346-01373: Dojindo), and 20ng / ml DMEM (Cat.#064-05384:WAKO) containing βFGF (Cat.#0 The culture was performed using Nacalai tesque (8459-64).

[0058] Mouse or human MSCs were cultured in 10 cm culture dishes (Cat .#664160-013: CELLSTAR) 1×10 6 The cells were seeded at a density of 100 cells / dish and cultured for 4 to 7 days, and subcultured when the cells were 80% confluent. For subculture, the medium was aspirated, serum components were removed with 1x PBS, and the cells were treated with 0.25% trypsin EDTA (Cat.#201-16945: Wako) for 2 minutes. Gently shake the dish. The cells were tapped, and as soon as the cells began to detach, a growth maintenance medium was added to recover the cells. After centrifugation at 250 × g for 5 minutes, the supernatant was removed by aspiration, and a growth maintenance medium was added to the precipitated cells. The cells were then plated in a new 10 cm culture dish at 1 × 10 6 The cells were seeded at 1 piece per dish.

[0059] Furthermore, OriCell TM Strain C57BL / 6 mouse MSCs (MUBMX-01001: Cyagen) had already been subcultured six times by the manufacturer at the time of purchase. The purchased cells are confirmed by the manufacturer to be CD29, CD44, CD31, and Sca-1 positive (>70%) and CD117 negative (<5%) before being shipped. The manufacturer also recommends using the cells for subculture 10 times or less, and does not guarantee the properties of the cells at the time of purchase after that number of subcultures. In addition, OriCell is used for the maintenance culture of the cells. TM The use of Mouse MSC Growth Medium (MUXMX90011: Cyagen) is recommended.

[0060] MSC suspension culture A TAITEC BR-40LF bio-shaker was used for shaking culture. A cell suspension of mouse or human MSCs was prepared in 20 ml of growth maintenance medium in a 125 ml Erlenmeyer flask (Cat.#431405: Corning). Mouse MSCs (OriCellTM When culturing mouse MSCs and purified mouse MSCs with shaking, the growth medium was supplemented with 20ng / ml βFGF (Cat.#064-05384 The number of cells per flask at the start of shaking culture was 100%. TM 1.0 x 10 mouse MSCs 7 5.0 × 10 purified mouse MSCs 5 1.0 × 10 for human MSCs 6 pcs or 1.0×10 7 The shaking culture was carried out at 37°C, 5% CO2, 85-95 rpm, and with a rotation amplitude of 40 mm, and the cells were incubated for 3-4 days. The medium was exchanged once. To exchange the medium, first, the entire medium containing the cells was placed in a 50 ml centrifuge tube (Cat. The cells were transferred to a flask (Cat. #TR2004:True Line) and centrifuged at 280G for 5 minutes at 4°C. After removing the supernatant, 20 ml of fresh medium was added using a 25 ml pipette (Cat. #760180:greiner bio-one), pipetted 2-3 times, and transferred to a flask. 10-14 days after the start of shaking culture, the formation of cell clusters was visible to the naked eye.

[0061] Shaking suspension culture of mouse MSCs using neural stem cell medium The medium for neural stem cells (see Non-Patent Documents 4, 11, and 12) used was Advanced DMEM / F12 (Cat. 12491015: Gibco) containing 1% P / S (Cat. #168-23191: Wako), 10 mM HEPES (Cat. #346-01373: Dojindo), 1xN2 (Cat. #17502-048: Gibco), 20 ng / ml EGF (Cat. #059-07873: WAKO), 20 ng / ml FGF (Cat. #064-05384: WAKO), and 1xB27 (Cat. #17504-044: Gibco). Mouse MSCs (Oricell TMA cell suspension of mouse MSCs (1.0 × 107 cells) was prepared in 20 ml of neural stem cell medium and placed in a 125 ml Erlenmeyer flask (Cat. #431405: Corning). The cell suspension was cultured at 37°C, 5% CO2, 85-95 r / min, and 40 mm amplitude using a TAITEC BR-40LF bio-shaker. The medium was replaced once every 3-4 days. To replace the medium, the entire medium containing the cells was transferred to a 50 ml centrifuge tube (Cat. #TR2004: True Line), and the cells were centrifuged at 280G for 5 minutes at 4°C. After removing the supernatant, 20 ml of new medium was added using a 25 ml pipette (Cat. #760180: Greiner bio-one), pipetted 2-3 times, and transferred to the flask. The formation of cell clusters was confirmed with the naked eye 10 to 14 days after the start of shaking culture.

[0062] Formation of MSC cell clusters using existing 3D suspension culture vessels Adherent cultured mouse or human MSCs were cultured at 3 × 10 6 Cell suspension concentration in cells / ml Kuraray 3D culture vessel (Elplasia Cat.#RB 500 40°C) The cells were seeded onto 10 NA plates (Kuraray). The same proliferation maintenance medium as that used for the above-mentioned MSC shaking suspension culture was used for the culture, and the medium was replaced every 3 to 4 days.

[0063] Reattachment and cell expansion of MSC clusters Cell clusters formed by shaking culture of mouse or human MSCs were left to attach to a plastic culture dish containing a growth maintenance medium. For human MSCs, the above-mentioned growth medium was used except that βFGF was removed. As a result, it was possible to expand mouse and human MSC cells. Many cells migrated from the periphery of the cell cluster attached to the culture dish, amplified, and spread over the culture surface of the culture dish. After cell migration and amplification, the main body of the cell cluster attached to the culture dish could be easily recovered by peeling off the bottom of the cell cluster with the tip of a 200 μl pipette tip. When the collected cell mass was placed in another culture dish, cell proliferation by cell migration was possible. 10cm culture dish (Cat.#664160-013:CELLSTAR) Or migrate to a 12-well plate (Cat.#665-180: CELLSTAR) The expanded cells were used for flow cytometry analysis or differentiation induction analysis. For differentiation induction analysis, 2-3 cell clusters were seeded in each well of a 12-well plate (see Differentiation Induction Method). For culture in a 10 cm culture dish, 8-12 cell clusters with a diameter of over 200 μm were placed at equal intervals across the culture dish.

[0064] Differentiation induction method, staining method (see non-patent document 6) For the induction of differentiation into osteoblasts and adipocytes, cells were cultured in a 12-well plate (Cat.#665-180: CELLSTAR) at 1×10 5 The cells were seeded at a concentration of 1000 cells / well. For cell cluster-derived migratory cells, 2-3 cell clusters were seeded in each well of a similar 12-well plate, and the cells that migrated and expanded from the cell clusters were used. The adhesion state was confirmed, and when the cells were 60-70% confluent (about 7 days of culture), the medium was changed to osteoblast differentiation induction medium (Cat.#PT3002: Lonza) and adipocyte differentiation induction medium (Cat.#PT3004: Lonza). The medium was then changed once every 3-4 days, and differentiation induction was performed for up to 21 days. During the induction period of about 2 weeks, the adherent cells and the formed lipid droplets sometimes floated and peeled off. In such cases, the culture was terminated at that point. Alkaline phosphatase (ALP) staining was used to confirm osteoblast differentiation, and oil red O staining was used to confirm adipocyte differentiation. Hematoxylin staining was not performed for either staining method.

[0065] To induce differentiation into chondrocytes, the cells were pelleted at 150G in a 15 ml centrifuge tube and cultured in chondrocyte induction medium (Cat. #PT3003: Lonza) supplemented with 10 ng / ml transforming growth factor-β3 (Cat. #PT4124: LONZA) and 500 ng / ml bone morphogenetic protein-6 (Cat. #6325-BM-020: R&D Systems). The medium was changed twice a week and the cells were cultured for 21 days. Differentiation of the induced chondrocytes was confirmed by toluidine blue staining (Cat. #209-14545: Wako). Matoxylin staining was not performed.

[0066] All stained cells were stained with 4% PFA (paraformaldehyde) (Cat.#163-20145 Fixation was performed using a 30 ml tube (Wako).

[0067] ALP staining (see non-patent document 6) A Histofine assay kit (Cat.#415161: Nichirei) was used for staining. The PFA was washed with PBS, and the reagents in the kit were mixed and adjusted just before staining. The staining solution was filtered through a 0.22 μm syringe filter (Cat.#SLGP033RS: Merk Millipore), and stained for 30 minutes, then washed with water. As a negative control (NC), MSCs before osteoblast differentiation were simultaneously stained with ALP.

[0068] Oil Red O staining (see Non-Patent Document 6) After washing the PFA with PBS, it was treated with 60% isopropyl alcohol for 1 minute, and stained for 30 minutes with Oil Red O staining solution (Cat.#40492:Muto Pure Chemicals) filtered with a 0.22μm syringe filter (Cat.#SLGP033RS:Merk Millipore). After staining, the staining solution was removed, and the section was treated again with 60% isopropyl alcohol for 1 minute, and then washed with water.

[0069] Toluidine blue staining (see Non-patent Document 6) The cell pellets after differentiation induction were embedded in paraffin and cut into sections on slides. They were deparaffinized with xylene, dexyleneized with 100% alcohol, treated with 80% alcohol, and then stained for 30 minutes with toluidine blue staining solution (Cat.#209-14545: Wako) filtered through a 0.22μm syringe filter (Cat.#SLGP033RS: Merk Millipore). After staining, they were dehydrated with 100% alcohol, treated with xylene, and then mounted with Marinol (Cat.#2009-3: Muto pure Chemicals).

[0070] Neuronal induction (see Non-Patent Document 4) The cell clumps cultured with shaking in neural stem cell medium were transferred to a medium containing 10% FBS (Cat.#SH30910.03:Hyclone) after removing EGF, FGF, and 1xB27 from the neural stem cell medium, and then treated with poly-L-ornithine (Cat.#163-27421:WAKO; 37℃, 12 hours), and then seeded (2-3 cell clumps / well) on a chamber slide (Cat.#SCS-NO8:MATSUNAMI) that had been treated with fibronectin (Cat.#062-05701:WAKO; 37℃, 12 hours). 7 days after seeding, the cell clumps The cells that migrated out were fixed with 4% PFA (Cat. #163-20145: WAKO) and used for fluorescent immunostaining observation.

[0071] Fluorescent immunostaining (see Non-patent Document 4) After washing the PFA-fixed sample with 1x PBS, it was treated with 0.3% Triton-X100 (Cat.#160-24751:WAKO) at room temperature for 5 minutes, and washed again with 1x PBS. The washed cells were treated with blocking buffer (1x PBS containing 0.01% Triton-X100 (Cat.#160-24751:WAKO) and 5% Bovine Serum Albumin (Cat.#23208:Thermoscientific)) for 30 minutes, and then primary staining (4°C, overnight) was performed using anti-βIII-Tublin antibody (Cat.#ab18207:abcom) diluted 500 times. After washing with 1x PBS, secondary staining (room temperature for 1 hour) was performed using Donkey Anti-Rabbit IgG H&L (Alexa Flour (registered trademark) 488; Cat#ab150073:abcom) diluted 1000 times. The cell samples were washed with 1×PBS and then plated on VECTASHIELD mounting medium with The images were mounted using DAPI (Cat#H-1200:VECTOR) and observed using a confocal laser scanning microscope (LSM780:Zeiss).

[0072] RT-PCR Total RNA was extracted using Trizol (Cat.#15596108: Invitrogen) and RNeasy Mini Kit (Cat.#74106: Quiagen) (extracted according to the Quiagen Kit protocol), and genomic DNA was removed using DNase I (Cat.#AM2222: Ambion). Reverse transcription was performed using the Reverse Transcription System (Cat.#A3500: Promega) following the protocol procedure, with random primers (Cat.#C118B: Promega) and AMV reverse transcriptase (Cat.#C118B: Promega). #M900B:Promega) and MgCl2 (Cat.#A351H:Promega) a) was used. For PCR, cDNA was amplified using Go Taq Green Master Mix (Cat.#M7123: Promega) (following the Promega protocol). PCR products were electrophoresed on a 1.0-1.5% agarose gel: mouse PDGFRα; 270bp, 60℃, 35 cycles F: 5'-TACATCATCCCCCTGCCAGA-3' (SEQ ID NO: 1) R: 5'-AAGGTTATCCCGAGGAGGCT-3' (SEQ ID NO: 2) GAPDH; 418bp, annealing at 67℃, 26 cycles F: 5'-CACCATGGAGAAGGCCGGGG-3' (SEQ ID NO: 3) R: 5'-GACGGACACATTGGGGGTAG-3' (SEQ ID NO: 4) OPN; 437bp, 62℃, 35 cycles F: 5'-TCACCATTCGGATGAGTCTG-3' (SEQ ID NO: 11) R: 5'-ACTTGTGGCTCTGATGTTCC-3' (SEQ ID NO: 12) OCN; 292bp, 62℃, 35 cycles F: 5'-AAGCAGGAGGGCAATAAGGT-3' (SEQ ID NO: 13) R: 5'-AGCTGCTGTGACATCCATAC-3' (SEQ ID NO: 14) Adipsin; 433bp, 64℃, 40 cycles F: 5'-ACTCCCTGTCCGCCCCTGAACC-3' (SEQ ID NO: 15 ) R: 5'-CGAGAGCCCCACGTAACCACACCT-3' (SEQ ID NO: 16) PPARγ; 460bp, 56℃, 35 cycles F: 5'-GTGCGATCAAAGTAGAACCTGC-3' (SEQ ID NO: 17) R: 5'-CCTATCATAAATAAGCTTCAATCG-3' (SEQ ID NO: 18) Agrican; 146bp, 64℃, 35 cycles F: 5'-CGCCACTTTCATGACCGAGA-3' (SEQ ID NO: 19) R: 5'-TCATTCAGACCGATCCACTGGTAG-3' (SEQ ID NO: 20) Sox9; 132bp, 61℃, 35 cycles F: 5'-CCTTCAACCTTCCTCACTACAGC-3' (SEQ ID NO:2 1) R: 5'-GGTGGAGTAGAGCCCTGAGC-3' (SEQ ID NO: 22) Col2A1; 121bp, 61℃, 35 cycles F: 5'-CCTCCGTCTACTGTCCACTGA-3' (SEQ ID NO: 23) R: 5'-ATTGGAGCCCTGGATGAGCA-3' (SEQ ID NO: 24) Nestin; 492bp, 60℃, 35 cycles F: 5'-AATGGGAGGATGGAGAATGGAC-3' (SEQ ID NO: 25) R: 5'-TAGACAGGCAGGGCTAGCAAG-3' (SEQ ID NO: 26) Twist; 225bp, 64℃, 35 cycles F: 5'-GGAGGATGGAGGGGGCCTGG-3' (SEQ ID NO: 27) R: 5'-TGTGCCCCACGCCCTGATTC-3' (SEQ ID NO: 28) Human Sox2; 151bp, 64℃, 40 cycles F: 5'-GGGAAATGGGAGGGGTGCAAAAGAGG-3' (SEQ ID NO: 5) R: 5'-TTGCGTGAGTGTGGATGGGATTGGTG-3' (SEQ ID NO: 6) Oct3 / 4: 144bp, 68℃, 40 cycles F: 5'-GACAGGGGGAGGGGAGGAGCTAGG-3' (SEQ ID NO: 7) R: 5'-CTTCCCTCCAACCAGTTGCCCCAAAC-3' (SEQ ID NO: 8) GAPDH; 613bp, 56℃, 35 cycles F: 5'-GTCAAGGCCGAGAATGGGAA-3' (SEQ ID NO: 9) R: 5'-GCTTCACCACCTTCTTGATG-3 (SEQ ID NO: 10)

[0073] result Figure 1: Cell cluster formation in mouse and human MSCs by shaking culture OriCell TM Mouse MSCs (MUBMX-01001: Cyagen) and human MSCs (purified from the bone marrow of a 19-year-old male) were repeatedly subcultured in adherent culture, and cells with low and high subculture numbers were prepared and subjected to shaking culture. Mouse MSCs could be subcultured in adherent culture up to 50 times after purchase (56 times counted before shipping). Human MSCs could be subcultured in adherent culture up to 20 times after donation (22 times counted before donation).

[0074] 1.0 x 10 in flask 6 Mouse MSCs (total number of subcultures before shipment: 12 ), or 1.0×10 7 Human MSCs (total number of subcultures from before donation: 6) were used. After two months of shaking culture, both mouse and human MSCs formed cell masses (Figure 1-a). Furthermore, under similar shaking culture conditions, mouse MSCs that had been subcultured 44 times since shipping and human MSCs that had been subcultured 22 times since donation were used for two months of shaking culture, and both mouse and human MSCs formed cell masses (not shown).

[0075] As mentioned above, the OriCell TMMouse MSCs had already been subcultured six times by the manufacturer at the time of purchase, and human-derived MSCs had already been subcultured twice at the time of donation. In this Example, hereafter, the number of subcultures refers to the continuous count number from before shipment and before donation (including the number of subcultures before shipment and before donation).

[0076] The number of cell clusters formed by shaking culture of human MSCs (passage number: 6-7 times) was 1.0 × 10 6 In the case of 14.3±2.0 pieces after 1 month, The number of cells at the start of shaking culture was 1.0 × 10 7 In the case of The number of cells was 15.0 ± 1.0 after 1 month and 11.7 ± 2.5 after 2 months (mean ± standard deviation of three experimental results). In other words, the number of cells at the start of shaking culture was 1.0 × 10 6 Pieces Even when the amount of the culture medium was increased by 10-fold, there was no significant difference in the number of cell aggregates formed.

[0077] The size of the cell clusters formed by shaking culture of human MSCs that had been subcultured 7 times was 1.0 × 10 6 In the case of this item, 433.3±103.3μm in one month At the start of shaking culture, the cell count was 1.0 × 10 7 In the case of this individual, the mean diameter was 783.3±248.3μm after one month and 833.3±1μm after two months. The result was 86.1 μm (mean value ± standard deviation of six experimental results).

[0078] The number of cell clusters formed by human MSCs with a high number of subcultures (19 times) after one month of shaking culture was 1.0 × 10 6 In the case of pieces, 14 pieces, 1.0 x 10 7 In the case of one experiment, the number of cells was 15 (result of one experiment). In addition, the size of the cell clusters formed by human MSCs that had been subcultured 19 times for one month under shaking culture was 1.0 × 10 6In the case of 500±126.5μm, 1.0×10 7 In this case, the mean value of six cell clusters was 766.7±206.6 μm (mean value of six cell clusters±standard deviation).

[0079] Figure 2: Effect of shaking culture on maintaining the differentiation potential of mouse MSCs We investigated the effect of shaking culture on maintaining the differentiation potential of mouse MSCs. TM At a relatively early stage, after only 11 passages, mouse MSCs retained the ability to differentiate into osteoblasts (positive ALP staining) and chondrocytes (positive toluidine blue staining), but had already lost the ability to differentiate into fat cells, showing negative results with Oil Red O staining (Figure 2-a: top).

[0080] However, OriCell, which has been subcultured many times (36 times) in adherent culture, TM Even in the case of mouse MSCs, when they were subjected to shaking culture for two months to form cell clusters, the cells that migrated and proliferated from the cell clusters not only retained their ability to differentiate into osteoblasts (ALP staining positive), but also retained or recovered their ability to differentiate into adipocytes (lipid droplets indicated by the arrow in Figure 2-a) (Figure 2-a: lower panel).

[0081] Mouse MSCs that co-express PDGFRα and Sca-1 on the cell surface are known to be high-quality MSCs that maintain an undifferentiated state (Non-Patent Documents 4 and 5). TM Mouse MSCs express PDGFRα + / Sca-1 + The co-positive fraction (co-positive fraction) was maintained at approximately 90% (not shown), but when subculture was repeated, this co-positive fraction decreased to 46.1% after 8 subcultures (Figure 2-b: left), and further decreased to 25.0% after 23 subcultures (not shown).

[0082] However, OriCell, which has been subcultured many times (44 times) in adherent culture, TM Even in mouse MSCs, by subjecting them to shaking culture for two months to form cell clusters, the co-positive fraction of cells that migrated and proliferated from the cell clusters recovered to 68.3% (Figure 2-b: right).

[0083] RT-PCR analysis showed that OriCell cells cultured by adhesion culture TM Mouse MSCs highly expressed the PDGFRα gene at the 9th passage, but not at the 25th passage. As the number of repetitions increased to 41, expression of the PDGFRα gene significantly decreased, suggesting the possibility that undifferentiated potential was lost.

[0084] However, OriCell, which has been subcultured 37 times, TM It was confirmed that even in mouse MSCs, PDGFRα gene expression was restored to a high level by subjecting them to shaking culture for two months to form cell masses (Figure 2-C).

[0085] Figure 3: Oricell cultured with shaking TM Mesodermal differentiation of mouse MSCs Oricell TM Mouse MSCs were used to prepare cells with a low adherent culture passage number (9 passages) and cells with a high adherent culture passage number (30 passages). After one month of shaking culture, they were induced to differentiate into mesodermal cells such as osteoblasts, chondrocytes, and adipocytes. The results are shown in Figure 3.

[0086] Both low-passage (9 passages) and high-passage (30 passages) adherent culture cells induced to differentiate after shaking culture showed the ability to differentiate into osteoblasts (ALP positive), chondrocytes (toluidine blue positive), and adipocytes (oil red O positive).

[0087] Figure 4: Tissue-specific gene expression analysis (RT-PCR) during differentiation induction into mesodermal cells Oricell TM Mouse MSCs were used to prepare cells with a low adhesion passage number (8-9 passages) and cells with a high adhesion passage number (30 passages). The expression of genes related to osteoblasts, chondrocytes, and adipocytes was analyzed using RT-PCR in cells that were induced to differentiate immediately after adhesion culture and in cells that were induced to differentiate by shaking culture after adhesion culture. When cells were induced to differentiate into osteoblasts for 21 days, the expression of osteoblast markers OCN and OPN was higher in shake-cultured cells than in adherent-cultured cells in cells with a low number of adherent-culture passages (passages 8 to 9).The expression of OPN was higher in shake-cultured cells than in adherent-cultured cells in cells with a high number of adherent-culture passages (passages 30) (Figure 4-a).

[0088] When cells were induced to differentiate into chondrocytes for 21 days, the expression of chondrocyte markers Agrican, Sox9, and Col2A1 was higher in shake-cultured cells than in adherent-cultured cells in cells with a low number of adherent-culture passages (passages 8 to 9).The expression of Agrican and Sox9 was higher in shake-cultured cells than in adherent-cultured cells in cells with a high number of adherent-culture passages (passages 30) (Figure 4-b).

[0089] When cells were induced to differentiate into adipocytes for 21 days, the cells cultured in adhesion did not express the adipocyte markers Adipsin and PPARγ, regardless of the number of passages. On the other hand, when cells with a low adhesion passage number (9 passages) were cultured with shaking, significant expression of Adipsin and PPARγ was observed, and when cells with a high adhesion passage number (30 passages) were cultured with shaking, expression of PPARγ was observed (Figure 4-c).

[0090] Figure 5: Effect of shaking culture on maintaining the differentiation potential of human MSCs We investigated the effect of shaking culture on maintaining the differentiation potential of human MSCs. After 20 passages in adherent culture, human MSCs showed the ability to differentiate into osteoblasts (ALP staining positive), but lost the ability to differentiate into adipocytes (Oil Red O staining negative) (Figure 5-a: top).

[0091] However, when human MSCs that had been subcultured six times in adherent culture were subjected to shaking culture for two months to form cell clusters, the cells that migrated and proliferated from the cell clusters retained not only the ability to differentiate into osteoblasts (ALP positive staining), but also a high ability to differentiate into adipocytes (Oil Red O positive staining) (Fig. 5-a: middle). In particular, the cells that migrated to the periphery of the cell clusters adhered to the culture dish formed many lipid droplets, indicating that they may be highly undifferentiated. Furthermore, when human MSCs that had been subcultured six times were subjected to adherent culture for the same two months as shaking culture, the number of subcultures reached 12, and the number of fat cells was 10. The ability to differentiate into fat cells had already been lost (negative for oil red O staining: not shown).

[0092] It is known that human MSCs expressing CD271 (LNGFR), CD90 (Thy-1), and CD106 (VCAM-1) on the cell surface are good quality MSCs that maintain an undifferentiated state (Non-Patent Document 7). Human MSCs subcultured 21 times in adherent culture highly expressed CD90 (Figure 5-b: upper left). Similarly, cells that migrated and proliferated from cell masses formed by shaking culture for 2 months of human MSCs subcultured 7 times in adherent culture maintained CD90 expression at a high level of 97.9% to 98.0% (Figure 5-b: lower left). On the other hand, the expression of CD271 in human MSCs subcultured 7 times in adherent culture had already decreased, and the expression did not recover even in shaking culture (not shown).

[0093] However, FACS analysis of CD106 (Non-Patent Document 7), a cell surface marker for high-quality MSCs, showed that expression had decreased to 6.0-7.5% in human MSCs that had been subcultured 21 times in adherent culture (Figure 5-b: upper right), whereas expression was maintained at 59.6%-87.1% in cells that had migrated and proliferated from cell clusters formed by shaking culture of human MSCs that had been subcultured 7 times in adherent culture for 2 months (Figure 5-b: lower right).

[0094] RT-PCR analysis showed that human MSCs cultured in adherent culture expressed the Oct3 / 4 gene, known as an undifferentiated stem cell marker, after 7 passages, but the expression decreased after 19 passages, suggesting the possibility of loss of undifferentiated potential. However, it was confirmed that even in human MSCs that had been passaged 19 times, the expression of the Sox2 and Oct3 / 4 genes, known as stem cell-related markers, was restored to a high level by forming cell clusters in shaking culture for 2 months (Figure 5-C).

[0095] Figure 6: Chondrogenic differentiation potential of human MSCs Using human MSCs, we prepared cells with a low number of adherent subcultures (passage number 9) and cells with a high number of adherent subcultures (passage number 19), and analyzed their differentiation potential into chondrocytes.

[0096] When cells with a small number of adherent subcultures (9 passages) that were induced to differentiate immediately after adhesion culture were induced to differentiate into cartilage for 21 days, pellet formation was observed, but only a few cells were positive for toluidine blue, which stains the cartilage matrix.On the other hand, when cells with a small number of adherent subcultures (9 passages) were induced to differentiate into cartilage after shaking culture, pellet formation was observed due to toluidine blue-positive cells (Figure 6-a).

[0097] When cells with many adherent subcultures (19 passages) that were induced to differentiate immediately after adhesion culture were induced to differentiate into cartilage for 21 days, no pellet formation was observed. On the other hand, when cells with many adherent subcultures (19 passages) were induced to differentiate into cartilage after shaking culture, pellet formation by toluidine blue-positive cells was observed (Figure 6-b).

[0098] Figure 7: Adherent culture of MSC cell clusters using three-dimensional suspension culture vessels (prior art) Human MSCs (subcultured 9 times) or OriCell were cultured in a low-adhesive three-dimensional culture vessel (Kuraray Elplasia RB 500 400 NA Plate), which is used in the existing method of forming MSC cell aggregates. TM Mouse MSCs (subculture number: 25th and 41st) were cultured at 3 × 10 6The results showed that In the case of human MSCs, they adhered to the culture dish, and no cell mass was formed even after 7 days (Fig. 7-a). On the other hand, in the case of mouse MSCs, as reported by Baraniak et al. (Non-Patent Document 10), cell masses were formed after 7 days of culture in the incubator (Fig. 7-b). The cell masses collected at this point were transferred back to the culture dish and left in an adhesive culture environment. As a result, the cell masses adhered to the culture dish and cells migrated around it, but the cell masses did not maintain their morphology and disappeared after 7 days, and the differentiation ability into adipocytes was lost (oil red O staining was negative and no lipid droplets were observed) (Fig. 7-c). b).

[0099] Figure 8: Oricell using a three-dimensional suspension culture vessel (prior art) TM Differentiation potential analysis of mouse MSC clusters Oricell cells were maintained in an adherent culture on a low-adhesion three-dimensional suspension culture vessel (Kuraray Elplasia RB 500 400 NA Plate). TM Mouse MSCs (subculture number: 25th and 41st) were cultured at 3 × 10 6 The cells were seeded at 100 cells / ml. After 7 days, the cells were formed. The cell aggregates were seeded onto a 12-well plate (Cat. #665-180: CELLSTAR, Greiner Bio-One) to transition to adherent culture, and the differentiation of cells that migrated from the adherent cell aggregates into osteoblasts and adipocytes was examined.

[0100] Oricell after 25 and 41 adherent subcultures TM Mouse MSCs were induced to differentiate into osteoblasts for 21 days. Both were positive for ALP, confirming their ability to differentiate into osteoblasts (Figure 8-a). On the other hand, Oricell after 25 and 41 adherent subcultures TM Mouse MSCs were induced to differentiate into adipocytes for 21 days, but no formation of Oil Red O-positive lipid droplets was observed (Figure 8-b).

[0101] Figure 9: Morphological maintenance and cell supply ability of shaking-cultured human MSC cell clusters Human MSCs that had been subcultured seven times in adherent culture were cultured with shaking for two months to form cell clusters, which were then placed in a culture dish. The next day, cells were observed to migrate around the cell clusters, and after seven days, the cells had proliferated to fill the culture dish (Figure 9-a: top). Furthermore, the re-adhered cell clusters maintained their morphology even after seven days, and it was possible to mechanically detach the cell clusters and place them in a new culture dish (first re-adhesion). The day after the cell clusters were placed again, cells were observed to migrate around the cell clusters as described above, and within 10 days, the cells had proliferated to fill the culture dish, and the cell clusters maintained their morphology (Figure 9-a: two photographs on the left in the middle row). The cell clusters were mechanically detached and placed in a new culture dish. Even when the cells were placed on the culture dish four more times (second to fifth re-adhesion), similar cell migration to the periphery of the cell clusters was observed, and within 14 days the cells had proliferated to fill the culture dish, and the cell clusters maintained their morphology (Figure 9-a: three photographs on the middle right to the bottom row).

[0102] Furthermore, by repeating the process of detaching the cell mass and re-adhering it to the culture dish three times, the migrated cells showed the ability to differentiate into osteoblasts (positive for ALP staining) and into adipocytes (positive for lipid droplets and Oil Red O staining), and therefore maintained their undifferentiated state (Figure 9-b). Therefore, it was found that the MSC cell mass according to the present invention has strong adhesive power between cells, and continues to supply undifferentiated MSCs to the culture dish without losing the morphology of the cell mass even when returned to an adhesive culture environment.

[0103] Figure 10: Cell clusters formed by shaking culture of purified mouse MSCs Purified mouse MSCs were also cultured in a shaking culture to produce OriCell cells. TM We examined whether the sorted mouse purified MSCs would form cell aggregates similar to those of mouse MSCs. After sorting, the mouse purified MSCs were subcultured twice in adherent culture to ensure sufficient cell numbers. These twice-subcultured mouse purified MSCs (5.0 × 10 5 100 cells / flask) were cultured with shaking for 2 months. OriCell TMA cell mass was formed similar to that of mouse MSC (not shown). When the formed cell mass was placed in a new culture dish, migration of cells to the periphery of the cell mass was observed the next day (Fig. 10-b: left), and the migrated cells proliferated to fill the culture dish within 10 days. Even during this time, the cell mass maintained its shape without losing its shape. Furthermore, when the migrated and proliferated cells were induced to differentiate into adipocytes, they produced lipid droplets 14 days later (Fig. 10-c), demonstrating that they retained the ability to differentiate into adipocytes.

[0104] Figure 11: Cell cluster formation using Oricell™ mouse MSC neural stem cell medium Similar shaking culture was performed using Oricell™ mouse MSCs in neural stem cell medium. We also examined whether cell clusters were formed in the case of schizophrenia. We also examined the differentiation potential of the resulting cell clusters into mesodermal cells (bone, fat, cartilage) and neural cells. Furthermore, we analyzed the expression of neural crest stem cell markers Nestin and Twist, and mesenchymal stem cell marker PDGFRα by RT-PCR.

[0105] When cells with few adherent subcultures (passages 9 times) were cultured with shaking in neural stem cell medium, the formation of cell clusters was observed within one month (Figure 11-a). Using this cell mass, we induced differentiation into osteoblasts, chondrocytes, and adipocytes, and observed the formation of ALP-positive osteoblasts, adipocytes showing oil red O-positive lipid droplets, and cartilage pellets formed by toluidine blue-positive cells (Figure 11-b). In addition, using this cell mass, we induced differentiation into nerve cells, and observed βIII-Tublin-positive nerve cells (Figure 11-c).

[0106] Furthermore, even with cells that had been subcultured many times (39 passages), it was possible to form cell clusters by using neural stem cell medium (Fig. 11-d). RT-PCR analysis showed that the cells in the cell clusters significantly expressed the neural crest stem cell markers Nestin and Twist, and the mesenchymal stem cell marker PDGFRα (Fig. 11-e).

[0107] summary As is clear from the above results, by using shaking culture, MSCs that had lost their undifferentiated state could be restored to cell masses of highly undifferentiated MSC populations. Furthermore, the MSC cell masses formed by shaking culture retained their undifferentiated state while also having a strong ability to maintain their morphology, and could be used to supply undifferentiated MSCs by reattaching them to a new culture dish. [Industrial Applicability]

[0108] According to the method of the present invention, MSCs are cultured from an adhesive environment using a completely new agitation and shaking culture method, which allows the formation of robust three-dimensional MSC masses (spheres) that maintain their differentiation potential and morphology.

[0109] This suggests that they may be useful as a stem cell pool that continues to supply stem cells that retain their undifferentiated state. In terms of application to regenerative medicine, it is expected that this cell mass will itself act as a scaffold to create space in the defective area and promote tissue regeneration by stem cells.

Claims

1. A method for maintaining the undifferentiated state of mesenchymal stem cells, characterized in that mesenchymal stem cells that have been subcultured two or more times in adherent culture are subjected to shaking suspension culture for ten days or more to form tough cell masses.

2. The method according to claim 1, wherein the shaking culture is carried out at a rotation speed of 20 to 200 rpm.

3. The method described in claim 1 or 2, wherein the shaking culture is performed with an amplitude of 10 to 40 mm.

4. A method according to any one of claims 1 to 3, wherein the mesenchymal stem cells subjected to shaking culture are cells that have lost the ability to differentiate into a specified cell, and the ability to differentiate into the specified cell is restored by shaking culture.

5. A cell mass obtained by the method described in any one of claims 1 to 4.

6. A method for producing cells differentiated from mesenchymal stem cells, comprising the steps of obtaining undifferentiated mesenchymal stem cells by the method described in any one of claims 1 to 4, and culturing the mesenchymal stem cells in a differentiation-inducing medium.

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