Method for maintaining undifferentiated state of mesenchymal stem cells using 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 Art

[0002] In the medical and dental fields, for substantial defects in bone tissue, reconstruction and implant treatments using artificial materials such as β-TCP or titanium, or autologous bone transplantation are performed. In treatments using artificial materials including autologous bone transplantation, absorption of the regenerated bone that occurs after treatment has been a problem (Non-Patent Documents 1 and 2). Also, in the field of bone regeneration, scaffolds or membranes for preventing invasion of granulation tissue are often necessary for space making during surgery, and their effects in the regeneration site must also be considered.

[0003] In recent years, research and treatment using autologous cells have also been carried out, and the representative thereof is mesenchymal stem cells (MSC) (hereinafter, in this specification, mesenchymal stem cells are abbreviated as MSC). [[ID=十七]]<ニューラルネットワークのモデルは、層の数、ノードの数、活性化関数、学習アルゴリズムなどのハイパーパラメータによって定義されます。

[0004] MSC was originally defined as a cell that adheres and proliferates by seeding bone marrow cells on a plastic culture dish and has the ability to differentiate into mesodermal tissues and cells such as fat, cartilage, and bone (Non-Patent Document 3).

[0005] Marker search of mouse bone marrow MSC was performed, and in 2009, by Morikawa et al. using a flow cytometer, PI / CD45 - / Ter119 - / Sca-1 + / PDGFRα + It was reported that mouse MSC was concentrated in the fraction (Non-Patent Documents 4 and 5). This purification technique has been publicly detailed by Houlihan et al. (Non-Patent Document 6). Human bone marrow MSC has been reported by Babaura et al. to be highly concentrated in the CD271 (LNGFR) / CD90 (Thy-1) + / CD90(Thy-1) + fraction (Non-Patent Document 7).

[0006] MSCs exist in various tissues, but bone marrow is generally the tissue from which a large number of MSCs can be reliably obtained. MSCs are defined as cells from bone marrow that adhere to and proliferate when seeded onto a plastic culture dish, and that can differentiate into osteoblasts, chondrocytes, and adipocytes (Non-Patent Literature 3), and are attracting attention as a cell source for regenerative medicine. However, repeated long-term adhesive culture reduces their proliferative and differentiation capabilities, which is a problem in clinical practice as it leads to differences in results between facilities and patients.

[0007] Specifically, it is known that conventional MSCs have limited proliferative capacity in adherent culture environments, and consequently lose their differentiation ability (Non-Patent Documents 8, 9). It is also known that purified MSCs gradually lose proliferative capacity in adherent culture environments (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 aggregates (spheres or spheroids) in a suspended state without adhering to the culture dish (Non-Patent Document 10). However, this method does not allow for long-term culture. There are no reports on what changes occur in cells when they are 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 [Overview of the project] [Problems that the invention aims to solve]

[0010] The problem that this invention aims to solve is culturing MSCs for a long period of time while maintaining their undifferentiated state. [Means for solving the problem]

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

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

[0013] Item 1. A method for culturing MSCs while maintaining their undifferentiated state, characterized by shaking the MSCs during culture.

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

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

[0016] Item 4. Perform subculturing two or more times, or follow the method described in any one of items 1 to 3.

[0017] Item 5. The MSCs subjected to shaking culture are cells that have lost the ability to differentiate into a predetermined cell type. The method according to any one of items 1 to 4, wherein the ability to differentiate into the specified cells is restored by shaking culture.

[0018] Item 6. A cell aggregate obtained by the method described in any one of items 1 to 5.

[0019] Item 7. A method for cell differentiation, comprising the step of culturing the cell aggregate described in Item 6 in a differentiation induction medium.

[0020] Item 8. A cell aggregate differentiated into the specified cells by the method described in Item 7. [Effects of the Invention]

[0021] According to the present invention, by performing shaking culture, MSCs can be guided to the formation of cell aggregates and cultured for a long period of time while maintaining their undifferentiated state. Furthermore, according to the present invention, when using MSCs that have lost their differentiation ability due to long-term conventional adherent culture, etc., they can be cultured by shaking. This also allows for the restoration of their differentiation potential. In the field of stem cells, to which this invention belongs, physical stimulation has generally been thought to induce differentiation. Therefore, the above-mentioned effect of this invention, which is that undifferentiated state can be maintained for a long period of time by performing shaking culture on MSCs, is unexpected and cannot be predicted from the prior art. [Brief explanation of the drawing]

[0022] [Figure 1] This shows cell aggregate formation in mouse and human MSCs after shaking culture (2 months). Figure 1 left: Mouse MSCs. Used after 12 adherent subcultures. Figure 1 right: Human MSCs. Used after 6 adherent subcultures. [Figure 2] This paper demonstrates the effect of shaking culture on maintaining the differentiation potential of mouse MSCs. Figure 2a, top panel: 11 adherent subcultures (Figure 2a, top left: osteoblasts, top center: chondrocytes, top right: adipocytes). Figure 2a, bottom panel: Shaking culture after 36 adherent subcultures (Figure 2a, bottom left: osteoblasts, bottom right: adipocytes). Figure 2b, left: 8 adherent subcultures. Figure 2b, right: Shaking culture after 44 adherent subcultures. Figure 2c: PDGFRα expression analysis (RT-PCR) of cell aggregates after 9, 25, and 41 adherent subcultures and 11 and 37 adherent subcultures followed by 2 months of shaking culture. [Figure 3] This shows the mesodermal differentiation potential of Oricell™ mouse MSCs cultured by shaking. Figure 3, left column: osteoblasts. Figure 3, center column: chondrocytes. Figure 3, right column: adipocytes. Figure 3, top row: 1 month of shaking culture after 9 adherent subcultures. Figure 3, bottom row: 1 month of shaking culture after 30 adherent subcultures. [Figure 4] The following shows gene expression analysis (RT-PCR) after differentiation induction into mesodermal cells. Figure 4a: After induction of osteogenic differentiation. Figure 4b: After induction of cartilage differentiation. Figure 4c: After induction of adipocyte differentiation. The numbers above Figures 4a, 4b, and 4c indicate the number of adherent passages. [Figure 5]This shows the effect of shaking culture on maintaining the differentiation potential of human MSCs. Figure 5a left column: Osteoblasts. Figure 5a right column: Adipocytes. Figure 5a top row: 20 adherent subcultures. Figure 5a middle row: Shaking culture after 6 adherent subcultures. Figure 5a bottom row: 20 adherent subcultures without induction of osteogenic differentiation. Figure 5b top row: 21 adherent subcultures. Figure 5b bottom row: Shaking culture after 7 adherent subcultures. Figure 5c: The numbers above Figure 5c indicate the number of adherent subcultures. [Figure 6] This shows the chondrogenic differentiation potential of human MSCs. Figure 6a, top panel: Cartilage (toluidine blue staining). 9 adherent subcultures. Figure 6a, bottom panel: Cartilage (toluidine blue staining). Shaking culture after 9 adherent subcultures. Figure 6b, top panel: Cartilage (toluidine blue staining). 19 adherent subcultures. No cartilage pellet formation. Figure 6b, bottom panel: Cartilage (toluidine blue staining). Shaking culture after 19 adherent subcultures. [Figure 7] The results of adhesion culture of MSC cell aggregates using the prior art three-dimensional suspension culture vessel are shown. Figure 7a: Human MSCs (used after 9 adhesion subcultures). 7 days of culture. Figure 7b: Mouse MSCs. Figure 7b top row: Used after 25 adhesion subcultures (2nd from the left: three-dimensional suspension culture vessel, immediately after seeding. 3rd from the left: three-dimensional suspension culture vessel, 7 days of culture. 4th from the left: reattached to culture dish, 7 days after reattachment. 5th from the left: reattached to culture dish, adipogenic differentiation induced). Figure 7b bottom row: Used after 41 adhesion subcultures (2nd from the left: three-dimensional suspension culture vessel, immediately after seeding. 3rd from the left: three-dimensional suspension culture vessel, 7 days of culture. 4th from the left: reattached to culture dish, 7 days after reattachment. 5th from the left: reattached to culture dish, adipogenic differentiation induced). [Figure 8] This shows the differentiation potential analysis of Oricell™ mouse MSC cell aggregates using a three-dimensional suspension culture vessel (prior technology). 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: adipocytes). [Figure 9]This shows the morphological maintenance and cell supply capacity of human MSC cell aggregates cultured by shaking. Figure 9a, top row, 1st from the left: Cell aggregate seeded into an adhesion culture dish, day 0. Figure 9a, top row, 2nd from the left: Day 1. Figure 9a, top row, 3rd from the left: Day 7. Figure 9a, top row, 4th and 5th from the left: Cell aggregate adhesion (1st time). Figure 9a, middle row, 1st from the left: Day 12. Figure 9a, middle row, 2nd from the left: Day 15. Figure 9a, middle row, 3rd from the left: Day 27. Figure 9a, middle row, 4th from the left: Day 44. Figure 9a, middle row, 5th from the left: Day 54. Figure 9a, bottom row, left: Day 57. Figure 9a, bottom row, right: Day 67. Figure 9b: Differentiation induction of migrating cells after 3rd re-adhesion (Figure 9b left: osteoblasts. Figure 9b right: adipocytes). [Figure 10] This shows cell aggregates formed by shaking culture of purified mouse MSCs. Figure 10a right: Purified mouse MSCs. Figures 10b and c: After two passaging cycles, shaking culture was performed, and after cell aggregate formation, the cell aggregates were reattached to a culture dish. Figure 10b shows the cells on day 1 after adhesion. Figure 10c shows adipocytes (lipid droplets) after differentiation induction. [Figure 11] This shows cell aggregate formation in Oricell™ mouse MSCs using neural stem cell culture medium. Figure 11a: Shaking culture for 1 month after 9 adherent passages. Figure 11b: Shaking culture for 1 month after 9 adherent passages (Figure 11b, first from the left: osteogenic differentiation. Figure 11b, second from the left: adipophilic differentiation. Figure 11b, third from the left: cartilage differentiation.) Figure 11c: Neural differentiation. Figure 11d: Shaking culture for 1 month after 39 adherent passages. Figure 11e: RT-PCR after 1 month of shaking culture (the number above Figure 11d indicates the passage number). [Modes for carrying out the invention]

[0023] A method for culturing MSCs while maintaining their undifferentiated state. This invention provides a method for culturing MSCs in cell aggregates while maintaining their undifferentiated state, characterized by shaking culture of MSCs.

[0024] The type of MSC used in the present invention is not particularly limited, and examples include cells derived from bone marrow, dental pulp, and adipose tissue. The animal species of the MSC is also not particularly limited, and examples 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 by culturing such MSCs by shaking. The culture vessel is not particularly limited and can be appropriately set according to the required amount of culture medium, the type of shaking method, etc. Examples of culture vessel shapes include Erlenmeyer flasks and seesaw-type bioreactor flasks (e.g., flasks with a polygonal (4- to 8-sided) bottom) as shown in Figure WO2015 / 064705. Examples of culture vessel capacities include 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 cell adhesion to the bottom surface of the inner wall of the vessel may be used, or a normal culture vessel that has not undergone such treatment may be used.

[0026] The type of shaking method is not particularly limited and examples include rotation, figure-eight, reciprocating, and seesaw type. In the present invention, rotation is preferred, but other shaking methods can be adopted. In the case of shaking culture by rotation, the rotation speed is not particularly limited, but typically it can be set in the range of 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 less likely for spheres to stick to the sides and bottom (bed) of the culture vessel.

[0027] In this specification, as a preferred embodiment, the oscillatory conditions such as the rotation speed and amplitude were described by taking the swirling type as an example. However, as long as the same physical stimulus can be applied to stem cells, the figure-eight type, reciprocating type, seesaw type, etc. can be adopted. For example, in the case of figure-eight type oscillatory culture, since the culture vessel is oscillated through two substantially circular orbits that touch at a single point, for each circle forming the figure-eight, the conditions can be set to be the rotation speed and amplitude exemplified in the above swirling type. Also, 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 within the range of 10 to 40 mm. For example, in the case of the seesaw type, the angle of oscillatory culture can be appropriately set within the range of 2° to 12°, and the oscillation period can be set within the range of 5 to 60 rpm.

[0028] The culture medium to be used is not particularly limited as long as it is a liquid medium suitable for culturing stem cells. Examples of such a culture medium include Minimum Essential Medium (MEM) medium, etc. The culture medium may also contain additives that are usually included during the culturing of normal stem cells as necessary. Specific examples of the additives include fetal bovine serum, amino acids (e.g., L-glutamine, L-alanyl-L-glutamine, etc.), antibiotics (e.g., penicillin, streptomycin, etc.). <{0000247}>serum), amino acids (e.g., L-glutamine, L-alanyl-L-glutamine, etc.), antibiotics (e.g., penicillin, streptomycin, etc.).

[0029] As a preferred mode of the culture of the present invention, subculture can be mentioned. In subculture, stem cells are collected before reaching the confluent state. For example, in the case of MSC, 1×10 3 ~ 1×10 7 cells (per ml), preferably 1×10 5 ~1×10 6 cells (per ml) approximately Seed cells into a fresh culture medium at a suitable temperature. In addition, in the culture of the present invention, it is preferable to change the culture medium as appropriate (for example, every 1 to 5 days, preferably every 3 to 4 days). According to the method of the present invention, MSCs can be subcultured in an undifferentiated state without losing their differentiation ability. In the present invention, subculture can be performed by shaking culture for, for example, one or more times, preferably three or more times, preferably five or more times. In addition, there is no particular upper limit to subculture by shaking culture in the present invention, but for example, subculture can be performed three times or less, preferably two or less times, preferably one or less.

[0030] The shaking culture time is preferably about 1 to 90 days (about 24 to 2160 hours), more preferably 10 to 75 days (about 240 to 1800 hours), and even more preferably 14 to 60 days (about 336 to 1440 hours). The shaking culture temperature in this process is not particularly limited, but is 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] The method of the present invention allows for the culture of MSCs while they remain in an undifferentiated state and possess differentiation potential. Furthermore, the shaking culture causes the MSCs to proliferate and aggregate, forming cell aggregates. Accordingly, the present invention also provides a method for forming (undifferentiated) cell aggregates, characterized by shaking culture of MSCs; and a method for inhibiting MSC differentiation, characterized by shaking culture of MSCs. The cell aggregates obtained by the method of the present invention have the ability to differentiate into various cells belonging to the mesoderm, but in a preferred embodiment, the cell aggregates have the ability to differentiate into osteoblasts, adipocytes, chondrocytes, and more preferably adipocytes. As described in the examples, conventional adherent culture results in the loss of the ability to differentiate into adipocytes, so this embodiment is preferred.

[0032] In this invention, undifferentiated state means that cells are in an undifferentiated state and possess the ability to differentiate. In this invention, the undifferentiated state of MSCs can typically be confirmed by whether or not they exhibit the ability to differentiate into both osteoblasts and adipocytes, as shown in the examples of this application. Furthermore, in this invention, "maintaining undifferentiated state" includes not only the state in which MSCs maintain the differentiation ability they initially possessed without losing it, but also the state in which the ability of MSCs to differentiate into at least one of the cells they can originally differentiate into (as described above) decreased or was lost during the culture process prior to shaking culture, but that differentiation ability was restored by shaking culture. Accordingly, in this invention, for example, as shown in the examples of this application, the state in which MSCs that have lost the ability to differentiate into a specific cell (e.g., adipocytes) as a result of repeated subculturing by methods other than shaking culture (e.g., adherent culture) have their differentiation ability restored by shaking culture is also included in the scope of "maintaining undifferentiated state." Accordingly, the present invention also provides a method for restoring the differentiation ability to a predetermined cell, characterized in that MSCs that have lost the ability to differentiate into a predetermined cell are cultured by shaking.

[0033] Furthermore, although the method of the present invention is characterized by shaking culture of MSCs, static culture may be combined with it as long as the effects of the present invention are obtained. For example, as shown in the examples of this application, after forming a cell aggregate that retains its undifferentiated state by performing shaking culture for a certain period of time, Cell aggregates may be subjected to static culture. Such static culture may be performed using either adherent or non-adherent culture. The culture medium, culture temperature, CO2 concentration, etc. in such static culture should be controlled by the aforementioned shaking method. Conditions similar to those for culture can be appropriately adopted. As the culture vessel, a non-adhesive culture vessel (e.g., a non-adhesive culture dish, non-adhesive well, non-adhesive flask, etc.) that has been treated to suppress cell adhesion to the bottom surface of the inner wall of the vessel may be used, or a normal culture vessel that has not undergone such treatment may be used. The number of subculturing cycles in static culture is not particularly limited as long as the effects of the present invention are obtained, but for example, in the present invention, subculturing can be performed 2 or more times, preferably 3 or more times, preferably 5 or more times. Also, when performing static culture, the upper limit of subculturing in static culture is not particularly limited, but for example, subculturing can be performed 5 times or less, preferably 3 times or less, preferably 1 time or less. In such embodiments, the static culture time is preferably about 0.1 to 60 days, and preferably 1 to 30 days. Preferably, 3 to 14 days is preferred. In addition, in this embodiment of the present invention, the static culture time is preferably about 2.4 to 1440 hours, more preferably about 24 to 720 hours, and more preferably about 72 to 336 hours.

[0034] Cell aggregates that retain their undifferentiated state The present invention provides cell aggregates obtained by the culture method of the present invention described above. In this specification, a cell aggregate refers to a mass of cells having a three-dimensional extent, rather than a group of cells that have proliferated planarly along the bottom and / or walls of the inner wall of a culture vessel by adherent culture or the like. A cell aggregate can also be referred to as a sphere, spheroid, etc. The cell aggregate of the present invention exhibits the effect of remaining undifferentiated and retaining its differentiation ability even after long-term culture. In this invention, all cells constituting the cell aggregate may maintain their undifferentiated state, but it is sufficient that cells that maintain their undifferentiated state are included in the cell aggregate to the extent that it can be used as a stem cell pool that continuously supplies stem cells that maintain their undifferentiated state; or for applications such as regenerative medicine.

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

[0036] In the description of the method of the present invention mentioned above, swirling shaking culture was cited as a preferred type of shaking method for obtaining cell aggregates. However, the scope of "cell aggregates obtained by swirling shaking culture" in the present invention may, for example, include not only cell aggregates actually obtained by swirling shaking culture, but also cell aggregates that are in an undifferentiated state and have not lost their differentiation potential, as long as they have been cultured by other methods of shaking.

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

[0038] Differentiation induction method The present invention provides a method for cell differentiation, which includes the step of culturing the cell aggregate described above in a differentiation induction medium.

[0039] The method of the present invention can induce differentiation from MSCs into cells belonging to the mesoderm. Examples of cells belonging to the mesoderm and mesenchymal systems include adipocytes, osteoblasts, chondrocytes, Examples include osteocytes, cardiomyocytes, tendinocytes, dental pulp cells, odontoblasts, etc., with osteoblasts and adipocytes being preferred. Furthermore, the cell aggregate of the present invention can also be applied to the culture of purified MSCs (Non-Patent Literature 4) containing neural crest-derived cells, and examples of cells belonging to the neural crest include 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] For the culture medium used in this process, any medium suitable for inducing differentiation into the desired cell type can be used as appropriate. Examples of such media include DMEM medium (e.g., sodium pyruvate-free DMEM medium from Nacalai Tesque); αMEM medium (e.g., MEM Alpha (1×) medium from Gibco); Mesenchymal stem cell growth medium from Takara Bio; and MSCGM from 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 are some examples. These culture media may be used individually or in combination of two or more types.

[0041] In this process, the culture medium may contain a differentiation inducer for the desired cell type.

[0042] Examples of agents that promote differentiation into adipocytes include insulin, dexamethasone, and 3- Examples include isobutyl-1-methylxanthine. These adipocyte differentiation-inducing agents may be used individually or in combination of two or more.

[0043] Examples of agents that promote differentiation into osteoblasts include ascorbic acid, ascorbic acid-2-phosphate, β-glycerophosphate, dexamethasone, hydrocortisone hemisuccinate, statins, isoflavone derivatives, and 3-benzothiepine derivatives. Compounds such as epin-2-carboxamide, helioxanthine derivatives TH (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide), phenamyl (3,5-diamino-6-chloro-N-[imino(phenylamino)methyl]pyrazine-2-carboxamide, harmine and its analogs, acerogenin and its analogs, resveratrol, etc.); 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-inducing agents may be used individually or in combination of two or more.

[0044] Examples of agents that promote differentiation into chondrocytes include BMP-6, TGF-β3, dexamethasone, and ascorbic acid. These chondrocyte differentiation-promoting agents may be used individually or in combination of two or more.

[0045] Examples of differentiation-promoting agents for cardiomyocytes include KY03I, KY02111, and βFGF. These differentiation-promoting agents for cardiomyocytes may be used individually or in combination of two or more.

[0046] Examples of differentiation-promoting agents for tendinocytes include PDGF and VEGF. These differentiation-promoting agents for tendinocytes may be used individually or in combination of two or more.

[0047] Differentiated cell mass The present invention provides a cell aggregate differentiated into predetermined cells by the differentiation induction method of the present invention described above.

[0048] The differentiated cell aggregate according to the present invention uses undifferentiated cell aggregate obtained by the method of the present invention described above as a raw material. As described above, the undifferentiated cell aggregate of the present invention, which is used as a raw material, differs from conventional MSC cell aggregates and exhibits the unexpected effect of remaining undifferentiated and retaining its differentiation potential even after long-term subculturing. Therefore, it is clear that the cell aggregate differentiated into desired cells using such undifferentiated cell aggregate as a raw material is also different from conventional cell aggregates.

[0049] The embodiments of the present invention will be described in more detail below with reference to the following examples, and the effects and benefits of the present invention will be illustrated. These examples are for illustrative and illustrative purposes only, and the present invention is not limited to these examples. [Examples]

[0050] Experimental method The experiment was conducted according to the following method.

[0051] Purification sorting of mouse MSCs (see Non-Patent Document 6) The femurs and tibias of five 4-week-old male C57 / BL6 cubs (CLEA JAPAN) were crushed in a mortar and pestle, and HBSS was prepared by adding 2% FBS (Cat.#SH30910.03:Hyclone), 10mM HEPES (Cat.#346-01373:Dojindo), and 1% Penicillin / Streptomycin (P / S:Cat.#168-23191,Wako). + (Cat.#14025134:Gibco) (hereafter HBSS+ The bone was suspended in a prepared solution, and red blood cells were removed. The crushed bone was enzymatically treated at 37°C for 1 hour with a solution of 0.2% collagenase (Cat.#034-10533:Wako) in 10 mM HEPES (Cat.#346-01373:Dojindo) and 1% P / S (Cat.#168-23191:Wako) in DMEM (Cat.#08459-64:Nacalai tesque). The enzymatically treated bone was then subjected to cell sizing of 40 μm in diameter. The cells were passed through a strainer (Cat.#352340:Falcon), centrifuged at 280G for 7 minutes at 4°C, the supernatant was removed, and the cells were collected. Typically, 1 × 10⁶ cells are obtained from one organism. 7 ~ 3 x 10 7 Individual cells can be harvested. 1 x 10⁶ cells harvested. 7 Each piece in 1 ml of HBSS + adjustment solution Suspend in liquid and add 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), and APC-conjugated PDGFRα (APA5:Cat.#17-14). Add 2 μl each of the 01-81 antibody (0.2 mg / ml, eBioscience) and the FITC-conjugated Sca-1 (Ly6A / E:Cat.#11-5981-85, 0.5 mg / ml, eBioscience) antibody, and let stand for 30 minutes in a light-shielded environment at 4°C. Next, it was centrifuged at 280G for 7 minutes at 4°C, resulting in 1 × 10⁻⁶ 7 Cell suspension at 1 ml / cell and To be HBSS + Prepare the solution using the prepared solution to obtain a final concentration of 1 μg / ml of propidium iodide (PI). After labeling dead cells with staining (Cat.#169-26281, WAKO), PI was measured 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 adherent culture: Cells cultured adherently in a 10cm culture dish (Cat.#664160-013:CELLSTAR) were washed twice with PBS, then detached using 1 ml of Cell Dissociation Buffer (Cat.#13151014:Gibco) and subjected to HBSS. + Add the prepared solution and incubate at 280G for 5 minutes at 4°C. The heart was separated, the supernatant was removed, and the precipitated cells were collected.

[0053] Cell aggregate samples formed by shaking culture: Considering the difficulty in separating and recovering each cell or cell surface antigen in the cell aggregate into single cells without damaging them, An average of 10 cell aggregates were placed in a 10 cm culture dish (Cat.#664160-013:CELLSTAR) and left to stand for 7 days. Cells that migrated from the cell aggregates 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 allowed to stand for 2-3 minutes to detach the cells. Subsequently, 9 ml of HBSS was added. + Add the prepared solution and bake at 280G for 5 minutes, 4 The cells were centrifuged at °C, the supernatant was discarded, and the precipitated cells were collected. The collected cells were 1 × 10⁶. 7 Individual 1 ml HBSS + Suspend in the prepared solution and add APC-conjugated Sca-1(L 2 μl of antibody (y6A / E:Cat.#11-5981-85, 0.5 mg / ml, eBioscience) was added to each sample, and the mixture was left to stand for 30 minutes in a light-shielded environment at 4°C. Next, it was incubated at 280G for 7 minutes. After minutes, centrifuge at 4°C and measure 1 × 10 7 HBSS to produce a cell suspension of 1 ml / cell +The cells were prepared using a special solution and analyzed using Aria III (BD Bioscience). For the analysis of cells derived from cell aggregates, no subculturing was performed; cells that migrated from the cell aggregates were used directly for analysis.

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

[0055] FACS analysis of human MSCs Similar to the analysis of mouse cells, adherent cells were cultured in a 10 cm culture dish (Cat.#664160-013:CELLSTAR), washed twice with PBS, detached using 1 ml of Cell Dissociation Buffer (Cat.#13151014:Gibco), and then subjected to 9 ml of HBSS. + Add the prepared solution, centrifuge at 280G for 5 minutes, and then centrifuge at 4°C. The supernatant was removed and the cells were collected. For cell aggregate analysis, an average of 10 cell aggregates were placed in a 10 cm culture dish (Cat.#664160-013:CELLSTAR) for 7 days. Cells that migrated from the cell aggregates attached to the culture dish were washed twice with PBS, and the cells were detached using 1 ml of Cell Dissociation Buffer (Cat.#13151014:Gibco) and then subjected to 9 ml of HBSS. + Add the prepared solution and centrifuge at 280G for 5 minutes at 4°C. The supernatant was removed and the cells were collected. Note that in the analysis of cells derived from cell aggregates, no subculturing was performed; cells that migrated from the cell aggregates were used directly for analysis. In both the case of adherent culture and adherent culture derived from cell aggregates, 7 × 10 cells were obtained from one 10 cm culture dish. 6 ~1 × 107 Individual cells were recoverable. 1 × 10⁶ cells recovered 7 Each piece in 1 ml of HBSS + Suspend in the prepared solution, (PE-conjugated LNGFR:Cat.#130-091-885, 5.5 μg / ml, Miltenyi Biotec), FITC-conjugated Thy-1 Add 2 μl each of the following antibodies: Cat.#328110, 0.2 mg / ml, Biolegend, and APC-conjugated CD106 (VCAM-1):Cat.#305810, 0.5 mg / ml, Biolegend. Incubate in a light-shielded environment at 4°C for 30 minutes. Next, it was centrifuged at 280G for 5 minutes at 4°C, resulting in 1 × 10⁻⁶ 7 Cell suspension at 1 ml / cell and To be HBSS + Prepare with the prepared solution, Aria III (BD Bioscience The analysis was performed using ).

[0056] Adhesion culture of mouse and human MSCs OriCell TM Strain C57BL / 6 Mouse MSCs (MUBMX-0) 1001:CYAGEN) and purified mouse MSCs were maintained in MEM-α+GlutaMAX-I (Cat.#32561-10) 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: Culture was performed using Gibco).

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

[0058] Mouse or human MSCs are cultured in a 10cm culture dish (Cat) at 37°C and 5% CO2. .#664160-013:CELLSTAR) 1×10 6 Cells were seeded at a concentration of one cell per dish and cultured for 4-7 days, then subcultured when 80% confluence was reached. For subculture, the culture medium was aspirated, serum components were removed with 1×PBS, and the cells were treated with 0.25% trypsin EDTA (Cat.#201-16945:Wako) for 2 minutes. Gently remove from dish. After tapping and the cells began to detach, growth maintenance medium was immediately added and the cells were collected. After centrifugation at 250×g for 5 minutes, the supernatant was aspirated and removed. Growth maintenance medium was added to the precipitated cells and the cells were transferred to a new 10cm culture dish in 1×10⁶ cells. 6 I sowed the seeds one per dish.

[0059] Furthermore, OriCell TM Strain C57BL / 6 mouse MSCs (MUBMX-01001:Cyagen) had already undergone 6 subculturing cycles at the manufacturer upon purchase. Furthermore, purchased cells are shipped after being checked by the manufacturer for being positive for CD29, CD44, CD31, and Sca-1 (>70%) and negative for CD117 (<5%). The manufacturer also recommends using the cells for 10 or fewer subculturing cycles, and does not guarantee the same cell properties as at the time of purchase for subsequent subculturing cycles. Additionally, OriCell is used for cell maintenance culture. TM The use of Mouse MSC Growth Medium (MUXMX90011:Cyagen) is recommended.

[0060] MSC Shaking Suspension Culture A TAITEC BR-40LF bio-shaker was used for shaking culture. A cell suspension of mouse or human MSCs, prepared in 20 ml of growth maintenance medium, was placed in a 125 ml Erlenmeyer flask (Cat.#431405:Corning). (Mouse MSCs (OriCell))TM During shaking culture of mouse MSCs and purified mouse MSCs, 20 ng / ml βFGF (Cat.#064-05384) is added to the growth maintenance medium. WAKO was added. The number of cells per flask at the start of shaking culture was OriCell TM Mouse MSC 1.0 x 10 7 Individual, purified mouse MSCs, 5.0 x 10 5 Individual, 1.0 × 10⁶ human MSCs 6 individual or 1.0 × 10 7 The cells were adjusted to be individual. Shaking culture was performed at 37°C, 5% CO2, 85-95 r / min, with an amplitude of 40 mm while swirling, for 3-4 days. The culture medium was changed once. The culture medium change involved first transferring the entire culture medium containing the cells into a 50 ml centrifuge tube (Cat. The culture medium was transferred to #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), and the mixture was pipetted 2-3 times before being transferred to the flask. Cell aggregates were observed with the naked eye 10-14 days after the start of shaking culture.

[0061] Mouse MSC Shaking Suspension Culture in Neural Stem Cell Medium For the culture medium for neural stem cells (see Non-Patent Literature 4, Non-Patent Literature 11, and Non-Patent Literature 12), we used Advanced DMEM / F12 (Cat.12491015:Gibco) containing 1% P / S (Cat.#168-23191:Wako), 10 mM HEPES (Cat.#346-01373:Dojindo), 1×N2 (Cat.#17502-048:Gibco), 20 ng / ml EGF (Cat.#059-07873:WAKO), 20 ng / ml FGF (Cat.#064-05384:WAKO), and 1×B27 (Cat.#17504-044:Gibco). Mouse MSC (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). These cell suspensions were cultured with shaking at 37°C, 5% CO₂, 85~95 r / min, and amplitude 40 mm using a TAITEC BR-40LF bio-shaker. The culture medium was changed every 3-4 days. For the culture medium change, the entire medium containing the cells was first transferred to a 50 ml centrifuge tube (Cat.#TR2004: True Line), and the cells were centrifuged at 280 G 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), and the mixture was pipetted 2-3 times before being transferred to the flask. Cell aggregates were observed with the naked eye 10 to 14 days after the start of shaking culture.

[0062] Formation of MSC cell aggregates using existing three-dimensional suspension culture vessels Adherent cultured mouse or human MSCs, 3 × 10 6 Cell concentration of cell suspension (cells / ml) Kuraray 3D culture vessel (Elplasia Cat.#RB 500 40 Seeds were seeded on 0 NA Plate (Kuraray). For cultivation, the same growth maintenance medium as for the MSC shaking suspension culture described above was used, and the medium was changed every 3-4 days.

[0063] Re-adhesion and cell amplification of MSC cell aggregates Cell aggregates formed by shaking culture of mouse or human MSCs were placed in a plastic culture dish containing growth maintenance medium to allow them to adhere. For human MSCs, a culture medium with βFGF removed from the aforementioned growth medium was used. As a result, cell amplification of both mouse and human MSCs was possible. Many cells migrated from around the cell aggregates attached to the culture dish, amplified, and spread across the culture surface. After cell migration and amplification, the cell aggregates attached to the culture dish could be easily recovered by peeling off the bottom surface using the tip of a 200 μl pipette. Yes, it was possible. When the recovered cell aggregate was placed back into a separate culture dish, cell amplification through similar cell migration was possible. (10cm culture dish (Cat.#664160-013:CELLSTAR)) Alternatively, they can migrate to a 12-well plate (Cat.#665-180:CELLSTAR). The amplified cells were used for either 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 evenly spaced across the entire culture dish.

[0064] Differentiation induction method, staining method (see non-patent document 6) In the adhesion culture, cells are placed in a 12-well plate (Cat.#665-180:CELLSTAR) in 1 × 10⁶ units to induce differentiation into osteoblasts and adipocytes. 5 Cells were seeded at a concentration of cells / well. For migratory cells derived from cell aggregates, 2-3 cell aggregates were seeded in each well of a similar 12-well plate, and cells that migrated and amplified from the cell aggregates were used. Confluence of seeded cells After confirming adhesion, the culture medium was changed to osteoblast differentiation induction medium (Cat.#PT3002:Lonza) and adipocyte differentiation induction medium (Cat.#PT3004:Lonza) when the cells reached 60-70% confluence (approximately 7 days of culture). The medium was then changed every 3-4 days thereafter, and differentiation induction was carried out for a maximum of 21 days. During the induction period, adherent cells and formed lipid droplets sometimes floated and detached after about 2 weeks. 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 in either staining method.

[0065] To induce differentiation into chondrocytes, cells were pelleted in a 15 ml centrifuge tube at 150 G 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 culture was performed for 21 days with medium changes twice a week. 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 treated with 4% PFA (paraformaldehyde) (Cat.#163-20145) Fixation was performed using :Wako).

[0067] ALP staining (see non-patent document 6) A Histofine assay kit (Cat.#415161: Nichirei) was used for staining. PFAs were washed with PBS, and the reagents in the kit were mixed and prepared immediately before staining. The staining solution was filtered through a 0.22 μm syringe filter (Cat.#SLGP033RS: Merk Millipore) and used for staining for 30 minutes, followed by rinsing with water. For the negative control (NC), MSCs before osteoblast differentiation induction 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 then stained for 30 minutes with oil red O staining solution (Cat.#40492: Muto Pure Chemicals) filtered through a 0.22 μm syringe filter (Cat.#SLGP033RS: Merk Millipore). After staining, the staining solution was removed, and the sample was treated again with 60% isopropyl alcohol for 1 minute, followed by washing with water.

[0069] Toluidine blue staining (see Non-Patent Document 6) Cell pellets after differentiation induction were embedded in paraffin, and sections were prepared on glass slides. Deparaffinization was performed with xylene and dexylene with 100% alcohol, followed by treatment with 80% alcohol. The sections were then stained for 30 minutes with toluidine blue stain (Cat.#209-14545: Wako) filtered through a 0.22 μm syringe filter (Cat.#SLGP033RS: Merk Millipore). After staining, the sections were dehydrated with 100% alcohol, treated with xylene, and mounted with Marinol (Cat.#2009-3: Muto pure Chemicals).

[0070] Neuronal cell induction (see Non-Patent Document 4) Cell aggregates cultured with shaking in neural stem cell medium were transferred to a medium from which EGF, FGF, and 1×B27 had been removed and 10% FBS (Cat.#SH30910.03:Hyclone) had been added. After treatment with poly-L-ornithine (Cat.#163-27421:WAKO; 37℃, 12 hours), the cells were seeded onto chamber slides (Cat.#SCS-NO8:MATSUNAMI) treated with fibronectin (Cat.#062-05701:WAKO; 37℃, 12 hours) (2-3 cell aggregates / well). Cell aggregates were seeded 7 days after seeding. Cells that migrated from the substrate were fixed using 4% PFA (Cat.#163-20145:WAKO) and used for immunofluorescence staining observation.

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

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

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

[0074] 1.0 × 10 in the flask 6 Individual mouse MSCs (total number of subculturing cycles since shipment: 12) ), or 1.0 × 10 7 Using one human MSC (total number of subculturing cycles before donation: 6) Under these conditions, both mouse and human MSCs formed cell aggregates after two months of shaking culture (Figure 1-a). Furthermore, under the same shaking culture conditions, both mouse MSCs with 44 subcultures prior to shipment and human MSCs with 22 subcultures prior to donation formed cell aggregates after two months of shaking culture (not shown).

[0075] As mentioned above, the OriCell used this time... TMThe mouse MSCs had already undergone six subculturing cycles at the manufacturer upon purchase, while the human-derived MSCs had already undergone two subculturing cycles upon provision. In this example, the subculturing count will hereafter refer to the continuous count from before shipment and before provision (including the number of subculturing cycles before shipment and before provision).

[0076] The number of cell aggregates formed by human MSCs (passages: 6-7 times) under shaking culture is equal to the number of cells at the start of shaking culture, which is 1.0 × 10⁶. 6 In the case of 1, the number after 1 month is 14.3 ± 2.0, 2 The number of cells was 10.7 ± 2.1 after 1 month. The number of cells at the start of shaking culture was 1.0 × 10⁶. 7 In the case of one The number of cells was 15.0 ± 1.0 after one month and 11.7 ± 2.5 after two months (mean ± standard deviation of the results of three experiments). In other words, the number of cells at the start of shaking culture was 1.0 × 10⁶. 6 Individual Even when the amount was increased tenfold, there was no significant difference in the number of cell clumps formed.

[0077] The size of the cell aggregate formed by shaking culture of human MSCs that had been subcultured 7 times was 1.0 × 10⁶ cells at the start of shaking culture. 6 In the case of one individual, the average monthly growth rate is 433.3 ± 103.3 μm. The cell volume was 533.3 ± 150.6 μm after 2 months. The cell count at the start of shaking culture was 1.0 × 10⁶ 7 In the case of one sample, the size was 783.3 ± 248.3 μm after one month and 833.3 ± 1 μm after two months. The size was 86.1 μm (mean ± standard deviation of the results from 6 experiments).

[0078] The number of cell aggregates formed by human MSCs with a high number of passages (19) after 1 month of shaking culture was 1.0 × 10⁶ 6 In the case of 14 pieces, 1.0 × 10 7 In the case of 15 cells, the result was 15 cells (result of one experiment). Furthermore, the size of the cell aggregate formed by human MSCs with 19 passages after 1 month of shaking culture was 1.0 × 10⁶ cells, based on the number of cells at the start of shaking culture. 6In the case of individual particles, the thickness is 500 ± 126.5 μm, and the size is 1.0 × 10⁻⁶. 7 In the case of a single cell cluster, the size was 766.7 ± 206.6 μm (mean ± standard deviation of 6 cell clusters).

[0079] Figure 2: Effects of shaking culture on maintaining the differentiation potential of mouse MSCs The effect of shaking culture on maintaining the differentiation ability of mouse MSCs was investigated. TM Mouse MSCs retained the ability to differentiate into osteoblasts (ALP staining positive) and chondrocytes (toluidine blue staining positive) at a relatively low stage of 11 passages, but had already lost the ability to differentiate into adipocytes and showed negative staining for oil red O (Figure 2-a: top panel).

[0080] However, OriCell underwent many subculturing cycles (36 times) using adherent culture. TM Even in mouse MSCs, subjecting them to shaking culture for two months to induce cell aggregate formation allowed cells that migrated and proliferated from the cell aggregates to retain and recover not only the ability to differentiate into osteoblasts (ALP staining positive) but also the ability to differentiate into adipocytes (lipid droplets indicated by the arrows in Figure 2-a) (Figure 2-a: bottom panel).

[0081] Mouse MSCs that co-express PDGFRα and Sca-1 on their cell surface are known to be high-quality MSCs that maintain an undifferentiated state (Non-patent documents 4, 5). OriCells that have been passaged 7 times in adherent culture. TM Mouse MSCs are PDGFRα + / Sca-1 + The co-positive fraction retained approximately 90% (not shown), but when subculturing was repeated, this co-positive fraction decreased to 46.1% after 8 subculturing cycles (Figure 2-b: left panel), and further decreased to 25.0% after 23 subculturing cycles (not shown).

[0082] However, OriCell underwent many subculturing cycles (44 times) using adherent culture. TM Even in mouse MSCs, subjecting them to shaking culture for two months to induce cell aggregate formation restored the co-positive fraction of cells that migrated and proliferated from the cell aggregates to 68.3% (Figure 2-b: right panel).

[0083] RT-PCR analysis results showed that OriCell cultured in adherent culture TM Mouse MSCs showed high expression of the PDGFRα gene at 9 passages, but at 25 passages... As the number of cycles increases to 41, the expression of the PDGFRα gene decreases significantly, suggesting that undifferentiated potential may be lost.

[0084] However, OriCell, which has undergone 37 subculturing cycles... TM Even in mouse MSCs, it was confirmed that PDGFRα gene expression could be restored to a high level by subjecting them to shaking culture for two months to induce cell aggregate formation (Figure 2-C).

[0085] Figure 3: Oricell cultured by shaking TM Mesodermal differentiation of mouse MSCs Only TM Using mouse MSCs, cells with a low number of passages (9 passages) and cells with a high number of passages (30 passages) were prepared and cultured with shaking for one month. The results of differentiation induction into mesodermal cells, namely osteoblasts, chondrocytes, and adipocytes are shown in Figure 3.

[0086] Both cells with a low number of passages (9 passages) and cells with a high number of passages (30 passages) that were differentiated 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. Only TM Using mouse MSCs, we prepared cells with a low number of adhesion passages (8-9 passages) and cells with a high number of passages (30 passages). We analyzed the expression of genes related to osteoblasts, chondrocytes, and adipocytes in cells that underwent differentiation induction immediately after adhesion culture and in cells that underwent differentiation induction after shaking culture following adhesion culture, respectively, using RT-PCR. When osteoblasts were differentiated for 21 days, cells with fewer adherent culture passages (8-9 passages) showed higher expression of the osteoblast markers OCN and OPN in shaken cultured cells compared to adherent cultured cells. In cells with more adherent culture passages (30 passages), OPN expression was higher in shaken cultured cells compared to adherent cultured cells (Figure 4-a).

[0088] When chondrocytes were differentiated for 21 days, cells with fewer adherent culture passages (8-9 passages) showed higher expression of the chondrocyte markers Agrican, Sox9, and Col2A1 in shaking cultured cells compared to adherent cultured cells. In cells with more adherent culture passages (30 passages), shaking cultured cells showed higher expression of Agrican and Sox9 compared to adherent cultured cells (Figure 4-b).

[0089] When adipocytes were differentiated into adipocytes for 21 days, adherent cultured cells did not express the adipocyte markers Adipsin and PPARγ, regardless of the number of passages. On the other hand, cells with a low number of adherent passages (9 passages) showed marked expression of Adipsin and PPARγ when cultured with shaking, while cells with a high number of adherent passages (30 passages) showed PPARγ expression when cultured with shaking (Figure 4-c).

[0090] Figure 5: Effects of shaking culture on maintaining the differentiation potential of human MSCs The effect of shaking culture on maintaining the differentiation potential of human MSCs was investigated. In adherent culture, human MSCs showed the ability to differentiate into osteoblasts (ALP staining positive) after 20 passages, but lost the ability to differentiate into adipocytes (oil red O staining negative) (Figure 5-a: top panel).

[0091] However, when human MSCs that had been passaged six times in adherent culture were subjected to two months of shaking culture to form cell aggregates, the cells that migrated and proliferated from these cell aggregates retained not only the ability to differentiate into osteoblasts (ALP staining positive) but also a high ability to differentiate into adipocytes (Oil Red O staining positive) (Figure 5-a: middle panel). In particular, cells that migrated around the cell aggregates adhered to the culture dish formed many lipid droplets, suggesting a high degree of undifferentiated state. Furthermore, when human MSCs that had been passaged six times were subjected to adherent culture for the same two months as shaking culture, the number of passages reached 12, and lipid The ability to differentiate into fat had already been lost (negative for oil red O staining: not shown).

[0092] Human MSCs expressing CD271 (LNGFR), CD90 (Thy-1), and CD106 (VCAM-1) on their cell surface are known to be high-quality MSCs that maintain an undifferentiated state (Non-Patent Literature 7). Human MSCs that underwent 21 passages in adherent culture showed high CD90 expression (Figure 5-b: upper left). Similarly, cells that migrated and proliferated from cell aggregates formed by 2 months of shaking culture of human MSCs that had undergone 7 passages in adherent culture also 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 that had undergone 7 passages in adherent culture had already decreased, and its expression did not recover even in shaking culture (not shown).

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

[0094] RT-PCR analysis revealed that human MSCs cultured using adherent culture expressed the Oct3 / 4 gene, known as an undifferentiated stem cell marker, at 7 passages. However, this expression decreased at 19 passages, suggesting a possible loss of undifferentiated potential. Nevertheless, even in human MSCs that had undergone 19 passages, 2 months of shaking culture to induce cell aggregate formation restored high levels of expression for Sox2 and Oct3 / 4 genes, known as stem cell-related markers (Figure 5-C).

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

[0096] When cells with a low number of passages (9 passages) that were differentiated immediately after adherent culture were differentiated into cartilage for 21 days, pellet formation was observed, but only a small number of cells were positive for toluidine blue, which stains cartilage matrix. On the other hand, when cells with a low number of passages (9 passages) were cultured by shaking before being differentiated into cartilage, pellet formation by toluidine blue-positive cells was observed (Figure 6-a).

[0097] When cells with a high number of passages (19 passages) that were immediately differentiated after adherent culture were differentiated into cartilage for 21 days, no pellet formation was observed. On the other hand, when cells with a high number of passages (19 passages) that were cultured by shaking were differentiated into cartilage, pellet formation by toluidine blue-positive cells was observed (Figure 6-b).

[0098] Figure 7: Adherent culture of MSC cell aggregates using a three-dimensional suspension culture vessel (prior art) Human MSCs (passaged 9 times) or OriCells maintained in adherent culture in a low-adhesion three-dimensional culture vessel (Kuraray Elplasia RB 500 400 NA Plate), which is used in existing methods for forming MSC cell aggregates. TM Mouse MSCs (passages: 25 and 41) were divided into 3 × 10⁶ 6Seeds were sown at a rate of 1 / ml. As a result, In the case of MSCs, the cells adhered to the culture dish, and cell aggregate formation was not achieved even after 7 days (Figure 7-a). On the other hand, in mouse MSCs, as reported by Baraniak et al. (Non-Patent Literature 10), cell aggregates formed after 7 days of culture in the incubator (Figure 7-b). When the cell aggregates collected at this point were transferred back to the culture dish and left to stand in an adherent culture environment, the cell aggregates adhered to the culture dish and cells migrated around them, but the cell aggregates did not maintain their shape and disappeared after 7 days, indicating that the ability to differentiate into adipocytes was lost (negative for oil red O staining and no lipid droplets were observed) (Figure 7- b).

[0099] Figure 8: Oricell using a three-dimensional suspension culture vessel (prior art) TM Analysis of the differentiation potential of mouse MSC cell aggregates Oricells maintained in adherent culture in a low-adhesion three-dimensional suspension culture vessel (Kuraray Elplasia RB 500 400 NA Plate). TM Mouse MSCs (passages: 25 and 41) were divided into 3 × 10⁶ 6 Seeds were sown at a rate of 1 / ml. Formation occurred after 7 days. The cell aggregates were seeded into 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 subculture cycles TM Mouse MSCs were induced to differentiate into osteoblasts for 21 days, and both showed ALP positivity, confirming their ability to differentiate into osteoblasts (Figure 8-a). On the other hand, Oricell cells after 25 and 41 adherent subculturing cycles were also shown. TM When mouse MSCs were differentiated into adipocytes for 21 days, no formation of oil red O-positive lipid droplets was observed (Figure 8-b).

[0101] Figure 9: Morphological retention and cell supply capacity of human MSC cell aggregates cultured by shaking. Human MSCs subcultured 7 times by culturing were subjected to shaking culture for 2 months, and the cell aggregates formed were placed on a culture dish. As a result, cell migration around the cell aggregates was observed the next day, and after 7 days, the cells grew to fill the culture dish (Fig. 9-a: upper row). Also, the reattached cell aggregates maintained their morphology even after 7 days, and it was possible to mechanically detach the cell aggregates and place them on a new culture dish (first reattachment). The day after the cell aggregates were placed again, cell migration around the cell aggregates was observed as described above, and within 10 days, the cells grew to fill the culture dish, and the cell aggregates maintained their morphology (Fig. 9-a: middle row, left 2 photos). Even when this process of mechanically detaching the cell aggregates and placing them on a new culture dish was repeated 4 more times (second to fifth reattachments), cell migration around the cell aggregates was similarly observed, and within 14 days, the cells grew to fill the culture dish, and the cell aggregates maintained their morphology (Fig. 9-a: middle row, right 3 photos - lower row). Also, by repeating the process of detaching this cell aggregate and reattaching it to the culture dish 3 times, the migrated cells showed the ability to differentiate into osteoblasts (positive for ALP staining) and the ability to differentiate into adipocytes (positive for lipid droplets and Oil Red O staining). Therefore, it was revealed that they maintained their undifferentiated state (Fig. 9-b). Therefore, it was found that the MSC cell aggregates according to the present invention have a strong adhesive force between cells, and even when returned to the adherent culture environment again, they continuously supply undifferentiated MSCs to the culture dish without collapsing the morphology of the cell aggregates.

[0102]

[0103] Figure 10: Cell aggregates formed by shaking culture of purified mouse MSCs Whether cell aggregates similar to those in the case of mouse MSCs were formed was also examined by subjecting purified mouse MSCs to shaking culture. Purified mouse MSCs after sorting were subcultured 2 times by adherent culture to secure the number of cells. As a result of subjecting these 2-subcultured purified mouse MSCs (5.0×10 TM cells / flask) to shaking culture for 2 months, 5 OriCell TM ​The cells formed a cell aggregate similar to that of mouse MSCs (not shown). When the formed cell aggregate was placed in a new culture dish, cell migration around the cell aggregate was observed the following day (Figure 10-b: left panel), and the migrating cells proliferated to fill the culture dish within 10 days. During this process, the cell aggregate maintained its shape without losing its form. Furthermore, when the migrating and proliferating cells were induced to differentiate into adipocytes, they produced lipid droplets after 14 days (Figure 10-c), demonstrating that they retained the ability to differentiate into adipocytes.

[0104] Figure 11: Cell aggregate formation of Oricell™ mouse MSCs using neural stem cell culture medium. Oricell™ mouse MSCs were cultured using the same shaking method in neural stem cell culture medium. We investigated whether cell aggregates would form in these cases. We also examined the differentiation potential of the obtained cell aggregates into mesodermal cells (bone, fat, cartilage) and nerve cells. Furthermore, we analyzed the expression of neural crest stem cell markers Nestin and Twist, and the mesenchymal stem cell marker PDGFRα using RT-PCR.

[0105] Cells with few adherent passages (9 passages) were cultured with shaking in neural stem cell culture medium, and cell aggregate formation was observed within one month (Figure 11-a). When this cell aggregate was used to induce differentiation into osteoblasts, chondrocytes, and adipocytes, the formation of cartilage pellets consisting of ALP-positive osteoblasts, adipocytes showing oil red O-positive lipid droplets, and toluidine blue-positive cells was observed (Figure 11-b). Furthermore, when this cell aggregate was used to induce differentiation into neurons, βIII-Tublin-positive neurons were observed (Figure 11-c).

[0106] Furthermore, even with cells that had undergone many adherent passages (39 passages), cell aggregate formation was possible by using a culture medium for neural stem cells (Figure 11-d). RT-PCR analysis revealed that the cells in this cell aggregate significantly expressed Nestin and Twist, which are neural crest stem cell markers, and PDGFRα, a mesenchymal stem cell marker (Figure 11-e).

[0107] summary As is clear from the results above, by using shaking culture, we were able to restore MSCs that had lost their undifferentiated state back into cell aggregates of highly undifferentiated MSC populations. Furthermore, the MSC cell aggregates formed by shaking culture retain their undifferentiated state and possess robust morphological retention capabilities, allowing for the supply of undifferentiated MSCs by reattaching them to a new culture dish. [Industrial applicability]

[0108] According to the method of the present invention, by culturing MSCs in an adhesive environment using a completely new stirring and shaking culture method, it is possible to form robust, three-dimensional MSC spheres that maintain differentiation ability and morphology.

[0109] Therefore, it is suggested that it has potential as a useful stem cell pool that continuously supplies stem cells that maintain their undifferentiated state. Furthermore, from the perspective of applications in regenerative medicine, this cell aggregate is expected to act as a scaffold, creating space in the defective area and promoting tissue regeneration by stem cells.

Claims

1. A method for maintaining the undifferentiated state of mesenchymal stem cells, characterized by forming cell aggregates by subculturing mesenchymal stem cells that have been passed through two or more times using adherent culture for 10 days or more in a shaking suspension culture.

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

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

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

5. A cell aggregate obtained by the method according to 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 induction medium.

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