JP2022189188A - Method for producing mesenchymal stem cell - Google Patents

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 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 adrenocortical hormone.

Mesenchymal stem cells (MSCs) are a type of somatic stem cells present in the bone marrow of living organisms, etc., and are defined as adhesive cells that have the ability to differentiate into bone, cartilage, and adipocytes. Mesenchymal stem cells are considered to have an extremely low risk of becoming cancerous, and are highly promising as cell materials for use in regenerative medicine. In addition, mesenchymal stem cells accumulate at sites of tissue injury, release various humoral factors and exosomes, and have the function of regulating immune responses and anti-inflammatory effects, playing an important role in tissue repair and maintenance of homeostasis. is known to fulfill Therefore, mesenchymal stem cells themselves are also promising as cell preparations for treating immune diseases, inflammatory diseases, and the like.

However, since mesenchymal stem cells have different differentiation potential and growth/functional characteristics depending on the tissue from which they are derived, there is a demand for efficient production of mesenchymal stem cells that exhibit useful properties according to the purpose. had been

Neural crest cells are peculiar to vertebrates. They are cells that differentiate into a wide variety of cells such as pigment cells. It was known that neural crest cells can be produced by the methods disclosed in Patent Document 1 and Non-Patent Document 1, and that differentiation of mesenchymal stem cells can be induced therefrom (Non-Patent Document 2). A more efficient method of producing the was being investigated.

WO2020/230832

Fukuta M. et al., PLoS One. 2014 Dec 2;9(12):e 112291. Zhao C. and Ikeya M., Stem Cells Int. 2018 Jul 31; Article ID 9601623

An object of the present invention is to provide a medium composition, a method for producing mesenchymal stem cells, and the like for efficiently producing mesenchymal stem cells.

As a result of intensive studies on the above problems, the present inventors have found that mesenchymal stem cells can be efficiently induced from neural crest cells by using a medium composition containing adrenocortical hormones, and the medium composition We have found suitable concentrations of adrenocortical hormones in the product. Furthermore, the inventors have found 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 the production of drugs for repairing cartilage tissue. As a result, the present invention was completed.

That is, the present invention is as follows.
[1] A medium composition for inducing mesenchymal stem cells from neural crest cells, containing a basal medium and an adrenocortical hormone, wherein the converted concentration of the adrenocortical hormone in the medium composition is 1.25 μM or more. Medium composition.
[2] The medium composition of [1], wherein the adrenocortical hormone is a glucocorticoid or derivative thereof.
[3] The glucocorticoid or its derivative is at least one selected from the group consisting of cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, and beclomethasone propionate. 2].
[4] The medium composition of [2] or [3], wherein the glucocorticoid or derivative thereof is dexamethasone.
[5] The medium composition according to [1], wherein the converted concentration of adrenocortical hormone in the medium composition is 300 μM or less.
[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 of [8], wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells).
[10] A method for producing mesenchymal stem cells, comprising a step (step 1) of inducing mesenchymal stem cells by culturing neural crest cells in the medium composition according to any one of [1] to [7]. .
[11] The production method of [10], wherein the neural crest cells are derived from pluripotent stem cells.
[12] The production method of [11], wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells).
[13] The production method according to any one of [10] to [12], wherein the following steps are performed before step 1:
(Step A) A step of 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 production method of [13], wherein the extracellular matrix is laminin or fibronectin.
[15] The production method of [14], wherein the laminin is full-length laminin, laminin having an α2 chain, or laminin-211.
[16] The production method according to any one of [13] to [15], wherein the cell population obtained in step B is a cell population containing 70% or more neural crest cells.
[17] A production 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 adrenocortical hormone at an equivalent concentration of 1.25 μM or more and 300 μM or less,
A production method for producing mesenchymal stem cells from a cell population containing neural crest cells.
[18] The production method of [17], wherein the adrenocortical hormone is a glucocorticoid or a derivative thereof.
[19] The glucocorticoid or 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. 18].
[20] The production method of [18] or [19], wherein the glucocorticoid or derivative thereof is dexamethasone.
[21] A mesenchymal stem cell or a culture thereof obtained by the production method according to any one of [10] to [20].
[22] The mesenchymal stem cell or culture thereof according to [21], wherein the mesenchymal stem cell is characterized by high differentiation potential into chondrocytes.
[23] A method for producing chondrocytes, comprising the steps of:
(Step D) a step of obtaining a cell population containing mesenchymal stem cells by the production method according to any one of [10] to [20];
(Step E) A step of culturing the cell population containing the mesenchymal stem cells in a cartilage-inducing medium to obtain a cell population containing chondrocytes.
[24] Chondrocytes or a culture thereof obtained by the production method of [23].
[25] A drug for repairing cartilage tissue, containing the chondrocytes or culture thereof of [24].
[26] A method for producing a medicament for repairing cartilage tissue containing chondrocytes, the method comprising the steps of:
(Step D) a step of obtaining a cell population containing mesenchymal stem cells by the production method according to any one of [10] to [20];
(Step E) A step of culturing the cell population containing the mesenchymal stem cells in a cartilage-inducing medium to obtain a cell population containing chondrocytes.

According to the present invention, it is possible to provide a medium composition for efficiently inducing mesenchymal stem cells from neural crest cells. In addition, according to the present invention, there are provided an efficient method for producing mesenchymal stem cells, a method for producing chondrocytes, a drug for repairing cartilage containing the mesenchymal stem cells as an active ingredient, and repair of cartilage. A method, a method for producing a drug for repairing cartilage tissue, and the like can be provided.

FIG. 1 is a graph showing cell growth curves during induction of differentiation. FIG. 2 is a phase-contrast microscopic image of iNCCs whose differentiation was initiated with the medium composition of the present invention. FIG. 3 is a graph showing temporal changes in the expression levels of MSC markers in iNCCs for which differentiation induction was initiated with the medium composition of the present invention. The vertical axis represents relative values when the expression level in iPS cells is set to 1. The numbers on the horizontal axis represent the number of days after initiation of induction of differentiation into MSCs. FIG. 4 is a graph showing a cell growth curve of iNCCs for which differentiation induction was initiated with the medium composition of the present invention. The vertical axis represents the total number of mitotic divisions, and the horizontal axis represents the number of days after initiation of induction of differentiation into MSCs. FIG. 5 shows data showing the high differentiation potential of iMSCs obtained by inducing differentiation with the medium composition of the present invention into chondrocytes. The images show Alcian blue staining results 14 days after iMSCs were induced to differentiate into chondrocytes.

The present invention will be described in detail below.

1. Medium composition The present invention provides a medium composition for inducing mesenchymal stem cells from neural crest cells, containing a basal medium and an adrenocortical hormone, wherein the converted concentration of the adrenocortical hormone in the medium composition is 1.25.
A culture medium composition (hereinafter also referred to as the culture medium composition of the present invention) is provided that is at least μM.

The basal medium contained in the medium composition of the present invention can be a basal medium known per se, and is not particularly limited as long as it does not inhibit the induction of mesenchymal stem cells from neural crest cells. The basal medium includes, for example, IMDM medium, Medium199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen) , RPMI-base medium, StemFit (registered trademark), MCDB201 medium and mixed medium thereof. In this process,
Preferably, StemFit® medium is used. The medium may contain serum or may be serum-free. If necessary, the medium contains, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum replacement for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen May contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol (2ME), thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like.

The medium contains adrenocortical hormones. Adrenal cortical hormones include, for example, glucocorticoids and derivatives thereof, and the glucocorticoids and derivatives thereof include, for example, cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, Betamethasone, beclomethasone propionate. Among these, prednisolone, dexamethasone and betamethasone are preferred, and dexamethasone is especially preferred. One or more adrenal corticosteroids may be added to the medium, but one is preferred.

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) )-) is

It is a known synthetic adrenocortical hormone having a structure represented by Dexamethasone is commercially available and such commercial products can also be used in the present invention. Since differentiation of neural crest cells into mesenchymal stem cells is induced by using dexamethasone, mesenchymal stem cells can be produced with high efficiency by culturing neural crest cells in the medium composition of the present invention. be able to.

The concentration of adrenocortical hormone contained in the medium composition is not particularly limited as long as it is a concentration that can promote the differentiation of neural crest cells into mesenchymal stem cells. >50 nM (or >50 nM), >55 nM (or >55 nM), >60 nM (
or >60 nM), >65 nM (or >65 nM), >70 nM (or >70 nM), >75 nM (or >75 nM), >80 nM (or >80 nM), >85 nM ( or >85 nM), >90 nM (or >90 nM), >95 nM (or >95 nM), >100 nM (or >100 nM), >150 nM (or >150 nM), >200 nM ( or >200 nM), >250 nM (or >250 nM), >300 nM (or >300 nM), >350 nM (or >350 nM), >400 nM (or >400 nM), >450 nM ( or >450 nM), >500 nM (or >500 nM), >550 nM (or >550 nM), >600 nM (or >600 nM), >650 nM (or >650 nM), >700 nM ( or >700 nM), >750 nM (or >750 nM), >800 nM (or >800 nM), >850 nM (or >850 nM), >900 nM (or >900 nM), >950 nM ( or >950 nM) and ≤10 μM (or <10 μM), ≤9 μM (or <9 μM), ≤8 μM (or <8 μM), 7 μM
≤ (or <7 μM), ≤6 μM (or <6 μM), ≤5 μM (or <5 μM), 4
≤ 3 µM (or < 3 µM), ≤ 2 µM (or < 2 µM), ≤ 1 µM (or < 1 µM). When an adrenocortical hormone other than dexamethasone is used, it is preferably used at a concentration that exhibits the same glucocorticoid potency as dexamethasone at the above concentration. Too low corticosteroid concentrations in the medium may not be sufficient to promote culture maintenance and mesenchymal stem cell induction, whereas too high corticosteroid concentrations inhibit cell proliferation and Mesenchymal stem cells cannot be obtained efficiently. For example, when inducing differentiation into human mesenchymal stem cells, the concentration of adrenocortical hormone contained in the medium composition is, for example, 50 nM to 10 μM (or 50 nM to less than 10 μM, 50 nM to 10 μM, or 50 nM to 10 μM), preferably 60 nM to 5 μM (or 60 nM to less than 5 μM, 60 nM to 5 μM, or 60 nM to 5 μM) < μM),
More preferably 70 nM to 2 μM (or 70 nM or more and less than 2 μM, more than 70 nM and 2 μM or less, or 70
nM to less than 2 μM), still more preferably 100 nM to 1 μM (or 100 nM to less than 1 μM, more than 100 nM to less than 1 μM, or more than 100 nM and less than 1 μM).

The potency of adrenocortical hormones as glucocorticoids is 0.8 for cortisone, 4 for prednisolone, 5 for methylprednisolone, 5 for triamcinolone, 10 for paramethasone, 25-30 for dexamethasone, and 25 for betamethasone when hydrocortisone is 1. ~30. If there is a possibility that the potency of each adrenocortical hormone as a glucocorticoid differs from the above value depending on the manufacturer or storage conditions, it should be experimentally confirmed in advance. It is desirable to use one in good condition.

In addition, when betamethasone is used alone, the concentration of betamethasone contained in the medium composition is not particularly limited as long as it can promote the differentiation of neural crest cells into mesenchymal stem cells. >50 nM (or >50 nM), >55 nM (or >55 nM), >60 nM (
or >60 nM), >65 nM (or >65 nM), >70 nM (or >70 nM), >75 nM (or >75 nM), >80 nM (or >80 nM), >85 nM ( or >85 nM), >90 nM (or >90 nM), >95 nM (or >95 nM), >100 nM (or >100 nM), >150 nM (or >150 nM), >200 nM ( or >200 nM), >250 nM (or >250 nM), >300 nM (or >300 nM), >350 nM (or >350 nM), >400 nM (or >400 nM), >450 nM ( or >450 nM), >500 nM (or >500 nM), >550 nM (or >550 nM), >600 nM (or >600 nM), >650 nM (or >650 nM), >700 nM ( or >700 nM), >750 nM (or >750 nM), >800 nM (or >800 nM), >850 nM (or >850 nM), >900 nM (or >900 nM), >950 nM ( or >950 nM) and ≤10 μM (or <10 μM), ≤9 μM (or <9 μM), ≤8 μM (or <8 μM), 7 μM
≤ (or <7 μM), ≤6 μM (or <6 μM), ≤5 μM (or <5 μM), 4
≤ 3 µM (or < 3 µM), ≤ 2 µM (or < 2 µM), ≤ 1 µM (or < 1 µM). Too low concentrations of betamethasone in the medium may not be sufficient to promote culture maintenance and induction of mesenchymal stem cells, whereas too high concentrations of betamethasone inhibit cell proliferation and effectively intermittent cells. Leaf stem cells cannot be obtained. 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 to less than 10 μM, more than 50 nM to 10 μM , or more than 50 nM and less than 10 μM), preferably 60 nM to 5 μM (or more than 60 nM and less than 5 μM, more than 60 nM
5 μM or less, or 60 nM to less than 5 μM), more preferably 70 nM to 2 μM (or 70 nM to less than 2 μM, 70 nM to 2 μM, or 70 nM to less than 2 μM), even more preferably 100 nM to 1 μM (or 100 nM to 1 μM, or 100 nM to 1 μM, or 100 nM to 1 μM).

In addition, when prednisolone is used alone, the concentration of prednisolone contained in the medium composition is not particularly limited as long as it can promote the differentiation of neural crest cells into mesenchymal stem cells. ≥300 nM (or >300 nM), ≥330 nM (or >330 nM), ≥360 nM (or >360 nM), ≥390 nM (or >390 nM), ≥420 nM (or >420 nM), ≥450 nM (or >450 nM), ≥480 nM (or >480 nM), ≥510 nM (or >510 nM), ≥540 nM (or >540 nM), ≥570 nM (or >570 nM), >600 nM (or >600 nM), >900 nM (or >900 nM), >1.2 μM (or >1.2 μM), >1.5 μM (
or >1.5 μM), >1.8 μM (or >1.8 μM), >2.1 μM (or >2.1 μM), 2.4
≥ 2.7 µM (or >2.7 µM), ≥3 µM (or >3 µM), ≥3.3 µM (or >3.3 µM), ≥3.6 µM (or >3.6 µM), 3.9 ≥4.2 µM (or >3.9 µM), ≥4.5 µM (or >4.5 µM), 4.8 µM
≥ (or >4.8 µM), ≥5.1 µM (or >5.1 µM), ≥5.4 µM (or >5.4 µM), ≥5.7 µM (or >5.7 µM) and ≤70 µM (or <70 µM), ≤63 μM (or <63 μM), ≤56 μM (or <56 μM), ≤49 μM (or <49 μM), ≤42 μM (or <42 μM), ≤35 μM (or <35 μM), 28 μM or less (or 28
≤21 μM (or <21 μM), ≤14 μM (or <14 μM), ≤7 μM (or <7 μM). Too low a prednisolone concentration in the medium may not be sufficient to promote culture maintenance and mesenchymal stem cell induction, whereas too high a prednisolone concentration inhibits cell proliferation and effectively intermittent cells. Leaf stem cells cannot be obtained. 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 to 70 μM, or more than 300 nM to 70 μM). or 300 nM to less than 70 μM), preferably 360 nM to 35 μM (or 360 nM to less than 35 μM, 360 nM to 35 μM, or 360 nM to less than 35 μM), more preferably 420 nM to 14 μM (or 420 nM to 14 μM, 420 nM to 14 μM, or 420 nM to 14 μM), even more preferably 600 nM to 7 μM (or 600 nM to 7 μM, 600 nM to 7 μM, or more than 600 nM and less than 7 μM).

In one embodiment, the concentration of adrenocortical hormones contained in the medium composition is 50 nM or more and 10 μM or less for dexamethasone or betamethasone (or 50 nM or more and less than 10 μM or more than 50 nM when each one is used alone). 10 μM or less, or 50 nM to less than 10 μM), paramethasone is 100 nM to 30 μM (or 100 nM to less than 30 μM, 100 nM to 30 μM, or 100 nM to less than 30 μM), triamcinolone or methylprednisolone 250 nM to 60 μM (or 250 nM to 60 μM, 250 nM to 60 μM, or 250 nM to
< μM), prednisolone ≥300 nM and ≤70 μM (or ≥300 nM and <70 μM, >300 nM and ≤70 μM, or >300 nM and <70 μM), cortisone ≥1.50 μM and ≤375 μM ( or ≥1.50 μM and <375 μM, >1.50 μM and <375 μM, or >1.50 μM and <375 μM), and hydrocortisone is ≥1.250 μM and <300 μM (or or more than 1.250 μM and less than 300 μM).

In one embodiment, the concentration of adrenocortical hormones contained in the medium composition is 50 nM or more and 375 μM or less (or 50 nM or more and less than 375 μM, or 50 nM or more and 375 μM or less when each one is used alone).
μM or less, or more than 50 nM and less than 375 μM).

In one embodiment, when one or more adrenocortical hormones are used, the concentration of the adrenocortical hormone is multiplied by the potency of the glucocorticoid, and the sum of the concentrations is added to the one or more adrenocortical hormones. and the equivalent concentration is, for example, 1.25 μM or more (or 1.25 μM or more), 1.375 μM or more (or 1.375 μM or more), 1.5 μM or more (or 1.5 μM or more), 1.625 μM or more (or 1.625 μM or more μM), ≥1.75 μM (or >1.75 μM), ≥1.875 μM (or >1.875 μM), ≥2 μM (or >2 μM), ≥2.125 μM (or >2.125 μM)
, ≥2.25 μM (or >2.25 μM), ≥2.375 μM (or >2.375 μM), ≥2.5 μM (or >2.5 μM), ≥3.75 μM (or >3.75 μM), ≥5 μM (or >5 μM) , ≥6.25 μM (or >6.25 μM), ≥7.5 μM (or >7.5 μM), ≥8.75 μM (or >8.75 μM), ≥10 μM (or >10 μM), ≥11.25 μM (or >11.25 μM) ), ≥12.5 μM (or >12.5 μM), ≥13.75 μM (or >13.75 μM), ≥15 μM (or >15 μM), ≥16.25 μM (or >16.25 μM), ≥17.5 μM (or >17.5 μM) ), ≥18.75 μM (or >18.75 μM), ≥20 μM (or >20 μM), ≥21.25 μM (or >21.25 μM), ≥22.5 μM (or >22.5 μM), ≥23.75 μM (or >23.75 μM) ), ≤300 μM (or <300 μM), ≤270 μM (or <270 μM), ≤240 μM (or <240 μM), ≤210 μM (or <210 μM), ≤180 μM (or 180 μM) μM
150 μM or less (or 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 30 μM less than). For example, when using one or more adrenocortical hormones, the equivalent concentration of adrenocortical hormones is 1.25 μM to 300 μM (or 1.25 μM to less than 300 μM, 1.25 μM to 300 μM, or 1.25 μM to less than 300 μM). , preferably 1.5 μM to 150 μM (or 1.5 μM to 150 μM
less than 1.5 μM to 150 μM, or 1.5 μM to less than 150 μM), more preferably 17.5 μM to 60 μM (or 17.5 μM to less than 60 μM, 17.5 μM to 60 μM, or 17.5 μM to less than 60 μM), and It is more preferably 2.5 μM to 30 μM (or 2.5 μM or more and less than 30 μM, more than 2.5 μM and less than 30 μM, or more than 2.5 μM and less than 30 μM).

The medium composition of the present invention may contain serum. Serum is not particularly limited as long as it is animal-derived serum as long as it does not inhibit the induction of mesenchymal stem cells, but mammal-derived serum (eg, fetal bovine serum, human serum, etc.) is preferred. The concentration of serum may be within a concentration range known per se. Furthermore, when cultured mesenchymal stem cells are used for medical purposes, serum-free media should also be preferably used, as other animal-derived components may serve as a source of infection for blood-borne pathogens or as xenoantigens. obtain. If serum is not included, alternative serum additives (eg, Knockout Serum Replacement (KSR) (Invitrogen), Chemically-defined Lipid concentration (Gibco), etc.) may be used.

When mesenchymal stem cells induced and cultured by the medium composition of the present invention are used for medical purposes such as cell therapy, they may be infected with pathogens or become heterologous antigens. More preferably, the medium does not contain non-human animal-derived components.

By "stem cell" is meant an immature cell with self-renewal and differentiation/proliferation capacity. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation potential.
A pluripotent stem cell means a cell that has the ability to differentiate into all tissues and cells that constitute a living body. Multipotent stem cells refer to cells that have the ability to differentiate into multiple, but not all, types of tissues and cells. A unipotent stem cell means a cell that has the ability to differentiate into a specific tissue or cell.

Mesenchymal stem cells targeted in the present invention are a type of multipotent stem cells that can differentiate into adipocytes, osteocytes, chondrocytes, muscle cells, hepatocytes, nerve cells, etc. known as cells that are less likely to form The mesenchymal stem cells in the present invention are preferably positive for one or more mesenchymal stem cell markers (e.g., CD90, CD44, CD73, CD105, etc.), more preferably positive for the marker, and It may be negative for the expression of molecules that are not found to be expressed in foliar stem cells. Examples of molecules whose expression is not observed in mesenchymal stem cells include CD34, CD45, CD14, CD11b, CD79, CD19, HLA-DR and the like.

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, for example, rodents such as mice, rats, hamsters and guinea pigs, lagomorphs such as rabbits, pigs, cattle, goats and horses. , ungulates such as sheep, felines such as dogs and cats, and mesenchymal stem cells derived from primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees, preferably human-derived mesenchymal stem cells. stem cells.

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, e.g., bone morphogenetic proteins (preferably BMP-2 or BMP-4). ), basic fibroblast growth factor (bFGF), inhibin A or chondrogenesis stimulating active factor (CSA), collagenous extracellular matrices such as type I collagen (especially in gel form), and vitamins such as retinoic acid. A analog), preferably at a concentration that does not induce differentiation of mesenchymal stem cells (eg, differentiation into cartilage). In one embodiment, the medium may not contain factors that induce differentiation of mesenchymal stem cells other than adrenocortical hormones. In one embodiment, the medium may not contain factors other than dexamethasone that induce differentiation of mesenchymal stem cells.

The media composition of the present invention may contain scaffolding components. The scaffold components include laminin [including laminin α5β1γ1 (hereinafter, laminin 511), laminin α1β1γ1 (hereinafter, laminin 111), etc. and laminin fragments (laminin 511E8, etc.)], entactin, fibronectin, gelatin, vitronectin, synthmax (Corning Co.), extracellular matrices such as matrigel, etc., preferably laminin-511. These scaffold components are generally 0.001 μg/ml to 1000 μg/ml, preferably 0.01 μg/ml to 100 μg/ml, more preferably 0.1 μg/ml to 100 μg/ml.
μg/ml to 10 μg/ml, more preferably 0.1 μg/ml to 1 μg/ml, most preferably 0.2 μg/ml
to the medium at .

The medium composition of the present invention is used to induce differentiation of neural crest cells into mesenchymal stem cells.
"Neural Crest Cell (also called "NCC")" is a structure called the neural crest that is temporarily formed between the epidermal ectoderm and the neural plate during early vertebrate development. It refers to cells that are induced to various sites within the definitive body after the transition from mesenchymal to mesenchymal. The term "neural crest cells" as used herein includes not only cells collected from a living body, but also neural crest cells derived from pluripotent stem cells and subcultured cells thereof. In the present invention, the neural crest cells are not particularly limited in origin, and may be derived from any vertebrate, but neural crest cells derived from mammals are preferred. 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.

Neural crest cells may be biologically-derived neural crest cells, for example, neural crest-derived tissues in vivo (e.g., bone marrow, dorsal root ganglion, heart, cornea, iris, dental pulp, olfactory mucosa, etc.). can be manufactured from Neural crest cells derived from pluripotent stem cells can also be preferably used. As a method for obtaining neural crest cells from pluripotent stem cells, a method known per se can be used. One example is culturing pluripotent stem cells in a culture medium containing a TGFβ inhibitor and a GSK-3β inhibitor. A method of inducing differentiation is exemplified. Thus, by using a method known per se, it is possible to easily obtain neural crest cells, more specifically, cell populations containing neural crest cells.

Whether or not the thus obtained cell population 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. good. In addition, proteins present on the cell surface of neural crest cells such as CD271 protein (also referred to as “p75(NTR)”) can also be used as neural crest cell-specific markers. In the present invention, the neural crest cells are preferably derived from pluripotent stem cells, more preferably iPS cells.

In the present invention, pluripotent stem cells are any cells that are induced into intermediate mesoderm cells that have pluripotency capable of differentiating into many cells existing in the body and also have proliferative ability. is included. Examples of pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, cloned embryo-derived embryonic stem (ntES) cells obtained by nuclear transfer, spermatogonial stem cells (GS cells), and embryonic germ cells. (EG cells), induced pluripotent stem (iPS) cells, cultured fibroblasts, and pluripotent cells (Muse cells) derived from bone marrow stem cells. Preferred pluripotent stem cells are iPS cells, more preferably human iPS cells, from the viewpoint that they can be obtained without destroying embryos, ova, etc. in the manufacturing process.

Methods for producing iPS cells are known in the art, and can be produced by introducing reprogramming factors into arbitrary somatic cells. Here, the initialization factors are, for example, Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15 Genes or gene products such as -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 are exemplified, and these reprogramming factors may be used alone or in combination. Also good. Combinations of initialization factors include WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, WO2009/091659, WO2009/091659 101407、WO2009/102983、WO2009/114949、WO2009/117439、WO2009/126250、WO2009/126251、WO2009/126655、WO2009/157593、WO2010/009015、WO2010/033906、WO2010/033920、WO2010/042800、WO2010/050626、 WO2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO2010/111409, WO2010/111422, WO2010/115050, WO2010/124290, WO2010/147395, WO2010/147
(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, (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. :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. ), Nature. 474:225-9.

Somatic cells include, but are not limited to, fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, and primary cultured cells. , passaged cells, and cell lines are all included. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) blood cells (peripheral stem cells). blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosa cells, enterocytes, splenocytes, pancreatic cells (pancreatic exocrine cells) etc.), differentiated cells such as brain cells, lung cells, kidney cells and adipocytes.

The mammal from which somatic cells are collected is not particularly limited, but is preferably human.

INDUSTRIAL APPLICABILITY The present invention can provide mesenchymal stem cell differentiation promoting agents containing adrenocortical hormones. By culturing neural crest cells using a medium containing the differentiation-promoting agent, it is possible to increase induction efficiency into mesenchymal stem cells.

The differentiation-promoting agent of the present invention may consist only of adrenocortical hormones, but may also contain a physiologically acceptable carrier (e.g., physiological isotonic solution (physiological saline, the above-mentioned basal medium, glucose, etc.). adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), excipients, preservatives, stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), binders, It may also be provided as a composition containing a dissolution aid, a nonionic surfactant, a buffer (e.g., phosphate buffer, sodium acetate buffer), a preservative, an antioxidant, the above-mentioned additives, etc.). can.

The content of adrenocortical hormone contained in the differentiation-promoting agent of the present invention is such that when the differentiation-promoting agent of the present invention is added to a medium and used, the adrenocortical hormone concentration in the medium is It is preferably configured to contain a concentration sufficient to promote the induction of lineage stem cells.

The differentiation-promoting agent of the present invention is used in the form of an isotonic aqueous solution, powder, or the like, added to a medium, or the like.

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

In the kit, 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. Moreover, the kit may not necessarily contain all the components of the medium composition of the present invention, and may omit extremely easily available components such as water. When the component is in the form of powder, it can be used by dissolving in a buffer or the like at the time of use, if desired.

2. Method for Producing Mesenchymal Stem Cells The present invention provides a method for producing mesenchymal stem cells (hereinafter referred to as a method for producing mesenchymal stem cells), which comprises a step (step 1) of inducing mesenchymal stem cells by culturing neural crest cells in the medium composition of the present invention. , also referred to as the manufacturing method 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 adrenocortical hormones. According to the production method of the present invention, it is possible to efficiently produce mesenchymal stem cells. By culturing neural crest cells in a medium containing adrenocortical hormones, it becomes possible to efficiently differentiate neural crest cells into cells that retain their ability as mesenchymal stem cells.

The culture in step 1 can be suspension culture or adherent culture or a combination thereof. “Suspension culture” in the present invention refers to culturing cells (or aggregates of cells) while maintaining a state in which they are suspended in a culture solution. “Adhesive culture” refers to culture performed under conditions that allow cells (or aggregates of cells) to adhere to culture equipment and the like. In this case, the adhesion of cells means that a strong cell-substratum junction is formed between the cells or cell aggregates and the cultureware. More specifically, suspension culture refers to culture under conditions that do not form strong cell-substrate bonds between cells or cell aggregates and cultureware, etc., and adherent culture refers to cells or cells This refers to culturing under conditions that form a strong cell-substrate bond between the aggregates and the cultureware.

The incubator used for suspension culture is not particularly limited as long as it allows suspension culture, and can be appropriately determined by those skilled in the art. Such incubators include, for example, flasks, tissue culture flasks, culture dishes (dishes), petri dishes, tissue culture dishes, multidishes, microplates, microwell plates, micropores, multiplates, and multiwell plates. , chamber slides, petri dishes, tubes, trays, culture bags, Erlenmeyer flasks, spinner flasks or roller bottles. These incubators are preferably cell non-adhesive in order to enable suspension culture. As a non-cell-adhesive incubator, the surface of the incubator has been artificially treated (for example, superhydrophilic treatment such as MPC polymer, low protein adsorption treatment, etc.) for the purpose of reducing adhesion to cells. You can use things, etc. Rotation culture may be performed using a spinner flask, roller bottle, or the like. The culture surface of the incubator may be flat-bottomed or uneven.

The incubator used for adherent culture is not particularly limited as long as it allows adherent culture, and a person skilled in the art can appropriately select an incubator according to the culture scale, culture conditions, and culture period. It is possible to Such incubators include, for example, flasks, tissue culture flasks, culture dishes (dish), tissue culture dishes, multidishes, microplates, microwell plates, multiplates, multiwell plates, chamber slides, Petri dishes, Tubes, trays, culture bags, microcarriers, beads, stack plates, spinner flasks or roller bottles. These incubators are preferably cell-adhesive in order to allow adherent culture. Examples of cell-adhesive incubators include incubators whose surface has been artificially treated for the purpose of improving adhesion to cells, specifically surface-treated incubators, or An incubator whose inside is coated with a coating agent is included. Surface-treated culture vessels include culture vessels that have been surface-treated, such as by positive charge treatment. Coating agents include, for example, laminin [including laminin α5β1γ1 (hereinafter, laminin 511), laminin α1β1γ1 (hereinafter, laminin 111), etc. and laminin fragments (laminin 511E8, etc.)], entactin, collagen, fibronectin, gelatin, vitronectin, synth Examples include extracellular matrices such as Max (Corning) and Matrigel, and polymers such as polylysine and polyornithine.

Neural crest cells that can be used in step 1 are: 1. As described in the medium composition, it may be derived from a living organism or from a pluripotent stem cell. When neural crest cells derived from pluripotent stem cells are used, cells immediately after differentiation into neural crest cells may be used. may be used.
Purification as used herein refers to increasing the proportion of a specific type of cell (eg, neural crest cell) 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 to about 1×10 ⁷ cells/cm ² , preferably about 3×10 ² to about 5×10 ⁶ cells/cm ² , more preferably About 4×10 ² to about 2×10 ⁵ cells/cm ² , more preferably about 4×10 ² to about 1×10 ⁵ cells/cm ² , even more preferably about 3×10 ³ to about 1× It can be 10 ⁴ cells/cm ² .
In other embodiments, the concentration of neural crest cells to be cultured is from about 1×10 ² to about 1×10 ⁷ cells/cm ² , preferably from about 3×10 ² to about 5×10 ⁶ cells/cm ² , more preferably. is about 4×10 ² to about 2×10 ⁵ cells/cm ² , more preferably about 4×10 ² to about 1×10 ⁵ cells/cm ² , still more preferably about 1.5×10 ⁴ to about 3×10 5 cells/cm 2 . It can be ×10 ⁴ cells/cm ² .

Culture conditions such as culture temperature and CO ₂ concentration can be appropriately set. 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%.

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

In one embodiment, prior to step 1, the following steps:
(Step A) a step of 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.

In process A, 1. As described in the medium composition, a cell population containing neural crest cells can be obtained by a method known per se. Generally, in a cell population containing neural crest cells, the ratio of neural crest cells can vary greatly depending on the collected tissue and differentiation induction conditions. In one aspect of the present invention, the percentage of neural crest cells in a cell population containing neural crest cells is, for example, 1-95%, 1-90%, 1-85%, 1-80%, 1-75%, 1 ~70%, 1~65%, 1~60%, 1~55%, 1~50%, 1~45%, 1~40%, 1~35%, 1~30%, 1~25%, 1 It can be, but is not limited to, ~20%, 1-15%, or 1-10%. In another aspect, the percentage of neural crest cells in the cell population containing neural crest cells is, for example, 25-95%, 25-90%, 25-85%, 25-80%, 25-75%, It can be, but is not limited to, 25-70%, 25-65%, 25-60%, 25-55%, 25-50%, 25-45%, 25-40%, or 25-35%. In another aspect, the percentage of neural crest cells in the cell population containing neural crest cells is, for example, 50-95%, 50-90%, 50-85%, 50-80%, 50-75%, It can be, but is not limited to, 50-70%, 50-65%, or 50-60%. In another aspect, the percentage of neural crest cells in the cell population containing neural crest cells can be, for example, 70% or more, 75-95%, 75-90%, or 75-85%. is not limited to

The cell population containing neural crest cells obtained in step A may then be subjected to a step of purifying neural crest cells, and further subjected to a step of expanding (the purified neural crest cells). good. That is, step B can also be said to be a step of purifying and expanding neural crest cells. As used herein, the term "expansion culture" is a concept that includes culture for maintaining and/or growing desired cells, and preferably culture for growing desired cells.

The method for purifying neural crest cells is not particularly limited, and examples thereof include single cell conversion, sorting by a cell sorter using a fluorescently labeled neural crest cell-specific antibody, and magnetic beads bound to the antibody. and sorting using an affinity column on which the antibody is immobilized, but not limited thereto.

In the method of purifying by single cell formation, if necessary, the cells are subjected to mechanical dispersing treatment, dispersing treatment using enzymes such as collagenase and trypsin, and/or dispersing treatment using a chelating agent such as EDTA. It may be a cell state. In that case, a ROCK inhibitor may be added for the purpose of suppressing cell death. ROCK inhibitors are not particularly limited as long as they can inhibit the function of Rho-kinase (ROCK), and examples thereof include Y-27632, Fasudil/HA1077, H-1152, Wf-536, and derivatives thereof. be done. In addition, other known low-molecular-weight compounds can also be used as ROCK inhibitors (e.g., US Patent Application Publication Nos. 2005/0209261, 2005/0192304, 2004/0014755, 2004/0002508). , WO 2004/0002507, WO 2003/0125344, WO 2003/0087919, and WO 2003/062227, WO 2003/059913, WO 2003/062225, WO 2002/076976. 2004/039796).
In the method of purifying by sorting treatment, for example, a CD271-specific antibody can be used as the neural crest cell-specific antibody.

In the expansion culture in step B, culture conditions suitable for maintaining and/or growing neural crest cells can be used.

Culture conditions for expanding and culturing neural crest cells are not particularly limited as long as neural crest cells can be expanded and cultured, and culture conditions known per se can be used. One example is a method of culturing in a medium containing a TGFβ inhibitor, EGF (epidermal growth factor) and FGF2 (fibroblast growth factor 2).

A medium used for 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, Medium199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's
medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, StemPro34 (invitrogen), RPMI-base medium, StemFit (registered trademark) AK03N medium, and mixed media thereof. StemFit (registered trademark) medium is preferably used in this step. The medium may contain serum or may be serum-free. If necessary, the medium contains, for example, albumin, transferrin, Knockout Serum Replacement (KSR) (serum replacement for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen May contain one or more serum replacements such as precursors, trace elements, 2-mercaptoethanol (2ME), thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may also contain one or more substances such as growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, and the like.

In the present invention, a TGFβ inhibitor is a substance that inhibits signal transduction from binding of TGFβ to a receptor to SMAD, a substance that inhibits binding to the ALK family receptor, or a substance that inhibits SMAD by the ALK family. There is no particular limitation as long as it is a substance that inhibits phosphorylation. In the present invention, TGFβ inhibitors include, for example, Lefty-1 (exemplified by NCBI Accession No.: mouse: NM_010094, human: NM_020997), SB431542, SB202190 (R.K. 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, etc. are exemplified. The TGFβ inhibitor used for expansion culture of neural crest cells can preferably be SB431542.

The concentration of the TGFβ inhibitor such as SB431542 in the culture medium is not particularly limited as long as it inhibits ALK5, but is preferably 1 nM to 50 μM. 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, 50 μM but not limited to these. More preferably, it is 10 μM.

The concentration of EGF 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 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, but not limited to these. More preferably, it is 20 ng/ml.

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 ng/ml, 50 ng/ml, 60 ng/ml, 70 ng/ml, 80 ng/ml, 90 ng/ml, 100 ng/ml, but not limited to these. more preferably 20 ng/ml
is.

In addition, components other than those described above can be added to the medium as long as expansion culture of neural crest cells can be achieved.

The expansion culture of neural crest cells in step B may be either adherent culture or suspension culture, but is preferably performed by adherent culture.

Step B is characterized by using an extracellular matrix as a scaffold to achieve purification of neural crest cells.

As used herein, the term "scaffold" refers to (i) a cell-adhesive culture vessel, its material, a substance existing on the surface of the cell-adhesive culture vessel and constituting an adhesion site, and/or (ii) A substance that dissolves, disperses or suspends in a cell culture medium and forms a three-dimensional network in the cell culture medium, to which cells can adhere to the network.
Examples of such cell-adhesive incubators include incubators whose surface is artificially treated for the purpose of improving adhesion to cells, specifically surface-treated incubators, or , an incubator the inside of which is coated with a coating agent. Surface-treated culture vessels include culture vessels that have been surface-treated, such as by positive charge treatment.
Such scaffolds also include substances that coat the surface of culture vessels. Coating materials include laminin [including laminin α5β1γ1 (laminin 511), laminin α2β1γ1 (laminin 211), laminin α1β1γ1 (laminin 111), etc. and laminin fragments (laminin 511E8, etc.)], entactin, collagen, gelatin, vitronectin ), synthmax (Corning), extracellular matrices such as Matrigel, or polymers such as polylysine and polyornithine.
A substance that forms a three-dimensional network in such a cell culture medium exhibits the effect of uniformly suspending cells and/or tissues in a liquid culture medium. More specifically, low-molecular-weight compounds and high-molecular-weight compounds aggregate and self-organize via covalent bonds, ionic bonds, electrostatic interactions, hydrophobic interactions, Van der Waals forces, etc. to form nanofibers in liquid media. Nanofibers contained in the medium composition of the present invention include nanofibers obtained by refining a relatively large fiber structure made of a polymer compound by high-pressure treatment or the like. Although not bound by theory, in the medium composition of the present invention, nanofibers form a three-dimensional network that supports cells and tissues, thereby maintaining the cells and tissues in a suspended state.

In step B, the extracellular matrix used as a scaffold to achieve neural crest cell purification includes laminin or fibronectin, in particular laminin can be full length laminin, laminin with α2 chain or laminin 211.

Laminin is a glycoprotein that is a major component of basement membranes. Laminin is known to be involved in various cell functions such as cell adhesion, cell proliferation, metastasis and differentiation. Laminin is composed of heterotrimers with one each of α, β, and γ subunit chains. There are currently 5 types of α-subunit chains (α1, α2, α3, α4, α5), 3 types of β-subunit chains (β1, β2, β3), and 3 types of γ-subunit chains (γ1, γ2, γ3) Currently, 15 different laminin isoforms have been identified in humans, depending on the combination of these subunit chains. Laminin 211 that can be used in step B is α2
It may be laminin composed of subunit chains of chains, β1 chains, γ1 chains. The origin of laminin is preferably matched with the organism from which the neural crest cells are derived (for example, when human-derived neural crest cells are used, it is preferable to use human-derived laminin 211). It is known that laminin E8 fragment composed only of integrin-binding site has stronger cell adhesion activity than full-length laminin (Miyazaki T. et al., Nat Commun. 2012;3: 1236), the laminin 211 that can be used in step B can be the full length laminin 211 protein rather than a fragment. Laminin-211 may be prepared using a gene recombination technique known per se, or commercially available products may be used.

In step B, when the cell population is expanded in suspension culture, for example, laminin 211 is contained in the medium, and the floating cells use this as a scaffold to form aggregates, thereby obtaining a three-dimensional What is necessary is just to enable floating culture. Floating culture can be performed by a method known per se. One example is a method of expanding a cell population in suspension culture in a medium supplemented with laminin 211 while stirring the medium using a spinner flask or the like. Alternatively, a cell population can be expanded in suspension culture by combining polysaccharides (e.g., methylcellulose, xanthan gum, gellan gum, etc.) that have the effect of suspending cells when added to the medium, and laminin 211. In this embodiment, the cells spread three-dimensionally and proliferate in a state attached to nanofibers or in a sphere state. Cells adhere to the nanofibers and proliferate strongly using them as scaffolds, and as a result, the proliferated cells and cell clusters (spheres, etc.) are arranged on the nanofibers in the shape of grape clusters. Therefore, suspension culture of cells becomes possible. The concentration of laminin to be added may be appropriately set in consideration of various conditions such as the seeding density of the cell population and the concentration of the polysaccharide to be used in combination. In this specification, suspension culture means a culture method in which cells are not adhered to the surface of a culture vessel. Suspension culture may or may not be accompanied by physical agitation. Also, the cells to be cultured may be uniformly dispersed in the medium, or may be unevenly dispersed.

In step B, when expanding the cell population in adherent culture, the surface of the culture vessel is coated with laminin 211, for example. The coating amount of Laminin 211 is not particularly limited as long as the desired effects of the present invention can be obtained, and a generally recommended coating amount may be used. As an example, the coating amount of Laminin 211 is 0.1 ng/cm ² to 1000 ng/cm ² , preferably 0.5 ng/cm ² to 500 ng/cm ² , more preferably 1 ng/cm ² to 250 ng/cm 2 . ² , more preferably 2 ng/cm ² to 100 ng/cm ² , but not limited thereto.

In addition, although the culture period in step B may vary depending on the culture conditions, culture method, ratio of neural crest cells contained in the cell population, and the like, purification of neural crest cells is achieved in a relatively short period of time. Examples of culture periods include 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, and 1 to 13 days. days, 1-12 days, 1-11 days, 1-10 days, 1-9 days, 1-8 days, 1-7 days, 1-6 days, 1-5 days, 1-4 days, or 1- 3 days, but not limited to.

The culture temperature in step B is not particularly limited as long as neural crest cells can be cultured, but it is 30 to 40°C, preferably about 37°C. In addition, the CO ₂ concentration during culture in step B is not particularly limited as long as neural crest cells can be cultured, but is 2 to 5%, preferably about 5%.

In one embodiment, prior to step 1, the following steps:
(Step A) A step of 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 are performed.

In process A, 1. Neural crest cells can be obtained as described in the media composition . Neural crest cells can occur in the cell population used for manufacturing. Cell populations containing neural crest cells can vary greatly in the ratio of neural crest cells depending on the collected tissue and differentiation induction conditions. In one aspect of the present invention, the percentage of neural crest cells in a cell population containing neural crest cells is, for example, 1-95%, 1-90%, 1-85%, 1-80%, 1-75%, 1 ~70%, 1~65%, 1~60%, 1~55%, 1~50%, 1~45%, 1~40%, 1~35%, 1~30%, 1~25%, 1 It can be, but is not limited to, ~20%, 1-15%, or 1-10%. In another aspect, the percentage of neural crest cells in the cell population containing neural crest cells is, for example, 25-95%, 25-90%, 25-85%, 25-80%, 25-75%, It can be, but is not limited to, 25-70%, 25-65%, 25-60%, 25-55%, 25-50%, 25-45%, 25-40%, or 25-35%. In another aspect, the percentage of neural crest cells in the cell population containing neural crest cells is, for example, 50-95%, 50-90%, 50-85%, 50-80%, 50-75%, It can be, but is not limited to, 50-70%, 50-65%, or 50-60%. In another aspect, the percentage of neural crest cells in the cell population containing neural crest cells can be 60% or more, 70% or more, 80% or more, for example, 75-95%, 75-90%, or 75-85%, but is not limited to these.

In one embodiment, the cells are passaged as appropriate during step 1. Passaging is performed, for example, every 2-8 days or every 3-7 days after seeding. The passage interval is a period of time sufficient for the expansion of the cell aggregates, and a period shorter than the period during which the cell aggregates become too large and it becomes difficult for oxygen and nutrients to reach the cells inside the cell aggregates. preferably done.

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

In one embodiment, the cells are passaged as appropriate during step B or C. Passaging is performed, for example, every 2-8 days or every 3-7 days after seeding. The passage interval is a period of time sufficient for the expansion of the cell aggregates, and a period shorter than the period during which the cell aggregates become too large and it becomes difficult for oxygen and nutrients to reach the cells inside the cell aggregates. preferably done.

The number of passages during step B or C is, for example, 2 to 8 times (2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times), and 3 to 7 times. is preferred, more preferably 4 to 6 times. A portion of the cell population may be preserved as a frozen stock after an appropriate number of passages.

Transition from step B or C to step 1 is performed by exchanging the culture medium with the medium composition of the present invention. The timing of replacement may be after an appropriate number of passages, or may be during the final passage of step C. For example, 6 hours before the end of the last passage in step B or C is mentioned.

In one aspect, the present invention provides a production method for producing mesenchymal stem cells from a cell population containing neural crest cells,
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,
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, 1.25 μM or more and less than 300 μM, or 1.25 μM or more and less than 300 μM) in the presence of adrenocortical hormone culturing under
A production method for producing mesenchymal stem cells from a cell population containing neural crest cells is provided.

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

4. A method for producing chondrocytes.
The present invention provides a method for producing chondrocytes, including 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;
(Step E) A step of culturing the cell population containing the mesenchymal stem cells in a cartilage-inducing medium to obtain a cell population containing chondrocytes.

Chondrocytes mean cells that produce extracellular matrix that constitutes cartilage such as collagen, or progenitor cells that produce such cells. In the present application, the differentiation properties of chondrocytes are confirmed by Alcian blue staining. Alcian blue is a basic dye belonging to the phthalocyanine dyes, and includes sialylated mucosubstances (sialomucin), mucosubstances with sulfate groups (sulfomucin), chondroitin sulfate contained in cartilage and fibrous connective tissue, and hyaluronic acid. Staining acidic polysaccharides such as acidic mucopolysaccharides such as

Step D can be performed as described above for the method for producing mesenchymal stem cells of the present invention.

The medium used in step E can be prepared using a medium used for culturing mammalian cells as a basal medium. Examples of basal media 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 medium, Ham's F -12 medium, RPMI 1640 medium, Fischer's medium, mixed medium of these, etc., as long as it can be used for culturing mammalian cells, is not particularly limited.

In one aspect, the medium used in step E is serum-free medium. Serum-free medium means medium that does not contain unconditioned or unpurified serum. A medium containing purified blood-derived components or animal tissue-derived components is regarded as a serum-free medium.

The medium used in step E may contain serum replacement. Serum replacements may suitably contain, for example, albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3' thiol glycerol, or equivalents thereof. Such serum substitutes can be prepared, for example, by the method described in WO98/30679. In addition, commercially available serum substitutes can be used in order to more easily carry out the production method of the present invention. Examples of such commercially available serum substitutes include KSR (knockout serum replacement) (manufactured by Invitrogen), Chemically-defined Lipid concentration (manufactured by Gibco), and Glutamax (manufactured by Gibco).

The medium used in step E can contain other additives as long as the direction of differentiation of mesenchymal stem cells into chondrocytes is not impaired. Additives include, for example, insulin,
Iron sources (eg transferrin etc.), minerals (eg sodium selenate etc.), sugars (eg glucose etc.), organic acids (eg pyruvic acid, lactic acid etc.), serum proteins (eg albumin etc.), amino acids (eg L-glutamine etc.) ), reducing agents (eg, 2-mercaptoethanol, etc.), vitamins (eg, ascorbic acid, d-biotin, etc.), antibiotics (eg, streptomycin, penicillin, gentamicin, etc.), buffers (eg, HEPES, etc.), etc. , but not limited to.
The medium used in step E contains tumor necrosis factor-α (TNF-α), IL-1β and IL-17.
It preferably does not contain a substance that inhibits the differentiation of mesenchymal stem cells into chondrocytes.

In step E, the incubator used for culturing cells is not particularly limited as long as it can culture cells. Plates, microwell plates, multiplates, multiwell plates, microslides, chamber slides, petri dishes, tubes, trays, culture bags, roller bottles, and the like.

In step E, the incubator used for culturing the cells is preferably cell-adhesive. As long as the direction of differentiation of mesenchymal stem cells to chondrocytes is not impaired, the cell-adhesive culture vessel should be made hydrophilic for the purpose of improving the adhesion to cells on the surface of the culture vessel. They may be imparted or coated with any cell-supporting substrate such as extracellular matrix (ECM) or an artificial material that mimics their function.
Although it is not limited as long as the direction of differentiation of mesenchymal stem cells into chondrocytes is not impaired, examples of the ECM that coats the culture vessel include fibronectin, collagen, etc.
Fibronectin is preferred.

In step E, the mesenchymal stem cells can be cultured by a method known per se such as adhesion culture, suspension culture, and tissue culture, but adhesion culture is preferred.

Other culture conditions can be set as appropriate. For example, the culture temperature is not particularly limited as long as the desired effect can be achieved, but it is about 30-40°C, preferably about 37°C. The CO ₂ concentration is about 1-10%, preferably about 2-5%. Oxygen concentration is usually 1 to 40%, but can be appropriately selected according to culture conditions and the like.

The mesenchymal stem cells of the present invention are characterized by their high ability to differentiate into chondrocytes. High differentiation potential into chondrocytes means that the number of chondrocytes generated as a result of inducing differentiation of the mesenchymal stem cells of the present invention into chondrocytes is higher than that of other mesenchymal stem cells, specifically biologically-derived Mesenchymal stem cells, especially bone marrow-derived mesenchymal stem cells, were induced to differentiate into chondrocytes, resulting in a greater number of chondrocytes. More specifically, high differentiation potential into chondrocytes means that other mesenchymal stem cells are differentiated in a medium for inducing chondrocyte differentiation under similar (preferably identical) culture conditions. 1.5-fold or more, 2-fold or more, 2.5-fold or more, 3-fold or more, 3.5-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold It means 9 times or more, 10 times or more, 50 times or more, or 100 times or more.

5. Agent for Repairing Cartilage Tissue Further, the present invention provides an agent for repairing cartilage tissue, which contains the chondrocytes of the present invention or a culture thereof. "Culture" refers to the result obtained by culturing cells/cell populations in the production method of the present invention, and includes cells, media, and in some cases cell-secreted components.

Cartilage tissue is connective tissue composed of cartilage matrix and chondrocytes. Cartilage is divided into hyaline cartilage (articular cartilage, epiphyseal plate, costal cartilage, tracheal cartilage, laryngeal cartilage, etc.),
It is classified into fibrocartilage (sacroiliac joint, temporomandibular joint, sternoclavicular joint, intervertebral disc, pubic symphysis, joint meniscus, joint disc, etc.) and elastic cartilage (external auditory canal, eustachian tube, auricular cartilage, epiglottis cartilage, etc.). be. The agent of the present invention can be usefully used for repairing any of hyaline cartilage, fibrocartilage, and elastic cartilage.

As used herein, an agent for repairing cartilage tissue means 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 vivo where repair (regeneration) of cartilage tissue is desired, cartilage tissue is locally formed at the site of administration, resulting in repair of cartilage tissue. (regeneration) is achieved.

The chondrocytes obtained by the present invention themselves serve as transplant materials. Therefore, the chondrocytes can also be transplanted into patients as cell preparations.

In addition, mesenchymal cells obtained by the present invention are cultured on a substrate (scaffold) made of an artificial material such as biodegradable fibers to induce differentiation into chondrocytes to form a graft material. , can be ported.

The amount of cells obtained in step 1 seeded per unit surface area of the cell-contacting surface of the scaffold is, for example, 1×10 ⁴ to 1×10 ⁷ cells/cm ² , 2×10 ⁴ to 5×10 ⁵ cells. /cm ² , can be 5×10 ⁴ to 2×10 ⁵ /cm ² .

The cell culture time in step E in the presence of a scaffold is usually 6 hours or longer, preferably 12 hours or longer, and more preferably 18 hours or longer. Although the upper limit of culture time is not particularly limited, it is usually 72 hours or less, preferably 48 hours or less, and more preferably 30 hours or less.

The agent for repairing cartilage tissue of the present invention includes other compounds such as antibiotics, anti-inflammatory agents, immunosuppressants, cytokines, antiseptics, analgesics, stabilizers (antioxidants, ultraviolet absorbers, heat stabilizers, etc.), other therapeutic agents, and the like.

The agent for repairing cartilage tissue of the present invention is implanted according to an appropriate implantation method in accordance with medical practitioners' guidelines. For example, when the agent for repairing cartilage tissue of the present invention is to be implanted in a knee joint, the site of the joint to be implanted is cut with a scalpel or the like to open the joint cavity to create a cavity, and the cartilage of the present invention is inserted into the joint cavity. Implantation can be achieved by administering a drug to repair the tissue.

When a therapeutically effective amount of the agent for repairing cartilage tissue of the present invention is transplanted into a site requiring repair of cartilage tissue, the transplanted cells differentiate into chondrocytes, and the chondrocytes and chondrocytes produce Cartilage is repaired and a therapeutic effect is achieved by supplementing the defect site with the cartilage matrix.

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

The agent for repairing cartilage tissue of the present invention is suitable as a transplant material for treatment of cartilage damage. As used herein, cartilage damage refers to physical cartilage defect/degeneration/damage due to sports, accidents, etc., rheumatoid arthritis, knee osteoarthritis, hip osteoarthritis, osteosarcoma, femoral head necrolysis, The term is used in the sense of including defects/degeneration/damage of cartilage caused by diseases such as operculum dysplasia, meniscal injury, and traumatic arthritis.

Sites requiring cartilage tissue repair include, for example, articular cartilage, epiphyseal plate, costal cartilage, tracheal cartilage, laryngeal cartilage, sacroiliac joint, temporomandibular joint, and sternoclavicular joint, which have cartilage tissue defect/degeneration/damage. , intervertebral disc, pubic symphysis, joint meniscus, joint disc, external auditory canal, auditory tube, auricular cartilage and epiglottis cartilage.

6. Method for Repairing Cartilage Tissue The present invention comprises the step of transplanting a therapeutically effective amount of the chondrocytes and/or the agent for repairing cartilage tissue of the present invention into a site of a mammal in need of cartilage tissue repair. Methods of treating cartilage damage are provided, comprising:

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

As used herein, "effective amount" means that amount of active ingredient that produces the desired effect. As used herein, "therapeutically effective amount" means that amount of active ingredient that produces the desired therapeutic effect when administered to a subject. A therapeutically effective amount may be administered (implanted) at once or divided into multiple doses (implanted). The number of applications for transplantation depends on the disease and is determined by medical practitioners and guidelines. When transplanting multiple times, the interval is not particularly limited, but a period of several days to several weeks may be provided.

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

7. Method for Producing Agent for Restoring Cartilage Tissue The present invention provides a method for producing an agent for restoring cartilage tissue containing 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;
(Step E) A step of culturing the cell population containing the mesenchymal stem cells in a cartilage-inducing medium to obtain a cell population containing chondrocytes.
Steps D and E in the method for producing a drug for repairing cartilage tissue of the present invention can be performed 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. can.

In the method for producing a drug for repairing cartilage tissue of the present invention, neural crest cells that are induced to differentiate into mesenchymal stem cells, pluripotent stem cells when the neural crest cells are derived from pluripotent stem cells, When the potential stem cells are iPS cells, the individual that is the source of the somatic cells to be induced into the iPS cells is not particularly limited. These cells may be histocompatible to the extent that mesenchymal stem cells derived from the donor's cells are able to engraft the recipient.

For example, when the agent for repairing cartilage tissue of the present invention is used for repairing cartilage tissue in humans, these cells are the patient's own cells from the viewpoint of preventing graft rejection and/or GvHD. or obtained from another person with an HLA type that is identical or substantially identical to the patient's HLA type. As used herein, "substantially identical HLA
"Type" means that the donor's HLA type matches that of the patient to the extent that mesenchymal stem cells derived from the donor's cells can engraft when transplanted into a patient with the use of immunosuppressants, etc. means that Examples thereof include HLA types having the same major HLA (3 main loci of HLA-A, HLA-B and HLA-DR, or 4 loci including HLA-Cw).

The agent for repairing cartilage tissue of the present invention includes other compounds such as antibiotics, anti-inflammatory agents, immunosuppressants, cytokines, antiseptics, analgesics, stabilizers (antioxidants, ultraviolet absorbers, heat stabilizers, etc.), other therapeutic agents, and the like.

The present invention will be described in more detail in the following examples, but the present invention is not limited by these examples.

Example 1: Induction of differentiation from iNCCs (iPS cell-derived neural crest cells) to iMSCs (iPS cell-derived mesenchymal stem cells)
1. Differentiation induction from iPS cells to neural crest cells (iNCC)
201B7 (iPS portal) was used as an iPS cell line. iPS cells were seeded at 6500 cells/well on a 6-well plate coated with laminin 511-E8 fragment (iMatrix511, Nippi) and placed in StemFit (registered trademark) AK03N (Ajinomoto Co.) medium at 37°C. , and cultured for 5 days under 5% CO ₂ . Then, the medium is StemFit AK03N (solution A + solution B) with SB431542 (Stemgent, Inc., 10 μM) and CHIR99021 (Wako, 0.3 μM (condition 1) or 0.9 μM (condition 2)) in a medium, Differentiation into neural crest cells was induced for 14 days at 37°C and 5% CO ₂ . The percentage of neural crest cells at the end of induction was calculated by determining the percentage of cells with high CD271 protein expression using FACS. In addition, the gene expression status of neural crest cell markers was examined by RT-PCR. The primers used in the RT-PCR method are shown below.

Marker gene Primer ID Taqman Cat.No.
TFAP2a Hs00271528_CE A15629
SOX9 Hs01001343_g1 4331182
TWIST1 Hs01675818_s1 4331182
(using βactin as a reference gene (Hs01101944_s1, 4331182))

2. Purification and expansion culture of neural crest cells (iNCC) The cell population containing neural crest cells produced in 1 above was converted to single cells using TrypLE Select (Thermo Fisher). The single-celled cell population was seeded on a scaffold-coated 6-well plate and cultured under culture conditions suitable for expansion culture of neural crest cells. More specifically, StemFit AK03N (A solution + B solution), SB431542 (10 μM), Epithelium growth factor (Sigma
Chemical, 20 ng/mL) and StemFit AK03NC solution (Ajinomoto Co., Inc., 0.08%), and cultured at 37° C. under 5% CO ₂ for 9 to 10 days. After culturing for 9 to 10 days, when the confluency reached about 90%, the percentage of neural crest cells in the cell population cultured on the scaffold material and the expression status of neural crest cell gene markers were evaluated as described in 1. above. determined by the same method as

Laminin 211 (Biolamina, 6.3 ng/cm ² and 62.5 ng/cm ² ) was used as a scaffold.
The scaffold was coated on the 6-well plate surface by directly suspending it in the culture medium.

3. Induction of Differentiation from iNCCs to iMSCs Obtained iNCCs (iPSC-derived NCCs) were transferred to a 6-well plate coated with vitronectin-N (Thermo Fisher Scientific) at a density of 1.5 μg/cm ² at 5.0×10 ³ . Seeded at a density of cells/well and placed in the following differentiation medium (T3
medium) was cultured for 13 days at 37° C. under 5% CO ₂ conditions to induce iMSCs (iPSC-derived MSCs).
T3 medium: StemFit (registered trademark) For Mesenchymal Stem Cells (Ajinomoto Co., Inc.), 90 nM Dexamethasone (Sigma-Aldrich)
Surface antigen analysis of 3 MSC positive markers (CD90, CD44, CD73) and 1 negative marker (CD34) was performed on the induced iMSCs using FACS. Table 1 shows the analysis results.

The obtained cells were positive for CD90, CD44 and CD73 and negative for CD34, confirming iMSC induction.

Example 2: Dexamethasone Concentration Study iNCCs were seeded at a density of 1.5 μg/cm ² onto vitronectin-N coated 6-well plates at a density of 3.0×10 ⁴ cells/well and subjected to StemFit® For Dexamethasone was added to Mesenchymal Stem Cells at 50 nM, 100 nM, 1 μM, 10 μM and 50 μM to induce iMSCs under conditions of 37° C. and 5% CO ₂ .
FIG. 1 shows cell growth curves during induction of differentiation. In the group to which 50 μM dexamethasone was added, cell growth stopped after 13 days.

Example 3: Examination of types of adrenocortical 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.
For the induced iMSCs, FACS was used to identify 4 MSC-positive markers (CD90, CD44, CD73, CD105), 1 NCC-positive marker (MSC weakly positive marker) (CD271), and 2 MSC-negative markers (CD45 , CD34) surface antigen analysis was performed. Table 2 shows the analysis results.

Similar to dexamethasone, prednisolone had a limited effect even when added at 100 nM.
When the titer as glucocorticoid was uniformed to 666 nM, there was an effect of promoting differentiation.

Example 4: Induction of differentiation from iNCCs to iMSCs and induction of differentiation of the iMSCs into chondrocytes
1. Differentiation induction from iPS cells to iNCC
Human iPSCs (strain 1231A3) were transfected with laminin 511-E8 fragment (iMatrix-511, Nippi Co., Ltd.)
coated plates or dishes at a density of 3.6 × 10 ³ cells/cm ² and cultured in StemFit AK03N medium for 4 days. After that, the medium was added with 10 μM SB431542 (FUJIFILM Wako).
and StemFit Basic03 (AK03N without bFGF, Ajinomoto, Tokyo, Japan) containing 1 μM CHIR99021 (Axon Medchem, Reston, VA, USA) and cultured for 10 days to induce differentiation of the cells into iNCCs. Cell numbers were counted using a Countess II FL (Thermo Fisher Scientific). The medium was replaced every 2 days from day 0 (the day of medium replacement) to day 6 from the start of induction of differentiation into iNCCs, and every day from day 7 to day 10.

2. Purification and expansion of iNCC 1. The obtained cells were subjected to FACS analysis, CD271 ^high positive cells were seeded on fibronectin-coated plates at a density of 1 × 104 cells/ ^cm2 and treated with ¹⁰ μM SB431542, 20 ng/mL EGF (FUJIFILM Wako), and FGF2 ( The cells were cultured in Basic03 medium supplemented with FUJIFILM Wako). Medium was changed every 3 days. Upon passaging, cells were detached with Accutase (Innovative Cell Technologies, San Diego, CA, USA) and replated on fibronectin-coated plates at a density of ¹ × 104 cells/ ^cm2 . After two subcultures to increase the number of cells sufficiently, 5 × 10 ⁵ iNCCs were suspended in 500 μl of STEM-CELL BANKER GMP grade (Takara, Kusatsu, Japan) and placed in a BICELL freezing container ( Nippon Freezer, Tokyo, Japan) was used to produce frozen stocks of purified iNCC.

3. Differentiation induction from iNCC to iMSC2 . Frozen stocks obtained in were thawed, seeded onto fibronectin-coated plates at a density of 1 × 104 cells/ ^cm2 , and cultured in ^Basic03 supplemented with 10 μM SB431542, 20 ng/mL EGF, and FGF2. Six hours before the first subculture, the medium was changed to T1 medium, and the cells were then cultured in the same medium to induce differentiation into MSCs. Passaging was performed every 4 days using Accutase at a density of 1 × 10 ⁴ cells/cm ² . As a control, PRIME-EV MSC Expansion medium (manufactured by FUJIFILM Irvine Scientific, Tokyo Japan), which has been widely used for inducing differentiation from iNCCs to iMSCs, was used.

FIG. 2 shows phase-contrast microscopy images of the cells after replacement with the differentiation-inducing medium every two passages (every 8 days). The upper row is the result of using the T1 medium, and the lower row is the result of using the control medium. From Fig. 2, regardless of whether the T1 medium or the control medium was used, the morphology of the cells began to change about 4 days after the induction of differentiation, and after 8 days, they took on mesenchymal stem cell-like morphology, and thereafter changed their morphology. was maintained.

FIG. 3 shows the results of measuring the number of cells over time after replacement with the differentiation-inducing medium. In the group induced to differentiate with the control medium, cell proliferation was slow to start, and almost no increase in cell number was observed until 3 days after the initiation of differentiation induction. On the other hand, in the group induced to differentiate in T1 medium, the cells proliferated exponentially from the beginning, and continued to proliferate smoothly thereafter. As a result, during the entire measurement period (from the initiation of differentiation induction to 35 days later), the group induced to differentiate with the T1 medium always had more cells than the group induced to differentiate with the control medium.

Next, time-course changes in the expression levels of human MSC markers (CD44, CD73, CD90, and CD105) in cells after the initiation of differentiation induction were analyzed by quantitative RT-PCR (Thunderbird SYBR qPCR Mix (TOYOBO, Osaka, Japan). Analysis was performed using the QuantStudio 7 Flex real-time PCR system used (Applied Biosystems, Forester City, Calif., USA)).
T1 medium: StemFit (registered trademark) 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 FIG.

As shown in FIG. 3, the expression levels of CD44, CD73, and CD105 in iMSCs differentiated using T1 medium were comparable to those of iMSCs differentiated using control medium.

From the above results, it was confirmed that differentiation of iNCCs into iMSCs can be induced from iNCCs by using T1 medium, as in the conventional method. Furthermore, it was also revealed that more iMSCs can be obtained than when a general-purpose medium is used.

4. 3. Induction of iMSC differentiation into chondrocytes . 1.5×10 ⁵ iMSCs obtained in 5 μl of cartilage induction medium ((DMEM/F12, Thermo Fish
er Scientific), 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), 10 ng/mL TGF-β3 (WAKO), 100 ng/ After suspension with ml BMP7 (WAKO) and 1% (v/v) FBS (Thermo Fisher Scientific) medium), the cells were transferred to a fibronectin-coated 24-well plate. After 1 hour, the cartilage induction medium was added to a total volume of 1 mL, and cultured for 14 days while changing the medium every 2 days. Differentiation properties of 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 applying Alcian blue solution (1% Alcian blue (MUTO PURE CHEMICAL CO., LTD, Tokyo, Japan)) at 25°C for 1 hour. FIG. 5 shows the staining results.

As shown in Figure 5, the cartilage induction of iMSCs differentiated in T1 medium (left image in Figure 5) is higher than the cartilage induction of iMSCs differentiated in control medium (right image in Figure 5). , very prominently deep-dyed with alcian blue.
Therefore, it was shown that when iNCCs are differentiated into iMSCs using T1 medium, iMSCs with excellent differentiation potential into chondrocytes can be obtained.

The converted concentrations of dexamethasone 50 nM, 90 nM, 100 nM, 1 μM, 10 μM, and 50 μM in this example are 1.333 μM, 2.3994 μM, 2.666 μM, 26.66 μM, 266.6 μM, and 1333 μM, respectively ( converted to a dexamethasone titer of 26.66).
The converted concentrations of 100 nM and 666 nM prednisolone in this example are 400 nM and 2.664 μM, respectively (converted with the titer of prednisolone being 4). The converted concentration of 100 nM betamethasone in this example is 2.666 μM (converted based on the betamethasone titer of 26.66).

According to the present invention, it is possible to provide a medium composition for efficiently inducing mesenchymal stem cells from neural crest cells. In addition, according to the present invention, there are provided an efficient method for producing mesenchymal stem cells, a method for producing chondrocytes, a drug for repairing cartilage containing the mesenchymal stem cells as an active ingredient, and repair of cartilage. A method, a method for producing a drug for repairing cartilage tissue, and the like can be provided. Therefore, the present invention is extremely useful, for example, in the medical field.

Claims (26)

A medium composition for inducing mesenchymal stem cells from neural crest cells, containing a basal medium and adrenocortical hormone, wherein the converted concentration of the adrenocortical hormone in the medium composition is 1.25 μM or more. . The medium composition according to claim 1, wherein the adrenocortical hormone is a glucocorticoid or derivative thereof. 3. According to claim 2, wherein the glucocorticoid or 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. Media composition as described. 4. The medium composition according to claim 2 or 3, wherein the glucocorticoid or derivative thereof is dexamethasone. The medium composition according to any one of claims 1 to 4, wherein the converted concentration of adrenocortical hormone in the medium composition is 300 µM or less. The medium composition according to claim 4, wherein the concentration of dexamethasone in the medium composition is 10 µM or less. The medium composition according to any one of claims 1-6, wherein the basal medium is a serum-free medium. The medium composition according to any one of claims 1 to 7, wherein the neural crest cells are derived from pluripotent stem cells. The medium composition according to claim 8, wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells). 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 claims 1 to 7 (step 1). The production method according to claim 10, wherein the neural crest cells are derived from pluripotent stem cells. The production method according to claim 11, wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells). The manufacturing method according to any one of claims 10 to 12, wherein the following steps are performed before step 1:
(Step A) A step of 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 production method according to claim 13, wherein the extracellular matrix is laminin or fibronectin. 15. The production method according to claim 14, wherein the laminin is full-length laminin, laminin having an α2 chain, or laminin 211. Claims 13 to 13, wherein the cell population obtained in step B is a cell population containing 70% or more neural crest cells
16. The production method according to any one of 15. A production 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 adrenocortical hormone at an equivalent concentration of 1.25 μM or more and 300 μM or less,
A production method for producing mesenchymal stem cells from a cell population containing neural crest cells. 18. The production method according to claim 17, wherein the adrenocortical hormone is a glucocorticoid or a derivative thereof. 19. According to claim 18, wherein the glucocorticoid or 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. Method of manufacture as described. 20. The production method according to claim 18 or 19, wherein the glucocorticoid or derivative thereof is dexamethasone. A mesenchymal stem cell or a culture thereof obtained by the production method according to any one of claims 10 to 20. 22. The mesenchymal stem cell or culture thereof according to claim 21, wherein the mesenchymal stem cell is characterized by high differentiation potential into chondrocytes. A method of producing chondrocytes, comprising the steps of:
(Step D) a step of obtaining a cell population containing mesenchymal stem cells by the production method according to any one of claims 10 to 20;
(Step E) A step of culturing the cell population containing the mesenchymal stem cells in a cartilage-inducing medium to obtain a cell population containing chondrocytes. Chondrocytes or a culture thereof obtained by the production method according to claim 23. A drug for repairing cartilage tissue, comprising the chondrocyte according to claim 24 or a culture thereof. A method for producing a medicament for repairing cartilage tissue containing chondrocytes, the method comprising the steps of:
(Step D) a step of obtaining a cell population containing mesenchymal stem cells by the production method according to any one of claims 10 to 20;
(Step E) A step of culturing the cell population containing the mesenchymal stem cells in a cartilage-inducing medium to obtain a cell population containing chondrocytes.

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