Method for producing mesenchymal stem cells

Abstract

To provide a medium composition for efficiently producing a mesenchymal stem cell, a method for producing a mesenchymal stem cell, and an agent or the like for repairing cartilage.SOLUTION: The present invention provides a medium composition for inducing a mesenchymal stem cell from a neural crest cell, containing a minimal essential medium and corticosteroid. In the medium composition, the converted concentration of the corticosteroid is 1.25 μm or more. There is also provided a method for producing a mesenchymal stem cell, including the step of culturing a neural crest cell in the medium to induce a mesenchymal stem cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing mesenchymal stem cells, and more particularly to a method for producing mesenchymal stem cells using a medium composition containing a basal medium and an adrenocortical hormone. [Background technology]

[0002] Mesenchymal stem cells (MSCs) are a type of somatic stem cell found in the bone marrow and other tissues of the body. They are defined as adhesive cells with the ability to differentiate into bone, cartilage, and adipocytes. Mesenchymal stem cells are believed to have an extremely low risk of cancer and are highly promising cell sources for use in regenerative medicine. Furthermore, mesenchymal stem cells accumulate at the site of tissue injury and release various humoral factors and exosomes to regulate immune responses and anti-inflammatory effects, playing an important role in tissue repair and homeostasis. Therefore, mesenchymal stem cells themselves are promising cell preparations for the treatment of immune and inflammatory diseases.

[0003] However, since mesenchymal stem cells have different differentiation potential and proliferation / functional characteristics depending on the tissue from which they are derived, there has been a need to efficiently produce mesenchymal stem cells that exhibit useful properties according to the purpose.

[0004] Neural crest cells are cells unique to vertebrates, which transiently arise between the neural tube and presumptive epidermal ectoderm during early development, migrate within the embryonic body, and differentiate into a wide variety of cells such as cells of the peripheral nervous system, cells of head tissue, pigment cells, etc. It was known that neural crest cells could be produced by the methods disclosed in Patent Document 1 and Non-Patent Document 1, and that differentiation of mesenchymal stem cells could be induced from these cells (Non-Patent Document 2), but research had been conducted into methods for producing mesenchymal stem cells more efficiently. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2020 / 230832 [Non-patent literature]

[0006] [Non-Patent Document 1] Fukuta M. et al., PLoS One. 2014 Dec 2;9(12):e 112291. [Non-patent document 2] Zhao C. and Ikeya M., Stem Cells Int. 2018 Jul 31; Article ID 9601623 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a medium composition for efficiently producing mesenchymal stem cells, a method for producing mesenchymal stem cells, and the like. [Means for solving the problem]

[0008] As a result of extensive research into the above-mentioned problems, the present inventors have discovered that mesenchymal stem cells can be efficiently induced from neural crest cells by using a medium composition containing adrenocortical hormones, and have also discovered an appropriate concentration of adrenocortical hormones in the medium composition. They have also discovered that mesenchymal stem cells obtained by culturing neural crest cells in such a medium composition have a high ability to differentiate into chondrocytes and are useful for producing drugs for repairing cartilage tissue, thereby completing the present invention.

[0009] That is, the present invention is as follows. [1] A medium composition for inducing mesenchymal stem cells from neural crest cells, comprising a basal medium and a corticosteroid, wherein the equivalent concentration of the corticosteroid in the medium composition is 1.25 μM or more. [2] The medium composition according to [1], wherein the adrenal cortical hormone is a glucocorticoid or a derivative thereof. [3] The medium composition described in [2], wherein the glucocorticoid or a derivative thereof is at least one selected from the group consisting of cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone propionate. [4] The medium composition according to [2] or [3], wherein the glucocorticoid or a derivative thereof is dexamethasone. [5] As described in [1], the equivalent concentration of the adrenal cortical hormone in the medium composition is 300 μM or less. Medium composition. [6] The medium composition according to [4], wherein the concentration of dexamethasone in the medium composition is 10 μM or less. [7] The medium composition according to any one of [1] to [6], wherein the basal medium is a serum-free medium. [8] The medium composition according to any one of [1] to [7], wherein the neural crest cells are derived from pluripotent stem cells. [9] The medium composition according to [8], wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells). thing.

[10] A method for producing mesenchymal stem cells, comprising the step of inducing mesenchymal stem cells by culturing neural crest cells in the medium composition according to any one of [1] to [7] (step 1).

[11] The method of production described in

[10] , wherein the neural crest cells are derived from pluripotent stem cells.

[12] The method according to

[11] , wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells). method.

[13] The method according to any one of

[10] to

[12] , wherein the following step is carried out before step 1: (Step A) obtaining a cell population containing neural crest cells; and (Step B) A step of expanding and culturing the cell population obtained in step A using an extracellular matrix as a scaffold.

[14] The method according to

[13] , wherein the extracellular matrix is ​​laminin or fibronectin.

[15] The method of producing according to

[14] , wherein the laminin is full-length laminin, laminin having an α2 chain, or laminin 211.

[16] The method according to any one of

[13] to

[15] , wherein the cell population obtained in step B is a cell population containing neural crest cells in an amount of 70% or more.

[17] A method for producing mesenchymal stem cells from a cell population containing neural crest cells, comprising: the neural crest cells are cells induced to differentiate from pluripotent stem cells, the cell population is a cell population containing 70% or more of the neural crest cells, The cell population is cultured in the presence of a corticosteroid at a concentration of 1.25 μM or more and 300 μM or less. This includes the step of cultivating the A method for producing mesenchymal stem cells from a cell population containing neural crest cells.

[18] The method according to

[17] , wherein the adrenal cortical hormone is a glucocorticoid or a derivative thereof.

[19] The manufacturing method described in

[18] , wherein the glucocorticoid or a derivative thereof is at least one selected from the group consisting of cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone propionate.

[20] The glucocorticoid or its derivative is dexamethasone.

[18] Or The manufacturing method is described in

[19] .

[21] Mesenchymal stem cells or cultures thereof obtained by the production method according to any one of

[10] to

[20] .

[22] The mesenchymal stem cells or cultures thereof according to

[21] , wherein the mesenchymal stem cells are characterized by a high ability to differentiate into chondrocytes.

[23] A method for producing chondrocytes, comprising the steps of: (Step D) Cells containing mesenchymal stem cells are produced by the production method according to any one of

[10] to

[20] . obtaining a cell population; (Step E) culturing the cell population containing mesenchymal stem cells in a cartilage induction medium to produce a cell population containing chondrocytes. Obtaining a cell population.

[24] Chondrocytes or cultures thereof obtained by the manufacturing method described in

[23] .

[25] A drug for repairing cartilage tissue, comprising the chondrocytes or cultures thereof according to

[24] .

[26] A method for producing a medicament for repairing cartilage tissue, comprising chondrocytes, the method comprising the steps of: (Step D) Cells containing mesenchymal stem cells are produced by the production method according to any one of

[10] to

[20] . obtaining a cell population; (Step E) culturing the cell population containing mesenchymal stem cells in a cartilage induction medium to produce a cell population containing chondrocytes. Obtaining a cell population. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a medium composition for efficiently inducing mesenchymal stem cells from neural crest cells. Furthermore, according to the present invention, it is possible to provide an efficient method for producing mesenchymal stem cells, a method for producing chondrocytes, a drug for repairing cartilage tissue, a method for repairing cartilage tissue, a method for producing a drug for repairing cartilage tissue, and the like, which contain the mesenchymal stem cells as an active ingredient. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a graph showing the cell proliferation curve during differentiation induction. [Figure 2] FIG. 2 shows a phase-contrast microscope image of iNCCs whose differentiation was initiated in the medium composition of the present invention. [Figure 3] 3 is a graph showing the time course of expression levels of MSC markers in iNCCs whose differentiation was initiated using the medium composition of the present invention. The vertical axis represents the relative value when the expression level in iPS cells is set to 1. The numbers on the horizontal axis represent the number of days since the start of differentiation induction into MSCs. [Figure 4] 4 is a graph showing the cell proliferation curve of iNCCs whose differentiation was initiated using the medium composition of the present invention. The vertical axis represents the total number of divisions, and the horizontal axis represents the number of days since the start of differentiation induction into MSCs. [Figure 5]Figure 5 shows data demonstrating the high differentiation potential of iMSCs obtained by inducing differentiation using the medium composition of the present invention into chondrocytes. The image shows the results of Alcian blue staining 14 days after iMSCs were induced to differentiate into chondrocytes. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below.

[0013] 1. Medium Composition The present invention provides a medium composition for inducing mesenchymal stem cells from neural crest cells, which comprises a basal medium and a corticosteroid, wherein the corticosteroid concentration in the medium composition is 1.25 The present invention provides a medium composition having a saturation level of 0.1 μM or higher (hereinafter also referred to as the medium composition of the present invention).

[0014] The basal medium contained in the medium composition of the present invention can be any known basal medium, and is not particularly limited as long as it does not inhibit the induction of neural crest cells into mesenchymal stem cells. Examples of such basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, and Ham's F12 medium. RPMI 1640 medium, Fischer's medium, StemPro34 (Invitrogen), RPMI-base medium, StemFit (registered trademark), MCDB201 medium, and mixed media thereof. Preferably, StemFit® medium is used. The medium may contain serum. If necessary, the medium may contain, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, cholesterol, etc. Protein precursors, trace elements, 2-mercaptoethanol (2ME), thiolglycerol, etc. and may contain one or more of the following serum replacements: lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, and antioxidants. The composition may also contain one or more substances such as an anti-inflammatory agent, pyruvic acid, a buffer, inorganic salts, etc.

[0015] The medium contains an adrenocorticoid. Examples of the adrenocorticoid include glucocorticoids and derivatives thereof. Examples of the glucocorticoids and derivatives thereof include cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone propionate. Among these, prednisolone, dexamethasone, and betamethasone are preferred, with dexamethasone being particularly preferred. One or more types of adrenocorticoids may be added to the medium, but one type is preferred.

[0016] Dexamethasone (CAS number: 50-02-2, CA index name: Pregna-1,4-diene-3,20-dione, 9-fluoro-11,17,21-trihydroxy-16-methyl-, (11β,16α)-) has the following formula:

[0017] [ka]

[0018] Dexamethasone is a known synthetic adrenocortical hormone having the structure represented by the formula: Dexamethasone is commercially available, and such commercially available products can also be used in the present invention. The use of dexamethasone induces differentiation of neural crest cells into mesenchymal stem cells, and therefore mesenchymal stem cells can be produced with high efficiency by culturing neural crest cells in the medium composition of the present invention.

[0019] The concentration of the adrenal cortical hormone contained in the medium composition is not particularly limited as long as it is a concentration that can promote differentiation of neural crest cells into mesenchymal stem cells. For example, When used in a concentration of 50 nM or more (or more than 50 nM), 55 nM or more (or more than 55 nM), 60 nM or more ( or greater than 60 nM), 65 nM or greater (or greater than 65 nM), 70 nM or greater (or greater than 70 nM), 75 nM or greater (or greater than 75 nM), 80 nM or greater (or greater than 80 nM), 85 nM or greater (or greater than 85 nM), 90 nM or greater (or greater than 90 nM), 95 nM or greater (or greater than 95 nM), 100 nM or greater (or greater than 100 nM), 150 nM or greater (or or more than 150 nM), 200 nM or more (or more than 200 nM), 250 nM or more (or more than 250 nM), 300 nM or more (or more than 300 nM), 350 nM or more (or more than 350 nM), 400 nM or more (or more than 400 nM), 450 nM or more (or more than 450 nM), 500 nM or more (or more than 500 nM), 550 nM or more (or more than 550 nM), 600 nM or more (or more than 600 nM), 650 nM or more (or more than 650 nM), 700 nM or more (or more than 700 nM), 750 nM or more (or more than 750 nM), 800 nM or more (or more than 800 nM), 850 nM or more (or more than 850 nM), 900 nM or more (or more than 900 nM) nM or more), 950 nM or more (or more than 950 nM) and 10 μM or less (or less than 10 μM), 9 μM or less (or less than 9 μM), 8 μM or less (or less than 8 μM), 7 μM or less (or less than 7 μM), 6 μM or less (or less than 6 μM), 5 μM or less (or less than 5 μM), 4 μM or less (or less than 4 μM), 3 μM or less (or less than 3 μM), 2 μM or less (or less than 2 μM) ), and 1 μM or less (or less than 1 μM). When a corticosteroid other than dexamethasone is used, it is desirable to use it at a concentration that exhibits glucocorticoid potency equivalent to that of dexamethasone at the aforementioned concentration. If the corticosteroid concentration in the medium is too low, it may not be sufficient to maintain the culture or promote the induction of mesenchymal stem cells, while if the corticosteroid concentration is too high, it will suppress cell proliferation and make it difficult to efficiently obtain mesenchymal stem cells. For example, when inducing differentiation into human mesenchymal stem cells, when dexamethasone is used alone, the concentration of the corticosteroid contained in the medium composition is, for example, 50 nM to 10 μM (or 50 nM or more but less than 10 μM, more than 50 nM but less than 10 μM, or more than 50 nM but less than 10 μM), preferably 60 nM to 5 μM (or 60 nM or more but less than 5 μM, more than 60 nM but less than 5 μM, or more than 60 nM but less than 5 μM), More preferably, it is 70 nM to 2 μM (or 70 nM or more but less than 2 μM, or more than 70 nM but less than 2 μM, or 70 more preferably 100 nM to 1 μM (or 100 nM or more but less than 1 μM, or 100 nM to 1 μM, or 100 nM to 1 μM).

[0020] The potency of adrenal cortical hormones as glucocorticoids is 1, when hydrocortisone is taken as 1. Cortisone was 0.8, prednisolone was 4, methylprednisolone was 5, and triamcinolone was 6. The ratios are 5 for fluoxetine, 10 for paramethasone, 25-30 for dexamethasone, and 25-30 for betamethasone. If there is a possibility that the glucocorticoid potency of the adrenal cortical hormone may differ from the above value depending on the manufacturer or storage conditions, it is advisable to confirm this experimentally in advance and use one that is of good quality and has been stored in good condition and has the above value as the potency.

[0021] In addition, when betamethasone is used alone, the betamethasone contained in the medium composition The concentration of is not particularly limited as long as it is a concentration that can promote differentiation of neural crest cells into mesenchymal stem cells, but for example, it is 50 nM or more (or more than 50 nM), 55 nM or more (or more than 55 nM), 60 nM or more ( or greater than 60 nM), 65 nM or greater (or greater than 65 nM), 70 nM or greater (or greater than 70 nM), 75 nM or greater (or greater than 75 nM), 80 nM or greater (or greater than 80 nM), 85 nM or greater (or greater than 85 nM), 90 nM or greater (or greater than 90 nM), 95 nM or greater (or greater than 95 nM), 100 nM or greater (or greater than 100 nM), 150 nM or greater (or or more than 150 nM), 200 nM or more (or more than 200 nM), 250 nM or more (or more than 250 nM), 300 nM or more (or more than 300 nM), 350 nM or more (or more than 350 nM), 400 nM or more (or more than 400 nM), 450 nM or more (or more than 450 nM), 500 nM or more (or more than 500 nM), 550 nM or more (or more than 550 nM), 600 nM or more (or more than 600 nM), 650 nM or more (or more than 650 nM), 700 nM or more (or more than 700 nM), 750 nM or more (or more than 750 nM), 800 nM or more (or more than 800 nM), 850 nM or more (or more than 850 nM), 900 nM or more (or more than 900 nM) nM or more), 950 nM or more (or more than 950 nM) and 10 μM or less (or less than 10 μM), 9 μM or less (or less than 9 μM), 8 μM or less (or less than 8 μM), 7 μM or less (or less than 7 μM), 6 μM or less (or less than 6 μM), 5 μM or less (or less than 5 μM), 4 μM or less (or less than 4 μM), 3 μM or less (or less than 3 μM), 2 μM or less (or less than 2 μM) ), and 1 μM or less (or less than 1 μM). If the betamethasone concentration in the medium is too low, it may not be sufficient to maintain the culture or promote the induction of mesenchymal stem cells, while if the betamethasone concentration is too high, it will inhibit cell proliferation and make it difficult to efficiently obtain mesenchymal stem cells. For example, when inducing differentiation into human mesenchymal stem cells, the concentration of betamethasone contained in the medium composition is, for example, 50 nM to 10 μM (or 50 nM or more but less than 10 μM, or more than 50 nM but less than 10 μM, or or more than 50 nM and less than 10 μM), preferably 60 nM to 5 μM (or 60 nM or more and less than 5 μM, more than 60 nM 5 μM or less, or more than 60 nM but less than 5 μM), more preferably 70 nM to 2 μM (or 70 nM or more but less than 2 μM, more than 70 nM but less than 2 μM, or more than 70 nM but less than 2 μM), even more preferably 100 nM to 1 μM (or 100 nM or more but less than 1 μM, more than 100 nM but less than 1 μM, or more than 100 nM but less than 1 μM). )

[0022] In addition, when using a single type of prednisolone, the prednisolone contained in the medium composition The concentration of the ron is not particularly limited as long as it is a concentration that can promote differentiation of neural crest cells into mesenchymal stem cells, and examples thereof include 300 nM or more (or more than 300 nM), 330 nM or more (or more than 330 nM), 360 nM or more (or more than 360 nM), 390 nM or more (or more than 390 nM), 420 nM or more (or more than 420 nM), 450 nM or more (or more than 450 nM), 480 nM or more (or more than 480 nM), 510 nM or more (or more than 510 nM), 540 nM or more (or more than 540 nM), 570 nM or more (or more than 570 nM), 600 nM or more (or more than 600 nM), 900 nM or more (or more than 900 nM), 1.2 μM or more (or more than 1.2 μM), 1.5 μM or more ( or greater than 1.5 μM), 1.8 μM or greater (or greater than 1.8 μM), 2.1 μM or greater (or greater than 2.1 μM), 2.4 μM or more (or more than 2.4 μM), 2.7 μM or more (or more than 2.7 μM), 3 μM or more (or more than 3 μM), 3.3 μM or more (or more than 3.3 μM), 3.6 μM or more (or more than 3.6 μM), 3.9 μM or more (or more than 3.9 μM), 4.2 μM or more (or more than 4.2 μM), 4.5 μM or more (or 4.5 >μM), 4.8 μM or more (or more than 4.8 μM), 5.1 μM or more (or more than 5.1 μM), 5.4 μM or more (or more than 5.4 μM) ), 5.7 μM or more (or more than 5.7 μM), 70 μM or less (or less than 70 μM), 63 μM or less (or less than 63 μM), 56 μM or less (or less than 56 μM), 49 μM or less (or less than 49 μM), 42 μM or less (or less than 42 μM), 35 μM or less (or less than 35 μM), 28 μM or less (or less than 28 μM) Prednisolone concentrations in the medium are below 21 μM (or below 21 μM), below 14 μM (or below 14 μM), and below 7 μM (or below 7 μM). If the prednisolone concentration in the medium is too low, it may be difficult to maintain the culture or to produce mesenchymal stem cells. However, if the prednisolone concentration is too high, it may not be sufficient to promote the induction of mesenchymal stem cells, and if the prednisolone concentration is too high, it may inhibit cell proliferation, preventing efficient production of mesenchymal stem cells. For example, when inducing differentiation into human mesenchymal stem cells, the concentration of prednisolone contained in the medium composition is, for example, 300 nM to 70 μM (or 300 nM or more but less than 70 μM, greater than 300 nM to 70 μM, or greater than 300 nM to less than 70 μM), preferably 360 nM to 35 μM (or 360 nM or more but less than 35 μM, greater than 360 nM to 35 μM, or greater than 360 nM to less than 35 μM), more preferably 420 nM to 14 μM (or 420 nM or more but less than 14 μM, greater than 420 nM to 14 μM, or greater than 420 nM to less than 14 μM), and even more preferably 600 nM to 7 μM (or 600 nM or more but less than 7 μM, greater than 600 nM to 7 μM, or greater than 600 nM to less than 7 μM).

[0023] In one embodiment, the concentrations of the adrenocortical hormones contained in the medium composition are When used alone, dexamethasone or betamethasone is 50 nM or more and 10 μM or less (or 50 nM or more and less than 10 μM, more than 50 nM and 10 μM, or more than 50 nM and 10 μM), paramethasone is 100 nM or more and 30 μM or less (or 100 nM or more and less than 30 μM, more than 100 nM and 30 μM, or more than 100 nM and 30 μM), and triamcinolone or methylprednisolone is 250 nM or more and 60 μM or less (or 250 nM or more and less than 60 μM, more than 250 nM and 60 μM, or more than 250 nM and 60 μM). and prednisolone is 300 nM or more and 70 μM or less (or 300 nM or more and less than 70 μM). , greater than 300 nM and less than 70 μM, or greater than 300 nM and less than 70 μM), cortisone is greater than 1.50 μM and less than 375 μM (or greater than 1.50 μM and less than 375 μM, greater than 1.50 μM and less than 375 μM, or greater than 1.50 μM and less than 375 μM), and hydrocortisone is greater than 1.250 μM and less than 300 μM (or greater than 1.250 μM and less than 300 μM, greater than 1.250 μM and less than 300 μM, or greater than 1.250 μM and less than 300 μM).

[0024] In one embodiment, the concentrations of the adrenocortical hormones contained in the medium composition are When used alone, the concentration is 50 nM or more and 375 μM or less (or 50 nM or more and less than 375 μM, or more than 50 nM and 375 μM). μM or less, or greater than 50 nM but less than 375 μM).

[0025] In one embodiment, when one or more adrenocortical hormones are used, The concentration is calculated by multiplying the concentration by the potency of glucocorticoid, and the sum of these is the concentration of one type. The equivalent concentration is defined as a corticosteroid concentration of 1.25 μM or more, and the equivalent concentration is, for example, 1.25 μM or more (or more than 1.25 μM), 1.375 μM or more (or more than 1.375 μM), 1.5 μM or more (or more than 1.5 μM), 1.625 μM or more (or more than 1.625 μM), 1.75 μM or more (or more than 1.75 μM), or 1.875 μM or more. (or greater than 1.875 μM), 2 μM or greater (or greater than 2 μM), 2.125 μM or greater (or greater than 2.125 μM) , 2.25 μM or more (or more than 2.25 μM), 2.375 μM or more (or more than 2.375 μM), 2.5 μM or more (or more than 2.5 μM), 3.75 μM or more (or more than 3.75 μM), 5 μM or more (or more than 5 μM), 6.25 μM or more (or more than 6.25 μM), 7.5 μM or more (or more than 7.5 μM), 8.75 μM or more (or is greater than 8.75 μM), greater than 10 μM (or greater than 10 μM), greater than 11.25 μM (or greater than 11.25 μM), greater than 12.5 μM (or greater than 12.5 μM), greater than 13.75 μM (or greater than 13.75 μM), greater than 15 μM (or greater than 15 μM), greater than 16.25 μM (or greater than 16.25 μM), greater than 17.5 μM (or greater than 17.5 μM), greater than 18.75 μM (or greater than 18.75 μM), greater than 20 μM (or greater than 20 μM), greater than 21.25 μM (or greater than 21.25 μM), greater than 22.5 μM (or greater than 22.5 μM), greater than 23.75 μM (or greater than 23.75 μM), and less than 300 μM (or greater than 300 μM). less than μM), less than 270 μM (or less than 270 μM), more than 240 μM or less (or less than 240 μM), 210 μM or less (or less than 210 μM), 180 μM or less (or less than 180 μM less than 150 μM, 120 μM or less (or less than 120 μM), 90 μM or less (or less than 90 μM), 60 μM or less (or less than 60 μM), 30 μM or less (or less than 30 μM). For example, when one or more corticosteroids are used, the converted concentration of the corticosteroids is The degree of or more than 1.25 μM and less than 300 μM), preferably 1.5 μM to 150 μM (or 1.5 μM or more and 150 μM less than, greater than 1.5 μM and less than 150 μM, or greater than 1.5 μM and less than 150 μM), more preferably 17.5 μM to 60 μM (or 17.5 μM or more and less than 60 μM, greater than 17.5 μM and less than 60 μM, or greater than 17.5 μM and less than 60 μM), and even more preferably 2.5 μM to 30 μM (or 2.5 μM or more and less than 30 μM, greater than 2.5 μM and less than 30 μM, or greater than 2.5 μM and less than 30 μM).

[0026] The medium composition of the present invention may contain serum. The serum is not particularly limited as long as it is derived from an animal and does not inhibit the induction of mesenchymal stem cells; however, mammalian serum (e.g., fetal bovine serum, human serum, etc.) is preferred. The serum concentration may be within a range known per se. Furthermore, when using cultured mesenchymal stem cells for medical purposes, other animal-derived components may serve as a source of infection for blood-borne pathogens or as xenoantigens, so serum-free media may also be suitably used. When serum is not contained, serum substitutes (e.g., Knockout Serum Replacement (KSR) (Invitrogen), Chemically-defined Lipid concentrated (Gibco), etc.) may be used.

[0027] When mesenchymal stem cells induced and cultured using the medium composition of the present invention are used for medical purposes such as cell therapy, there is a possibility that they may become infected with pathogenic bacteria or become xenoantigens, so it is more preferable that the medium of the present invention does not contain components derived from non-human animals.

[0028] "Stem cells" refer to immature cells that have the ability to self-renew and differentiate / proliferate. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation potential. Pluripotent stem cells are cells that have the ability to differentiate into all tissues and cells that make up a living organism. Multipotent stem cells are cells that have the ability to differentiate into multiple types of tissues and cells, but not all types. Unipotent stem cells are cells that have the ability to differentiate into specific tissues and cells.

[0029] The mesenchymal stem cells of interest in the present invention are a type of multipotent stem cell that can differentiate into adipocytes, bone cells, chondrocytes, muscle cells, hepatocytes, nerve cells, etc., and are known to be cells with a low probability of forming tumors when transplanted into a living body. The mesenchymal stem cells of the present invention are preferably positive for one or more mesenchymal stem cell markers (e.g., CD90, CD44, CD73, CD105, etc.). More preferably, they may be positive for the marker and negative for the expression of molecules not expressed in mesenchymal stem cells, such as CD34, CD45, CD14, CD11b, CD79, CD19, and HLA-DR.

[0030] The medium composition of the present invention can be suitably used for inducing mesenchymal stem cells derived from any animal. Mesenchymal stem cells that can be induced and cultured using the medium composition of the present invention include mesenchymal stem cells derived from rodents such as mice, rats, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, horses, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees, and are preferably derived from humans.

[0031] The medium composition of the present invention contains factors that induce differentiation of mesenchymal stem cells (e.g., glucocorticoids, factors called transforming growth factor-β family, bone morphogenesis factors, etc.). Protein (preferably BMP-2 or BMP-4), basic fibroblast growth factor (bFGF), insulin The medium may contain hibin A or chondrogenesis-stimulating activator (CSA), a collagenous extracellular matrix such as type I collagen (especially in gel form), and a vitamin A analogue such as retinoic acid, preferably at concentrations that do not induce differentiation of mesenchymal stem cells (e.g., differentiation into cartilage). In one embodiment, the medium may be free of factors that induce differentiation of mesenchymal stem cells other than corticosteroids. In one embodiment, the medium may be free of factors that induce differentiation of mesenchymal stem cells other than dexamethasone.

[0032] The medium composition of the present invention may contain a scaffold component. Examples of the scaffold component include laminin [laminin]. Examples of scaffold components include laminin α5β1γ1 (hereinafter referred to as laminin 511), laminin α1β1γ1 (hereinafter referred to as laminin 111), and laminin fragments (such as laminin 511E8), as well as extracellular matrices such as entactin, fibronectin, gelatin, vitronectin, Synthemax (Corning), and Matrigel, with laminin 511 being preferred. These scaffold components are generally concentrated at 0.001 μg / ml to 1000 μg / ml, preferably 0.01 μg / ml to 100 μg / ml, and more preferably 0.1 μg / ml to 10 μg / ml, more preferably 0.1 μg / ml to 1 μg / ml, most preferably 0.2 μg / ml Add to the medium.

[0033] The medium composition of the present invention is used to induce differentiation of neural crest cells into mesenchymal stem cells. "Neural crest cells (also referred to as "NCCs")" refer to cells that de-epithelialize from the neural crest, a structure that temporarily forms between the epidermal ectoderm and the neural plate during early vertebrate development, and are then induced to various sites within the embryonic body after transition from epithelium to mesenchyme. As used herein, the term "neural crest cells" includes not only cells collected from a living body, but also neural crest cells derived from pluripotent stem cells and their subcultured cells. In the present invention, the origin of neural crest cells is not particularly limited and they may be from any vertebrate, but neural crest cells derived from mammals are preferred. Examples of such mammals include, but are not limited to, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans. Humans are preferred.

[0034] Neural crest cells may be derived from a living organism, and can be produced, for example, from neural crest-derived tissues in a living organism (e.g., bone marrow, dorsal root ganglia, heart, cornea, iris, dental pulp, and olfactory mucosa). Alternatively, neural crest cells derived from pluripotent stem cells can also be suitably used. Methods known per se can be used to obtain neural crest cells from pluripotent stem cells, and one example is a method in which pluripotent stem cells are cultured in a culture medium containing a TGFβ inhibitor and a GSK-3β inhibitor, followed by differentiation induction. Thus, by using methods known per se, neural crest cells, more specifically, cell populations containing neural crest cells, can be easily obtained.

[0035] Whether or not the cell population thus obtained contains neural crest cells can be determined by confirming the expression of one or more neural crest cell-specific marker genes, such as TFAP2a, SOX9, SOX10, TWISTI, and PAX3, by a method known per se. Furthermore, neural crest cell-specific marker genes such as CD271 protein (also called "p75(NTR)") can also be detected. In the present invention, neural crest cells are preferably derived from pluripotent stem cells, and more preferably from iPS cells. It is more preferable that:

[0036] In the present invention, pluripotent stem cells include any cells that have pluripotency, which allows them to differentiate into many cells present in the body, and that can be induced into intermediate mesodermal cells that also have proliferation ability. Pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES) cells, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, pluripotent cells derived from cultured fibroblasts and bone marrow stem cells. (Muse cells), etc. Preferred pluripotent stem cells are iPS cells, more preferably human iPS cells, from the viewpoint that they can be obtained without destroying embryos, eggs, etc. in the production process.

[0037] Methods for producing iPS cells are known in the art, and involve introducing reprogramming factors into any somatic cell. Here, examples of reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc. Examples of reprogramming factors include genes and gene products, and these reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D,et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat. Cell Biol. 11:197-203, RL Judson et al., (2009), Nat. Biotechnol., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et. al. (2011), Nature. 474:225-9.

[0038] Somatic cells include, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specifically, somatic cells include, for example, (1) neural stem cells, (1) Tissue stem cells (somatic stem cells) such as blood stem cells, mesenchymal stem cells, and dental pulp stem cells; (2) Tissue precursor cells (3) Blood cells (peripheral blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells Examples include differentiated cells such as meat cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0039] The mammal from which the somatic cells are collected is not particularly limited, but is preferably a human.

[0040] The present invention provides a differentiation promoter for mesenchymal stem cells, which contains adrenocortical hormone. By culturing neural crest cells in a medium containing the differentiation promoter, it is possible to increase the efficiency of inducing mesenchymal stem cells.

[0041] The differentiation promoter of the present invention may consist solely of adrenocortical hormones, but it can also be provided as a composition further containing a physiologically acceptable carrier (e.g., a physiological isotonic solution (e.g., physiological saline, the above-mentioned basal medium, an isotonic solution containing glucose or other auxiliary agents (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.)), excipient, preservative, stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), binder, solubilizer, non-ionic surfactant, buffer (e.g., phosphate buffer, sodium acetate buffer), preservative, antioxidant, the above-mentioned additives, etc.).

[0042] The content of the adrenocortical hormone contained in the differentiation promoter of the present invention is preferably configured so that when the differentiation promoter of the present invention is used by adding it to a culture medium, the concentration of the adrenocortical hormone in the culture medium is sufficient to promote the induction of mesenchymal stem cells.

[0043] The differentiation promoter of the present invention is used by adding it to a culture medium in the form of an isotonic aqueous solution or powder.

[0044] As described above, the medium composition of the present invention can be produced by adding a corticosteroid to a basal medium, but it can also be used in the form of a kit containing the corticosteroid and the basal medium. That is, the corticosteroid and the basal medium are separately combined and supplied in the form of a kit, and the user can prepare and use the medium composition of the present invention by adding the corticosteroid to the basal medium at the time of use.

[0045] In the kit, the components constituting the basal medium and other components may be provided separately. Both components may be the same or different and may be liquid or powder, and each component may be provided separately or several components may be provided in a mixed state. Furthermore, the kit does not necessarily contain all of the components of the medium composition of the present invention, and components that are very easily available, such as water, may be omitted. If the components are in powder form, they can be dissolved in a buffer solution or the like before use, as desired.

[0046] 2.Mesenchymal stem cell production method The present invention provides a method for producing mesenchymal stem cells (hereinafter also referred to as the production method of the present invention), which includes a step (step 1) of inducing mesenchymal stem cells by culturing neural crest cells in the medium composition of the present invention. In the production method of the present invention, differentiation into mesenchymal stem cells is induced by culturing neural crest cells in a medium composition containing a basal medium and a corticosteroid. The production method of the present invention makes it possible to efficiently produce mesenchymal stem cells. Culturing neural crest cells in a medium containing a corticosteroid makes it possible to efficiently differentiate neural crest cells into cells that retain the ability of mesenchymal stem cells.

[0047] The culture in step 1 may be suspension culture, adhesion culture, or a combination thereof. In the present invention, "suspension culture" refers to culturing while maintaining a state in which cells (or cell aggregates) are suspended in a culture solution. "Adhesion culture" refers to culturing carried out under conditions that allow cells (or cell aggregates) to adhere to culture equipment, etc. In this case, cell adhesion means that strong cell-substratum junctions are formed between the cells or cell aggregates and the culture equipment. More specifically, suspension culture refers to culture under conditions that do not allow strong cell-substrate bonds to be formed between cells or cell aggregates and cultureware, etc., and adhesion culture refers to culture under conditions that do not allow strong cell-substrate bonds to be formed between cells or cell aggregates and cultureware, etc. The cell aggregates are cultured under conditions that allow strong cell-substrate bonds to form between the cell aggregates and the cultureware. say.

[0048] The culture vessel used for suspension culture is not particularly limited as long as it is capable of suspension culture, and can be appropriately determined by a person skilled in the art. Examples of such culture vessels include flasks, tissue culture flasks, culture dishes (dishes), Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, Erlenmeyer flasks, spinner flasks, and roller bottles. These culture vessels are preferably non-cell-adhesive to enable suspension culture. Non-cell-adhesive culture vessels include those whose surface has been artificially treated to reduce adhesion to cells (for example, ultra-hydrophilic treatment such as MPC polymer, low protein adsorption treatment, etc.). Rotational culture may be performed using spinner flasks, roller bottles, etc. The culture surface of the incubator may be flat or uneven.

[0049] The culture vessel used for adherent culture is not particularly limited as long as it is capable of supporting adherent culture. Those skilled in the art can select the appropriate culture vessel depending on the scale, conditions, and duration of culture. Examples of such culture vessels include flasks, tissue culture flasks, culture dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, microcarriers, beads, stack plates, spinner flasks, and roller bottles. These culture vessels are preferably cell-adhesive to enable adherent culture. Examples of cell-adhesive culture vessels include those whose surfaces have been artificially treated to improve cell adhesion, specifically surface-treated culture vessels or those whose interiors are coated with a coating agent. Surface-treated culture vessels include culture vessels with surface treatments such as positive charge treatment. Examples of coating agents include laminin [laminin α5β1γ1 (hereinafter referred to as laminin 511), laminin α1β1γ1 (hereinafter referred to as laminin 111), etc.] and laminin Examples of the extracellular matrix include extracellular matrices such as entactin, collagen, fibronectin, gelatin, vitronectin, Synthemax (Corning), and Matrigel, as well as polymers such as polylysine and polyornithine.

[0050] Neural crest cells that can be used in step 1 include: 1. Medium Composition As described in 2. above, neural crest cells may be derived from either a living organism or pluripotent stem cells. When neural crest cells derived from pluripotent stem cells are used, cells immediately after differentiation into neural crest cells may be used, or, as described below, cells that have been further purified or that have undergone purification and expansion culture may be used. Here, purification refers to increasing the proportion of a particular type of cell (eg, neural crest cells) in a cell population (eg, a cell population containing neural crest cells). In one embodiment, the concentration of neural crest cells to be cultured is about 1×10 2 ~Approx. 1×107 cells / cm 2 , preferred Or about 3 x 10 2 ~Approx. 5×10 6 cells / cm 2 , more preferably about 4×10 2 ~about 2×10 5 cells / cm 2 , more preferably about 4×10 2 ~Approx. 1×10 5 cells / cm 2 , and even more preferably about 3×10 3 ~Approx. 1×10 4 cells / cm 2 It can be said that: In other embodiments, the concentration of neural crest cells cultured is about 1 x 10 2 ~Approx. 1×10 7 cells / cm 2 , good Preferably about 3 x 10 2 ~Approx. 5×10 6 cells / cm 2 , more preferably about 4×10 2 ~about 2×10 5 cells / cm 2 , more preferably about 4×10 2 ~Approx. 1×10 5 cells / cm 2 , and even more preferably about 1.5×10 4 ~Approx. 3×10 4 cells / cm 2 It can be said that:

[0051] Culture conditions such as culture temperature and CO2 concentration can be set appropriately. The culture temperature is, for example, about 30° C. to about 40° C., preferably about 37° C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0052] Examples of known markers for mesenchymal stem cells include CD90, CD44, CD73, and CD105. Therefore, in the cell population of mesenchymal stem cells obtained by the above method, the majority of the cells (for example, 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 100% of the cells in the cell composition) express any one of CD90, CD44, CD73, and CD105, preferably Preferably, the cell composition of the present invention is positive for any combination of two markers, more preferably a combination of three markers, and most preferably all markers. Furthermore, it is more preferable that the cell composition of the present invention does not express molecules that are not expressed in mesenchymal stem cells. Examples of molecules that are not expressed in mesenchymal stem cells include CD34 (expressed in hematopoietic stem cells), CD45 (expressed in hematopoietic stem cells), CD14 (expressed in monocytes and macrophages), and CD11b (expressed in monocytes and macrophages). CD79 (expressed on leukocytes, macrophages, NK cells, and granulocytes), CD81 (expressed on B cells), Examples of such molecules include CD19 (expressed in B cells), CD19 (expressed in B cells), and HLA-DR (expressed in dendritic cells, B cells, monocytes, and macrophages). Thus, in a preferred embodiment, the majority of the cells in the cell composition of the present invention (for example, 60%, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, and most preferably 100% of the cells in the cell composition) are negative for the expression of molecules not found to be expressed in mesenchymal stem cells.

[0053] In one embodiment, step 1 is preceded by the following step: (Step A) obtaining a cell population containing neural crest cells; and (Step B) A step of expanding and culturing the cell population obtained in step A using an extracellular matrix as a scaffold may be carried out.

[0054] In step A, 1. Medium Composition As described in A cell population containing neural crest cells can be obtained. Generally, the proportion of neural crest cells in a cell population containing neural crest cells can vary greatly depending on the tissue collected and the differentiation induction conditions. In one embodiment of the present invention, the proportion of neural crest cells in a cell population containing neural crest cells can be, for example, but is not limited to, 1 to 95%, 1 to 90%, 1 to 85%, 1 to 80%, 1 to 75%, 1 to 70%, 1 to 65%, 1 to 60%, 1 to 55%, 1 to 50%, 1 to 45%, 1 to 40%, 1 to 35%, 1 to 30%, 1 to 25%, 1 to 20%, 1 to 15%, or 1 to 10%. In another embodiment, the proportion of neural crest cells in a cell population containing neural crest cells may be, for example, 25 to 95%, 25 to 90%, 25 to 85%, 25 to 80%, 25 to 75%, 25 to 70%, 25 to 65%, 25 to 60%, 25 to 55%, 25 to 50%, 25 to 45%, 25 to 40%, or 25 to 35%. In another embodiment, the percentage of neural crest cells in a cell population containing neural crest cells can be, for example, but is not limited to, 50 to 95%, 50 to 90%, 50 to 85%, 50 to 80%, 50 to 75%, 50 to 70%, 50 to 65%, or 50 to 60%. In another embodiment, the percentage of neural crest cells in a cell population containing neural crest cells can be, for example, but is not limited to, 70% or more, 75 to 95%, 75 to 90%, or 75 to 85%.

[0055] The neural crest cell-containing cell population obtained in step A is then purified to produce neural crest cells. The purified neural crest cells may be subjected to a step of culture expansion, or the purified neural crest cells may be subjected to a step of culture expansion. In other words, step B can also be called a step of purifying and culturing neural crest cells. As used herein, "expansion culture" refers to a culture that maintains and / or grows desired cells. The concept encompasses the above, and preferably it can be a culture for growing desired cells.

[0056] Methods for purifying neural crest cells are not particularly limited, and examples include, but are not limited to, single-cell isolation, sorting using a cell sorter with a fluorescently labeled neural crest cell-specific antibody, sorting using magnetic beads to which the antibody is bound, and sorting using an affinity column to which the antibody is immobilized.

[0057] In the single cell purification method, cells may be mechanically dispersed as needed. The cells may be processed to a single cell state by treatment with erythrocytes, dispersion treatment with enzymes such as collagenase or trypsin, and / or dispersion treatment with a chelating agent such as EDTA. In this case, a ROCK inhibitor may be added to suppress cell death. The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho-kinase (ROCK), and examples thereof include Y-27632, Fasudil / HA1077, H-1152, Wf-536, and derivatives thereof. Other known small molecule compounds can also be used as ROCK inhibitors (see, for example, U.S. Patent Application Publication Nos. 2005 / 0209261, 2005 / 0192304, 2004 / 0014755, 2004 / 0002508, 2004 / 0002507, 2003 / 0125344, 2003 / 0087919, and International Publication Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796). In the method of purification by sorting treatment, for example, a CD271-specific antibody can be used as the neural crest cell-specific antibody.

[0058] In the expansion culture in step B, culture conditions suitable for the maintenance and / or proliferation of neural crest cells can be used.

[0059] The culture conditions for expanding neural crest cells are not particularly limited as long as they can be expanded, and any culture conditions known per se can be used. For example, the conditions include culturing neural crest cells in a culture medium containing a TGFβ inhibitor, EGF (epidermal growth factor), and FGF2 (fibroblast growth factor 2). An example of such a method is to culture the cells in the presence of a .

[0060] The medium used for the expansion culture of neural crest cells can be prepared using a medium used for culturing animal cells as a basal medium. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, and Dulbecco's modified Eagle's Examples of suitable medium include DMEM medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (Invitrogen), RPMI-base medium, StemFit (registered trademark) AK03N medium, and mixtures thereof. In this step, StemFit (registered trademark) medium is preferably used. The medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as albumin, transferrin, Knockout Serum Replacement (KSR) (a serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol (2ME), thioglycerol, or may contain lipids, amino acids, or the like. One or more substances such as acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts It may also contain

[0061] In the present invention, a TGFβ inhibitor is a substance that inhibits signal transduction that continues from the binding of TGFβ to its receptor to SMAD, and is not particularly limited as long as it is a substance that inhibits binding to the ALK family receptor or a substance that inhibits phosphorylation of SMAD by the ALK family. In the present invention, examples of TGFβ inhibitors include Lefty-1 (NCBI Accession No. NM_010094 for mouse and NM_020997 for human), SB431542, SB202190 (RK Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A-83-01 (WO 2009 / 146408), and derivatives thereof. The TGFβ inhibitor used for neural crest cell expansion may preferably be SB431542.

[0062] The concentration of TGFβ inhibitors such as SB431542 in the culture medium is the concentration that inhibits ALK5. Although not particularly limited, 1 nM to 50 μM is preferable, and examples thereof include, but are not limited to, 1 nM, 10 nM, 50 nM, 100 nM, 500 nM, 750 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, and 50 μM. More preferably, it is 10 μM.

[0063] The concentration of EGF in the medium is preferably 1 ng / ml to 100 ng / ml, for example, but not limited to, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, and 100 ng / ml, and more preferably 20 ng / ml.

[0064] The concentration of FGF2 in the medium is preferably 1 ng / ml to 100 ng / ml, for example, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, and 100 ng / ml, but is not limited to these. More preferably, 20 ng / ml is.

[0065] Furthermore, components other than those mentioned above can also be added to the medium, as long as expansion of neural crest cells can be achieved.

[0066] The expansion culture of neural crest cells in step B may be either adhesion culture or suspension culture. However, adherent culture is preferred.

[0067] In step B, neural crest cells are purified using an extracellular matrix as a scaffold. It is characterized by being used in this way.

[0068] As used herein, the term "scaffold" refers to (i) a cell-adhesive culture vessel, its material, or a substance present on the surface of the cell-adhesive culture vessel and constituting an adhesion site, and / or (ii) a substance dissolved, dispersed, or suspended in a cell culture medium that forms a three-dimensional network in the cell culture medium, to which cells can adhere. Such cell-adhesive culture vessels include those whose surfaces have been artificially treated to improve cell adhesion, specifically surface-treated culture vessels or culture vessels whose interiors are coated with a coating agent. Surface-treated culture vessels include culture vessels whose surfaces have been treated with a positive charge or the like. Such scaffolds also include substances that are coated on the surface of culture vessels, such as laminin (including laminin α5β1γ1 (laminin 511), laminin α2β1γ1 (laminin 211), laminin α1β1γ1 (laminin 111), and laminin fragments (laminin 511E8, etc.)), entactin, collagen, gelatin, vitronectin, Synthemax (Corning), and extracellular matrices such as Matrigel, and polymers such as polylysine and polyornithine. Such substances that form a three-dimensional network in a cell culture medium exhibit the effect of uniformly suspending cells and / or tissues in the liquid medium. More specifically, examples of nanofibers contained in the medium composition of the present invention include nanofibers formed in a liquid medium by aggregation and self-organization of low-molecular-weight compounds or high-molecular-weight compounds via covalent bonds, ionic bonds, electrostatic interactions, hydrophobic interactions, van der Waals forces, etc., or nanofibers obtained by micronizing relatively large fibrous structures made of high-molecular-weight compounds by high-pressure treatment, etc. Without being bound by theory, in the medium composition of the present invention, the nanofibers form a three-dimensional network that supports the cells and tissues, thereby maintaining the suspended state of the cells and tissues.

[0069] In step B, extracellular matrix is ​​used as a scaffold to achieve neural crest cell purification. The fibronectin may be laminin or fibronectin, and in particular the laminin may be full length laminin, laminin with an α2 chain, or laminin 211.

[0070] Laminin is a glycoprotein that is a major component of basement membranes. It is involved in cell adhesion. Laminin is known to be involved in various cellular functions such as cell proliferation, metastasis, and differentiation. Laminin is composed of a heterotrimer having one each of α, β, and γ subunit chains. Currently, five types of α subunit chains (α1, α2, α3, α4, α5), three types of β subunit chains (β1, β2, β3), and three types of γ subunit chains (γ1, γ2, γ3) are known to exist, and 15 types of laminin isoforms have been confirmed in humans depending on the combination of these subunit chains. Laminin 211 that can be used in step B is α2 It can be laminin composed of subunit chains of the β1 chain, β1 chain, and γ1 chain. The origin of laminin is preferably the same as that of the organism from which neural crest cells are derived (for example, when using human-derived neural crest cells, it is preferable to use human-derived laminin 211). It is known that a laminin E8 fragment consisting only of the integrin-binding site has stronger cell adhesion activity than full-length laminin (see Miyazaki T. et al., Nat Commun. 2012;3:1236). However, the laminin-211 that can be used in step B may be the full-length laminin-211 protein, rather than a fragment. Laminin-211 may be prepared using known genetic recombination techniques or may be commercially available. Commercially available products may also be used.

[0071] In step B, when the cell population is expanded in suspension culture, for example, laminin 211 may be added to the medium, and the floating cells may use this as a scaffold to form aggregates, thereby enabling three-dimensional suspension culture. Suspension culture can be performed by a method known per se. One example is a method in which the cell population is expanded in suspension culture in a medium containing laminin 211 while stirring the medium using a spinner flask or the like. Alternatively, laminin 211 can be used in combination with polysaccharides (e.g., methylcellulose, xanthan gum, gellan gum, etc.) that have the effect of suspending cells when added to the culture medium. Alternatively, cell populations can be expanded in suspension culture. In this embodiment, cells are dispersed in a three-dimensional spread and proliferate attached to the nanofibers or in the form of spheres. The cells adhere to the nanofibers and proliferate strongly, using them as a scaffold. As a result, the proliferated cells and cell aggregates (e.g., spheres) are strung together on the nanofibers in a grape-like cluster-like fashion. This enables cell suspension culture. The concentration of laminin to be added can be appropriately determined taking into consideration various conditions, such as the seeding density of the cell population and the concentration of the polysaccharide used in combination. In this specification, suspension culture refers to a culture method in which cells do not adhere to the surface of the culture vessel. Suspension culture may or may not involve physical agitation. Furthermore, the cultured cells may be uniformly or non-uniformly dispersed in the medium.

[0072] In step B, when the cell population is expanded by adhesion culture, for example, laminin 211 is coated on the surface of the culture vessel. The amount of laminin 211 to be coated is determined by the method of the present invention. There are no particular limitations on the amount of coating, as long as the desired effect is obtained, and a commonly recommended amount of coating may be used. For example, the amount of laminin 211 to be coated is 0.1 ng / cm 2 ~1000 ng / cm 2 , preferably 0.5 ng / cm 2 ~500 ng / cm 2 , more preferably 1 ng / cm 2 ~250 ng / cm 2 , more preferably 2 ng / cm 2 ~100 ng / cm 2 However, the present invention is not limited to these.

[0073] The culture period in step B may vary depending on the culture conditions, culture method, and the number of neural crest cells contained in the cell population. Although the culture period may vary depending on factors such as the proportion of cells, purification of neural crest cells can be achieved in a relatively short period of time. Examples of culture periods include, but are not limited to, 1 to 21 days, 1 to 20 days, 1 to 19 days, 1 to 18 days, 1 to 17 days, 1 to 16 days, 1 to 15 days, 1 to 14 days, 1 to 13 days, 1 to 12 days, 1 to 11 days, 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, or 1 to 3 days.

[0074] The culture temperature in step B is not particularly limited as long as neural crest cells can be cultured, but is 30 to 40°C, preferably about 37°C. There are no particular limitations as long as the medullary crest cells can be cultured, but the concentration is 2 to 5%, preferably about 5%.

[0075] In one embodiment, step 1 is preceded by the following step: (Step A) obtaining a cell population containing neural crest cells; and (Step C) A step of expanding and culturing the cell population obtained in step A using fibronectin as a scaffold is carried out.

[0076] In step A, 1. Medium Composition Neural crest cells can be obtained as described in Neural crest cells can be generated in the cell population used for production. The proportion of neural crest cells in a cell population containing neural crest cells can vary greatly depending on the tissue collected and the differentiation induction conditions. In one embodiment of the present invention, the proportion of neural crest cells in a cell population containing neural crest cells can be, for example, but is not limited to, 1 to 95%, 1 to 90%, 1 to 85%, 1 to 80%, 1 to 75%, 1 to 70%, 1 to 65%, 1 to 60%, 1 to 55%, 1 to 50%, 1 to 45%, 1 to 40%, 1 to 35%, 1 to 30%, 1 to 25%, 1 to 20%, 1 to 15%, or 1 to 10%. In another embodiment, the percentage of neural crest cells in a cell population containing neural crest cells can be, for example, but is not limited to, 25-95%, 25-90%, 25-85%, 25-80%, 25-75%, 25-70%, 25-65%, 25-60%, 25-55%, 25-50%, 25-45%, 25-40%, or 25-35%. In another embodiment, the percentage of neural crest cells in a cell population containing neural crest cells can be, for example, but is not limited to, 50-95%, 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, or 50-60%. In another aspect, the proportion of neural crest cells in a cell population containing neural crest cells may be 60% or more, 70% or more, or 80% or more, for example, 75-95%, 75-90%, or 75-85%, but is not limited to these.

[0077] In one embodiment, the cells are passaged as appropriate during step 1. Passage is performed, for example, every 2 to 8 days or every 3 to 7 days after seeding. The passage interval is preferably a period sufficient for the expansion of the cell aggregates, but shorter than the period during which the cell aggregates become too large and oxygen and nutrients become less likely to reach the cells inside the cell aggregates.

[0078] The number of passages during step 1 may be, for example, 0 to 20 (0, 1, 2, 3, 4, 5). Examples of the number of times the cell population is passaged include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 times, preferably 1 to 10 times, and more preferably 2 to 8 times. After an appropriate number of passages, a portion of the cell population may be preserved as a frozen stock.

[0079] In one embodiment, the cells are passaged as appropriate during step B or C. Passage is performed, for example, every 2 to 8 days or every 3 to 7 days after seeding. The passage interval is preferably a period sufficient for the expansion of the cell aggregates, but shorter than the period during which the cell aggregates become too large and oxygen and nutrients become less likely to reach the cells inside the cell aggregates.

[0080] The number of passages between steps B and C can be, for example, 2 to 8 times (2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times), preferably 3 to 7 times, more preferably 4 to 6 times. After passaging, a portion of the cell population may be stored as a frozen stock.

[0081] The transition from step B or C to step 1 is carried out by replacing the culture medium with the medium composition of the present invention. The replacement may be performed after an appropriate number of passages, or after the final passage of step C. It may be during the passage. For example, it may be 6 hours before the end of the last passage in step B or C. can be done.

[0082] In one aspect, the present invention provides a method for producing mesenchymal stem cells from a cell population containing neural crest cells, comprising: the neural crest cells are cells induced to differentiate from pluripotent stem cells, the cell population is a cell population containing 70% or more of the neural crest cells, The cell population was treated with adrenal glandular steroids at a concentration of 1.25 μM or more and 300 μM or less (1.25 μM to 300 μM, or 1.25 μM or more and less than 300 μM, more than 1.25 μM and 300 μM or less, or more than 1.25 μM and less than 300 μM). Culturing in the presence of a cortical hormone, A method for producing mesenchymal stem cells from a cell population containing neural crest cells is provided.

[0083] 3. Mesenchymal stem cells As described above, mesenchymal stem cells are produced by culturing neural crest cells in a medium composition containing adrenocortical hormone. The present invention also provides mesenchymal stem cells thus produced and cultures thereof (hereinafter also referred to as mesenchymal stem cells of the present invention and cultures thereof). The term "culture" refers to the result obtained by culturing cells / cell populations using the production method of the present invention, and includes cells, medium, and, in some cases, cell-secreted components, etc.

[0084] 4. Method for producing chondrocytes. The present invention provides a method for producing chondrocytes, which comprises the following steps: (Step D) A step of obtaining a cell population containing mesenchymal stem cells by the method for producing mesenchymal stem cells of the present invention. The course, (Step E) culturing the cell population containing mesenchymal stem cells in a cartilage induction medium to produce a cell population containing chondrocytes. Obtaining a cell population.

[0085] Chondrocytes refer to cells that produce extracellular matrix components that make up cartilage, such as collagen, or precursor cells that become such cells. In this application, the differentiation characteristics of chondrocytes are confirmed by Alcian blue staining. Alcian blue is a basic dye that belongs to the phthalocyanine dye family and stains acidic polysaccharides such as sialylated mucosubstances (sialomucins), mucosubstances with sulfate groups (sulfomucins), chondroitin sulfate contained in cartilage and fibrous connective tissue, and acidic mucopolysaccharides such as hyaluronic acid.

[0086] Step D can be carried out as described above in the method for producing mesenchymal stem cells of the present invention.

[0087] The medium used in step E is a medium used for culturing mammalian cells as a basal medium. The basal medium is not particularly limited as long as it can be used to culture mammalian cells, and examples thereof include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, α MEM medium, DMEM medium, Ham's medium, Ham's F-12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof.

[0088] In one embodiment, the medium used in step E is a serum-free medium. The term "serum-free medium" refers to a medium that does not contain unconditioned or unpurified serum. Media containing purified blood-derived components or animal tissue-derived components are also considered to be serum-free media.

[0089] The medium used in step E may contain a serum substitute. For example, the serum substitute may contain albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679. Furthermore, to more easily carry out the production method of the present invention, commercially available serum substitutes can be used. Examples of such commercially available serum substitutes include KSR (knockout serum replacement) (Invitrogen), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).

[0090] The medium used in step E is a medium that does not impair the directed differentiation of mesenchymal stem cells into chondrocytes. Other additives may be included to the extent that they are not included in the formulation. Examples of additives include insulin, Examples of suitable anti-inflammatory agents include, but are not limited to, iron sources (e.g., transferrin, etc.), minerals (e.g., sodium selenate, etc.), sugars (e.g., glucose, etc.), organic acids (e.g., pyruvic acid, lactic acid, etc.), serum proteins (e.g., albumin, etc.), amino acids (e.g., L-glutamine, etc.), reducing agents (e.g., 2-mercaptoethanol, etc.), vitamins (e.g., ascorbic acid, d-biotin, etc.), antibiotics (e.g., streptomycin, penicillin, gentamicin, etc.), and buffering agents (e.g., HEPES, etc.). The medium used in step E contains a variety of substances, such as tumor necrosis factor-α (TNF-α), IL-1β, and IL-17. It is preferable that the composition does not contain any substance that inhibits the differentiation of mesenchymal stem cells into chondrocytes.

[0091] In step E, the incubator used for cell culture is one that can culture cells. Examples of suitable containers include, but are not limited to, flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, and roller bottles.

[0092] In step E, the culture vessel used for culturing the cells is preferably cell-adhesive. As long as the differentiation of mesenchymal stem cells into chondrocytes is not impaired, the cell-adhesive culture vessel may be hydrophilic for the purpose of improving the adhesion of cells to the surface of the culture vessel, and may be made of any cell-supporting substrate such as an extracellular matrix (ECM) or any of its derivatives. They may be coated with artificial materials that mimic the function of the As long as the differentiation of mesenchymal stem cells into chondrocytes is not impaired, the ECM coating the culture vessel may include, but is not limited to, fibronectin, collagen, etc. Fibronectin is preferred.

[0093] In step E, the mesenchymal stem cells are cultured by a method known per se, such as adhesion culture, suspension culture, or tissue culture. The cells can be cultured by any method, but adhesion culture is preferred.

[0094] Other culture conditions can be set appropriately. For example, the culture temperature is not particularly limited as long as the desired effect can be achieved, but is about 30 to 40°C, preferably about 37°C. The CO2 concentration is about 1 to 10%, preferably about 2 to 5%. The oxygen concentration is usually 1 to 40%, but is selected appropriately depending on the culture conditions, etc.

[0095] The mesenchymal stem cells of the present invention are characterized by their high ability to differentiate into chondrocytes. "High ability to differentiate into chondrocytes" means that the number of chondrocytes produced as a result of inducing differentiation of the mesenchymal stem cells of the present invention into chondrocytes is greater than the number of chondrocytes produced as a result of inducing differentiation of other mesenchymal stem cells, specifically mesenchymal stem cells derived from living organisms, particularly bone marrow-derived mesenchymal stem cells. More specifically, "high ability to differentiate into chondrocytes" means that the number of chondrocytes produced as a result of inducing differentiation of other mesenchymal stem cells under similar (preferably identical) culture conditions in a chondrocyte differentiation-inducing medium is 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, or 3.5 times greater than the number of chondrocytes produced as a result of inducing differentiation of other mesenchymal stem cells under similar (preferably identical) culture conditions. 4x or more, 4.5x or more, 5x or more, 6x or more, 7x or more, 8x or more, 9x or more, 10x or more , 50 times or more, or 100 times or more higher.

[0096] 5. Drugs for repairing cartilage tissue Furthermore, the present invention provides a drug for repairing cartilage tissue, which contains the chondrocytes of the present invention or a culture thereof. The term "culture" refers to the result obtained by culturing cells / cell populations in the production method of the present invention, and includes cells, medium, and, in some cases, cell-secreted components, etc.

[0097] Cartilage tissue is a connective tissue composed of cartilage matrix and chondrocytes. Depending on the properties of the cartilage matrix, cartilage can be divided into hyaline cartilage (articular cartilage, epiphyseal plate, costal cartilage, tracheal cartilage, laryngeal cartilage, etc.), Cartilage is classified into fibrocartilage (sacroiliac joint, temporomandibular joint, sternoclavicular joint, intervertebral disc, pubic symphysis, meniscus, articular disc, etc.) and elastic cartilage (external auditory canal, Eustachian tube, auricular cartilage, epiglottis cartilage, etc.) The agent of the present invention can be useful in repairing hyaline cartilage, fibrocartilage, and elastic cartilage.

[0098] In this specification, the term "agent for repairing cartilage tissue" refers to an agent used for repairing (regenerating) cartilage tissue. When the agent for repairing cartilage tissue of the present invention is administered to a site in a living body where cartilage tissue repair (regeneration) is desired, cartilage tissue is locally formed at the administration site, thereby achieving repair (regeneration) of cartilage tissue.

[0099] The chondrocytes obtained by the present invention can themselves be used as transplant materials, and therefore can be transplanted into patients as a cell preparation.

[0100] Furthermore, the mesenchymal cells obtained by the present invention can be cultured on a substrate (scaffold) made of an artificial material such as biodegradable fiber to induce differentiation into chondrocytes, forming a transplant material that can then be transplanted.

[0101] The amount of cells obtained in step 1 seeded per unit surface area of ​​the cell contact surface of the scaffold is, for example, 1 × 10 4 ~1×10 7 cells / cm 2 , 2 × 10 4 ~5×10 5 cells / cm 2 , 5×10 4 ~2×10 5 pieces / cm 2 It could be.

[0102] The culture time of the cells in the presence of the scaffold in step E is usually 6 hours or more, preferably 12 hours or more, more preferably 18 hours or more. The upper limit of the culture time is not particularly limited, but is usually 72 hours or less, preferably 48 hours or less, more preferably 30 hours or less.

[0103] The drug for repairing cartilage tissue of the present invention may further contain other compounds, such as antibiotics, anti-inflammatory agents, immunosuppressants, cytokines, preservatives, analgesics, stabilizers (antioxidants, UV absorbers, heat stabilizers, etc.), other therapeutic agents, etc.

[0104] The cartilage repair agent of the present invention is transplanted by a medical professional according to an appropriate transplantation method in accordance with guidelines. For example, when transplanting the cartilage repair agent of the present invention into a knee joint, an incision is made in the transplantation site of the joint with a scalpel or the like, the joint cavity is opened to form a cavity, and the cartilage repair agent of the present invention is administered into the joint cavity for transplantation.

[0105] When a therapeutically effective amount of the drug for repairing cartilage tissue of the present invention is transplanted into a site requiring cartilage tissue repair, the transplanted cells differentiate into chondrocytes, and the chondrocytes and the cartilage matrix produced by the chondrocytes complement the defective site, thereby repairing the cartilage and achieving a therapeutic effect.

[0106] The agent for repairing cartilage tissue of the present invention can be administered directly to the site requiring cartilage tissue repair. For example, when the site requiring cartilage tissue repair is articular cartilage, the agent of the present invention can be administered directly to the joint cavity of the articular cartilage.

[0107] The agent for repairing cartilage tissue of the present invention is suitable as a transplant material for treating cartilage damage. In this specification, cartilage damage is used to mean physical cartilage loss / degeneration / damage caused by sports, accidents, etc., as well as cartilage loss / degeneration / damage caused by diseases such as rheumatoid arthritis, knee osteoarthritis, hip osteoarthritis, osteosarcoma, femoral head necrosis, acetabular dysplasia, meniscus injury, and traumatic arthritis.

[0108] Examples of sites requiring cartilage tissue repair include articular cartilage, epiphyseal plates, costal cartilage, tracheal cartilage, laryngeal cartilage, sacroiliac joints, temporomandibular joints, sternoclavicular joints, intervertebral discs, pubic symphysis, menisci, articular discs, external auditory canals, Eustachian tubes, auricular cartilage, and epiglottis cartilage, which have defects, degeneration, or damage to cartilage tissue.

[0109] 6. Cartilage tissue repair method The present invention provides a method for treating cartilage damage, which comprises the step of transplanting a therapeutically effective amount of the above-mentioned chondrocytes and / or the agent for repairing cartilage tissue of the present invention into a site of a mammal requiring cartilage tissue repair.

[0110] The agent for repairing cartilage tissue of the present invention can be used for transplantation into, for example, rodents such as mice, rats, hamsters, guinea pigs, etc., lagomorphs such as rabbits, ungulates such as pigs, cows, goats, horses, sheep, etc., carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc. The agent for repairing cartilage tissue of the present invention is preferably for transplantation into primates or rodents, more preferably for transplantation into humans.

[0111] As used herein, "effective amount" refers to the amount of an active ingredient that produces a desired effect. As used herein, "therapeutically effective amount" refers to the amount of an active ingredient that produces a desired therapeutic effect when administered to a subject. The therapeutically effective amount may be administered (transplanted) once or in multiple divided doses. The number of transplants to be administered is determined by medical professionals and guidelines depending on the disease. When multiple transplants are performed, the interval between each is not particularly limited, but may be several days to several weeks.

[0112] The range of disease sites to which the agent for repairing cartilage tissue of the present invention can be applied is appropriately selected depending on the target disease, the species, age, sex, weight, symptoms, etc. of the animal to be administered.

[0113] 7.Method for producing a drug for repairing cartilage tissue The present invention provides a method for producing a medicament for repairing cartilage tissue, which contains chondrocytes, comprising the following steps: (Step D) A step of obtaining a cell population containing mesenchymal stem cells by the method for producing mesenchymal stem cells of the present invention. The course, (Step E) culturing the cell population containing mesenchymal stem cells in a cartilage induction medium to produce a cell population containing chondrocytes. Obtaining a cell population. Steps D and E in the method for producing a drug for repairing cartilage tissue of the present invention can be carried out as described above for the method for producing mesenchymal stem cells of the present invention and / or the method for producing chondrocytes of the present invention.

[0114] In the method of the present invention for producing a drug for repairing cartilage tissue, the individual that serves as the source of the neural crest cells that are induced to differentiate into mesenchymal stem cells, the pluripotent stem cells if the neural crest cells are derived from pluripotent stem cells, or the somatic cells that are induced into iPS cells if the pluripotent stem cells are iPS cells, is not particularly limited; for example, when producing a drug of the present invention for administration to an animal in need of cartilage tissue repair, these cells may be tissue compatible to the extent that mesenchymal stem cells derived from donor cells can be engrafted in the recipient.

[0115] For example, when the agent for repairing cartilage tissue of the present invention is used for repairing cartilage tissue in humans, from the viewpoint of preventing graft rejection and / or GvHD, these cells may be cells of the patient himself / herself, or may be cells collected from another person having an HLA type that is identical or substantially identical to the HLA type of the patient. "Type" refers to the HLA type of the donor that may affect the donor's HLA type when transplanted into a patient who is taking immunosuppressants, etc. This means that the cells match those of the patient to such an extent that mesenchymal stem cells derived from the cells can be engrafted. For example, the HLA type may be identical to that of the patient for the main HLA (the three major loci of HLA-A, HLA-B, and HLA-DR, or four loci including HLA-Cw).

[0116] The drug for repairing cartilage tissue of the present invention may further contain other compounds, such as antibiotics, anti-inflammatory agents, immunosuppressants, cytokines, preservatives, analgesics, stabilizers (antioxidants, UV absorbers, heat stabilizers, etc.), other therapeutic agents, etc.

[0117] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way. [Example]

[0118] Example 1: Induction of differentiation from iNCC (iPS cell-derived neural crest cells) to iMSC (iPS cell-derived mesenchymal stem cells) 1. Differentiation of iPS cells into neural crest cells (iNCCs) The iPS cell line used was 201B7 (iPS portal). iPS cells were seeded at 6,500 cells / well on a 6-well plate coated with laminin 511-E8 fragment (iMatrix511, Nippi) and cultured in StemFit® AK03N (Ajinomoto Co., Inc.) medium at 37°C under 5% CO for 5 days. The medium was then changed to StemFit AK03N (Solution A + Solution B) supplemented with SB431542 (Stemgent, Inc., 10 μM) and CHIR99021 (Wako, 0.3 μM (Condition 1) or 0.9 μM (Condition 2)). The cells were then cultured at 37°C under 5% CO. The differentiation of neural crest cells was induced for 14 days. The percentage of cells expressing high levels of protein was determined using FACS. In addition, the gene expression of neural crest cell markers was examined by RT-PCR. The primers used for RT-PCR are shown below.

[0119] Marker gene Primer ID Taqman Cat. No. TFAP2a Hs00271528_CE A15629 SOX9 Hs01001343_g1 4331182 TWIST1 Hs01675818_s1 4331182 (βactin was used as the reference gene (Hs01101944_s1, 4331182))

[0120] 2. Purification and expansion of iNCCs The cell population containing neural crest cells prepared in step 1 above was isolated into single cells using TrypLE Select (Thermo Fisher). The single cell population was then cultured on a 6-well plate coated with a scaffold. The cells were seeded onto plates and cultured under conditions suitable for the expansion of neural crest cells. More specifically, StemFit AK03N (solution A + solution B) was supplemented with SB431542 (10 μM), epithelium growth factor (Sigma-Aldrich), and 10 μM HCl. The cells were cultured for 9-10 days at 37°C under 5% CO2 in a medium supplemented with 20 ng / mL of PEG-400 (Ajinomoto Chemical Co., Ltd.) and StemFit AK03N C solution (Ajinomoto Co., Inc., 0.08%). After 9-10 days of culture, the confluency reached approximately 90%. At this point, the proportion of neural crest cells in the cell population cultured on the scaffold and the expression status of neural crest cell gene markers were determined using the same method as in 1 above.

[0121] The scaffold material was laminin 211 (Biolamina, 6.3 ng / cm 2 and 62.5 ng / cm 2 ) was used. The scaffold material was coated onto the surface of a 6-well plate by suspending it directly in culture medium. I did it.

[0122] 3. Differentiation of iNCCs into iMSCs The obtained iNCCs (iPSC-derived NCCs) were cultured at 1.5 μg / cm 2 5.0 × 10 cells / well on a vitronectin-N (Thermo Fisher Scientific)-coated 6-well plate at a density of 3 The cells were seeded at a density of 100 cells / well and cultured in the following differentiation medium (T3 The cells were cultured in a medium at 37°C and 5% CO2 for 13 days to induce iMSCs (iPSC-derived MSCs). T3 medium: StemFit® For Mesenchymal Stem Cells (Ajinomoto Co., Inc.), 90 nM Dexamethasone (Sigma-Aldrich) The induced iMSCs were subjected to surface antigen analysis using FACS for three MSC positive markers (CD90, CD44, CD73) and one negative marker (CD34). The analysis results are shown in Table 1.

[0123] [Table 1]

[0124] The obtained cells were positive for CD90, CD44, and CD73, but negative for CD34, confirming their induction as iMSCs.

[0125] Example 2: Dexamethasone concentration study iNCCs were treated with 1.5 μg / cm 2 3.0 x 10 cells onto a vitronectin-N coated 6-well plate at a density of 4 The cells were seeded at a density of 100 cells / well, and iMSCs were induced by adding StemFit® For Mesenchymal Stem Cells with 50 nM, 100 nM, 1 μM, 10 μM, or 50 μM dexamethasone at 37°C and 5% CO2. The cell proliferation curve during differentiation induction is shown in Figure 1. In the group to which 50 μM dexamethasone was added, cell proliferation stopped after the 13th day.

[0126] Example 3: Examination of types of adrenal cortical hormones iMSCs were induced according to Example 1, except that 100 nM dexamethasone, 100 nM betamethasone, 100 nM prednisolone, and 666 nM prednisolone were used. The induced iMSCs were analyzed for surface antigens using FACS for four MSC-positive markers (CD90, CD44, CD73, CD105), one NCC-positive marker (weakly positive for MSCs) (CD271), and two MSC-negative markers (CD45, CD34). The analysis results are shown in Table 2.

[0127] [Table 2]

[0128] Prednisolone, like dexamethasone, had limited effect even when added at 100 nM. When the glucocorticoid potency was adjusted to 666 nM, it had the effect of promoting differentiation.

[0129] Example 4: Induction of differentiation from iNCC to iMSC and induction of differentiation of the iMSC to chondrocytes 1. Differentiation of iPS cells into iNCCs Human iPSCs (1231A3 strain) were cultured in a culture medium containing laminin 511-E8 fragment (iMatrix-511, Nippi Co., Ltd.). 3.6 x 10 3 cells / cm 2 The cells were seeded at a density of 10 μM and cultured in StemFit AK03N medium for 4 days. After that, the medium was supplemented with 10 μM SB431542 (FUJIFILM Wako). The cells were then cultured for 10 days in StemFit Basic03 (AK03N, Ajinomoto, Tokyo, Japan) containing 1 μM bFGF and 1 μM CHIR99021 (Axon Medchem, Reston, VA, USA) to induce differentiation into iNCCs. The cells were counted using a Countess II FL (Thermo Fisher Scientific). The medium was changed from day 0 (the day the medium was changed) to day 10 after the start of differentiation into iNCCs. The media was changed every two days from the first day until the sixth day, and every day from the seventh to the tenth day.

[0130] 2. Purification and Expansion of iNCCs The cells obtained in 1. were subjected to FACS analysis to detect CD271 high Positive cells were identified as fibronectin-coated 1 x 10 on the cutting plate 4 cells / cm 2The cells were seeded at a density of 1 × 10 and cultured in Basic03 medium supplemented with 10 μM SB431542, 20 ng / mL EGF (FUJIFILM Wako), and FGF2 (FUJIFILM Wako). The medium was changed every 3 days. For subculture, the cells were detached with Accutase (Innovative Cell Technologies, San Diego, CA, USA) and cultured at 1 × 10 4 cells / cm 2 The cells were then replated onto fibronectin-coated plates at a density of 5 × 10. After two subcultures to sufficiently increase the cell number, the cells were reseeded at a density of 5 × 10. 5 iNCCs were suspended in 500 μl of STEM-CELL BANKER GMP grade (Takara, Kusatsu, Japan) and frozen using a BICELL freezing container (Nippon Freezer, Tokyo, Japan) to obtain purified iNCCs. Frozen stocks were made.

[0131] 3. Differentiation of iNCCs into iMSCs Thaw the frozen stock obtained in step 2 and add 1 x 10 4 cells / cm 2 The cells were seeded onto fibronectin-coated plates at a density of 1 × 10 and cultured in Basic03 supplemented with 10 μM SB431542, 20 ng / mL EGF, and FGF2. Six hours before the first passage, the medium was replaced with T1 medium, and the cells were subsequently cultured in the same medium to induce differentiation into MSCs. 4 cells / cm 2 The cells were cultured every 4 days using Accutase at a density of 1000 x g. As a control, PRIME-EV MSC Expansion medium (FUJIFILM Irvine Scientific, Tokyo, Japan), which has been widely used to induce differentiation of iNCCs into iMSCs, was used. Japan) was used.

[0132] Figure 2 shows phase contrast microscope images of cells after switching to differentiation-inducing medium, taken every two passages (every 8 days). The upper row shows the results when T1 medium was used, and the lower row shows the results when control medium was used. Figure 2 shows that whether T1 medium or control medium was used, the cells The morphology began to change, and after 8 days, the cells took on a mesenchymal stem cell-like form, which was maintained thereafter. .

[0133] Figure 3 shows the results of measuring the number of cells over time after switching to differentiation-inducing medium. In the group induced to differentiate with control medium, cell proliferation began slowly, with almost no increase in cell number observed until three days after the start of differentiation induction. In contrast, in the group induced to differentiate with T1 medium, proliferation was exponential from the beginning and continued smoothly thereafter. As a result, throughout the entire measurement period (up to 35 days after the start of differentiation induction), the group induced to differentiate with T1 medium consistently had a higher number of cells than the group induced to differentiate with control medium.

[0134] Next, the time course of changes in the expression levels of human MSC markers (CD44, CD73, CD90, and CD105) in the cells after the start of differentiation induction was analyzed using quantitative RT-PCR (QuantStudio 7 Flex real-time PCR system (Applied Biosystems, Forester City, CA, USA) using Thunderbird SYBR qPCR Mix (TOYOBO, Osaka, Japan)). T1 medium: StemFit® For Mesenchymal Stem Cells (Ajinomoto Co., Inc.), 90 nM Dexamethasone (Sigma-Aldrich), 0.2 μg / mL iMatrix-511 (Nippi Co., Ltd.) The primer sequences used are shown in Table 3, and the results of quantitative PCR are shown in Figure 4.

[0135] [Table 3]

[0136] As shown in Figure 3, the expression of CD44, CD73, and CD105 in iMSCs induced to differentiate using T1 medium was significantly reduced. The expression levels were all comparable to those of iMSCs induced to differentiate using control medium.

[0137] These results confirmed that T1 medium can induce differentiation of iNCCs into iMSCs, as previously reported. Furthermore, it was revealed that more iMSCs could be obtained than when using standard medium.

[0138] 4. Induction of iMSC differentiation into chondrocytes 1.5 x 10 obtained in 3. 5 iMSCs were cultured in 5 μl of chondrogenic induction medium (DMEM / F12, ThermoFish) The cells were suspended in a medium containing 1% (v / v) ITS + premix (Corning, Corning, NY, USA), 0.17 mM AA2P (Sigma, St. Louis, MO, USA), 0.35 mM proline (Sigma), 0.1 μM dexamethasone (Sigma), 0.15% (v / v) glucose (Sigma), 1 mM sodium pyruvate (Thermo Fisher Scientific), and 2 mM GlutaMAX (Thermo Fisher Scientific), supplemented with 10 ng / mL LTGF-β3 (WAKO), 100 ng / ml BMP7 (WAKO), and 1% (v / v) FBS (Thermo Fisher Scientific) and then transferred to a fibronectin-coated 24-well plate. After 1 hour, the chondrogenic induction medium was added to a total volume of 1 mL. The medium was then changed every 2 days and the cells were cultured for 14 days. The differentiation characteristics of the cells were confirmed by Alcian blue staining. The staining was performed by fixing the cells with 4% paraformaldehyde (PFA) (FUJIFILM Wako) for 30 minutes, rinsing with phosphate-buffered saline (PBS), and then treating them with Alcian blue solution (1% Alcian blue (MUTO PURE CHEMICAL CO., LTD., Tokyo, Japan)) at 25°C for 1 hour. The staining results are shown in Figure 5.

[0139] As shown in Figure 5, iMSCs differentiated in T1 medium and induced to form cartilage were stained much more intensely with Alcian blue (Figure 5, left image) than iMSCs differentiated in control medium and induced to form cartilage (Figure 5, right image). Therefore, it was demonstrated that when iNCCs are induced to differentiate into iMSCs using T1 medium, iMSCs with excellent differentiation ability into chondrocytes can be obtained.

[0140] In this example, dexamethasone was used at 50 nM, 90 nM, 100 nM, 1 μM, 10 μM, and 50 μM. The converted concentrations were 1.333 μM, 2.3994 μM, 2.666 μM, 26.66 μM, 266.6 μM, and 1333 μM, respectively (based on the titer of dexamethasone being 26.66). In this example, the equivalent concentrations of 100 nM and 666 nM prednisolone are 400 nM and 2.664 μM, respectively (based on the prednisolone titer of 4). In this example, the equivalent concentration of 100 nM betamethasone is 2.666 μM (based on the betamethasone titer of 26.66). [Industrial Applicability]

[0141] According to the present invention, a medium composition for efficiently inducing mesenchymal stem cells from neural crest cells can be provided. Furthermore, according to the present invention, it is possible to provide an efficient method for producing mesenchymal stem cells, a method for producing chondrocytes, a drug for repairing cartilage tissue, a method for repairing cartilage tissue, a method for producing a drug for repairing cartilage tissue, and the like, which contain the mesenchymal stem cells as an active ingredient. Therefore, the present invention is extremely useful, for example, in the medical field.

Claims

1. A medium composition for inducing mesenchymal stem cells from neural crest cells, comprising a basal medium and at least one corticosteroid selected from the group consisting of dexamethasone, prednisolone, and betamethasone, wherein the equivalent concentration of the corticosteroid in the medium composition is 1.25 μM or more and 300 μM or less.

2. The medium composition described in claim 1, wherein the adrenal cortical hormone is dexamethasone.

3. 3. The medium composition according to claim 1, wherein the concentration of dexamethasone in the medium composition is 10 μM or less.

4. The medium composition according to any one of claims 1 to 3, wherein the basal medium is a serum-free medium.

5. The medium composition according to any one of claims 1 to 4, wherein the neural crest cells are derived from pluripotent stem cells.

6. The medium composition according to claim 5, wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells).

7. A method for producing mesenchymal stem cells, comprising the step (Step 1) of culturing neural crest cells in the medium composition according to any one of claims 1 to 4 to induce mesenchymal stem cells.

8. The method of claim 7, wherein the neural crest cells are derived from pluripotent stem cells.

9. The method of claim 8, wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells).

10. The method according to any one of claims 7 to 9, wherein the following steps are carried out before step 1: (Step A) obtaining a cell population containing neural crest cells; and (Step B) A step of expanding and culturing the cell population obtained in step A using an extracellular matrix as a scaffold.

11. The method according to claim 10, wherein the extracellular matrix is ​​laminin or fibronectin.

12. The method of claim 11, wherein the laminin is full-length laminin, laminin having an α2 chain, or laminin 211.

13. The method of any one of claims 10 to 12, wherein the cell population obtained in step B is a cell population containing 70% or more neural crest cells.

14. A method for producing mesenchymal stem cells from a cell population containing neural crest cells, comprising: the neural crest cells are cells induced to differentiate from pluripotent stem cells, the cell population is a cell population containing 70% or more of the neural crest cells, Culturing the cell population in the presence of at least one adrenocortical hormone selected from the group consisting of dexamethasone, prednisolone, and betamethasone at an equivalent concentration of 1.25 μM to 300 μM. A method for producing mesenchymal stem cells from a cell population containing neural crest cells.

15. The manufacturing method described in claim 14, wherein the adrenal cortical hormone is dexamethasone.

16. A method for producing chondrocytes, comprising the steps of: (Step D) obtaining a cell population containing mesenchymal stem cells by the production method according to any one of claims 7 to 15; (Step E) A step of culturing the cell population containing mesenchymal stem cells in a cartilage induction medium to obtain a cell population containing chondrocytes.

17. A method for producing a medicament for repairing cartilage tissue, comprising chondrocytes, the method comprising the steps of: (Step D) obtaining a cell population containing mesenchymal stem cells by the production method according to any one of claims 7 to 15; (Step E) A step of culturing the cell population containing mesenchymal stem cells in a cartilage induction medium to obtain a cell population containing chondrocytes.

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