JP2023100226A - Method for producing differentiated smooth muscle cell - Google Patents

Abstract

To provide a method for producing a differentiated smooth muscle cell having a contractile ability from a mesenchymal stem cell.SOLUTION: The present invention provides a method for producing a differentiated smooth muscle cell from a mesenchymal stem cell, including the steps of (1) culturing a mesenchymal stem cell in a medium containing an MEK inhibitor and a transforming growth factor β1 and (2) recovering a cell population with the proportion of cells expressing desmin and/or smooth muscle myosin heavy chains being 20% or more.SELECTED DRAWING: None

Description

Application for application of Article 30, Paragraph 2 of the Patent Act (1) Web abstract collection of the 20th Annual Meeting of the Japanese Society for Regenerative Medicine https://site2. convention. co. jp/20jsrm/program/index. html Posting date: March 5, 2021 (2) The 20th General Assembly of the Japanese Society for Regenerative Medicine, held entirely online https://site2. convention. co. jp/20jsrm/ Posting date March 11, 2021

The present invention relates to a method for producing differentiated smooth muscle cells and use of the produced differentiated smooth muscle cells.

Smooth muscle cells are found in various hollow organs such as the gastrointestinal tract, ureter, trachea, and blood vessels, and play very important roles in various organs, such as participating in contraction/dilation and peristalsis. Therefore, especially in regenerative medicine, these cells are necessary for the production of various artificial organs such as major organs such as respiratory tracts, digestive tracts, bladders, blood vessels, and uterus.

Although a large amount of cells are required for organ regeneration, the procedure for isolating smooth muscle cells from living organisms is complicated, and the amount of smooth muscle cells to be collected is limited. Therefore, induction of differentiation from stem cells is essential. In addition, most mature smooth muscle cells are differentiated (contractile) smooth muscle cells with strong contractility, but under normal culture conditions, they are transformed into dedifferentiated (synthetic) smooth muscle cells with weak contractility. convert it. To date, there have been several reports on methods for inducing differentiation from mesenchymal stem cells to smooth muscle cells, but no definite methods have been demonstrated, and methods for inducing differentiation of differentiated smooth muscle cells are unknown. .

The inventors of the present application have succeeded in creating an artificial esophagus with a bio-3D printer using a three-dimensional culture prepared from esophageal smooth muscle cells and bone marrow mesenchymal stem cells, and the survival rate after transplantation into rats and after surgery Effectiveness was confirmed (Non-Patent Document 1). However, the quality of the artificial esophagus produced was unstable, and there were problems such as unstable strength and lack of contractile function.

Takeoka Y, Matsumoto K, Taniguchi D, Tsuchiya T, Machino R, Moriyama M, Oyama S, Tetsuo T, Taura Y, Takagi K, Yoshida T, Elgalad A, Matsuo N, Kunizaki M, Tobinaga S, Nonaka T, Hidaka S , Yamasaki N, Nakayama K, Nagayasu T. PLoS ONE (2019) 14(3), e0211339.

An object of the present invention is to provide a method for producing differentiated smooth muscle cells having contractility from mesenchymal stem cells.

The present invention includes the following inventions in order to solve the above problems.
[1] A method for producing differentiated smooth muscle cells from mesenchymal stem cells, comprising the steps of (1) culturing mesenchymal stem cells in a medium containing a MEK inhibitor and transforming growth factor β1, and (2) ) A production method comprising the step of recovering a cell population in which the ratio of cells expressing desmin and/or smooth muscle myosin heavy chain is 20% or more.
[2] In step (1), culture in a medium containing MEK inhibitor and transforming growth factor β1 for at least 4 days, then culture in medium containing transforming growth factor β1 but not MEK inhibitor. , the manufacturing method according to the above [1].
[3] The production method according to [1] or [2], wherein the culture period in step (1) is 3 weeks or more and 5 weeks or less.
[4] The above [1] to [3], wherein the mesenchymal stem cells are mesenchymal stem cells obtained by inducing differentiation from mesenchymal stem cells or pluripotent stem cells collected from bone marrow or adipose tissue. The manufacturing method according to any one of the above.
[5] The production method according to any one of [1] to [4] above, wherein the mesenchymal stem cells are mesenchymal stem cells obtained from a subject in need of smooth muscle cell transplantation.
[6] The production method according to any one of [1] to [5], wherein the MEK inhibitor is PD98059.
[7] A step of producing differentiated smooth muscle cells by the production method according to any one of [1] to [6] above, and a step of three-dimensionally culturing the obtained differentiated smooth muscle cells to form spheroids. A method for producing smooth muscle cell spheroids, comprising:
[8] The above-mentioned [7], wherein in the three-dimensional culture, the differentiated smooth muscle cells are mixed with one or more cells selected from nerve cells, vascular cells, and fibroblasts to form spheroids. manufacturing method.
[9] Differentiated smooth muscle cells obtained by the production method according to any one of [1] to [6] above, or smooth muscle obtained by the production method according to [7] or [8] above A pharmaceutical composition for treating smooth muscle damage, containing cell spheroids.

INDUSTRIAL APPLICABILITY The present invention can provide a method for producing differentiated smooth muscle cells having contractility from mesenchymal stem cells. Differentiated smooth muscle cells produced by the production method of the present invention and spheroids formed from the differentiated smooth muscle cells can be used as smooth muscle cells for transplantation to a living body for treatment of smooth muscle injury.

FIG. 1 shows that in Example 1, rat bone marrow-derived mesenchymal stem cells (BM-MSCs) were cultured under six culture conditions (groups 1 to 6), and differentiated smooth muscle cell markers (α-smooth muscle actin, Desmin, calponin, smooth muscle myosin heavy chain) were observed by immunostaining. Scale bar indicates 100 μm. FIG. 2 shows that rat BM-MSCs were cultured in a medium containing PD98059 and TGF-β1 for 1 week in Example 2, followed by differentiated smooth muscle cell markers (α-smooth muscle actin, desmin, calponin, smooth muscle myosin heavy chain). ) was observed by immunostaining. Scale bar indicates 200 μm. FIG. 3 shows that rat BM-MSCs were cultured in a medium containing PD98059 and TGF-β1 for 2 weeks in Example 2, and then differentiated smooth muscle cell markers (α-smooth muscle actin, desmin, calponin, smooth muscle myosin heavy chain ) was observed by immunostaining. Scale bar indicates 200 μm. FIG. 4 shows that rat BM-MSCs were cultured in a medium containing PD98059 and TGF-β1 for 3 weeks in Example 2, and then differentiated smooth muscle cell markers (α-smooth muscle actin, desmin, calponin, smooth muscle myosin heavy chain ) was observed by immunostaining. Scale bar indicates 200 μm. FIG. 5 shows that in Example 2, rat BM-MSCs were cultured in a medium containing PD98059 and TGF-β1 for 4 weeks, followed by differentiation of differentiated smooth muscle cell markers (α-smooth muscle actin, desmin, calponin, smooth muscle myosin heavy chain). ) was observed by immunostaining. Scale bar indicates 200 μm. FIG. 6 shows rat BM-MSCs cultured in medium containing PD98059 and TGF-β1 for 1 week, 2 weeks, 3 weeks, 4 weeks, or TGF-β1 after culturing in medium containing PD98059 and TGF-β1 for 1 week. The expression of differentiated smooth muscle cell markers (α-smooth muscle actin, desmin, calponin, smooth muscle myosin heavy chain) was observed by immunostaining for cells cultured for an additional 3 weeks in a medium containing, and each positive cell rate was compared. This is the result. Scale bar indicates 200 μm. 7 is a diagram showing an overview of the collagen-based cell contraction assay in Example 3. FIG. FIG. 8 shows, in Example 3, cell-free (group 1), rat dermal fibroblasts cultured in basal medium (DF, group 2), rat smooth muscle cells cultured in basal medium (SMC, group 3), basal Rat mesenchymal stem cells (MSCs, group 4) cultured in medium, PD98059 and rat BM- This is the result of performing a cell contraction assay on MSCs (group 5). ET-1(+) and ET-1(-) indicate the presence or absence of endothelin-1 addition. FIG. 9 shows the results of comparing the contraction area of collagen gels containing no cells or each cell in the cell contraction assay of Example 3. FIG.

[Method for Producing Differentiated Smooth Muscle Cells]
The present invention provides a method for producing differentiated smooth muscle cells from mesenchymal stem cells (hereinafter referred to as "the production method of the present invention"). The production method of the present invention may include the following steps (1) and (2).
(1) culturing mesenchymal stem cells in a medium containing a MEK inhibitor and transforming growth factor β1 (hereinafter referred to as “TGF-β1”), and (2) desmin and/or smooth muscle A step of recovering a cell population in which the percentage of cells expressing myosin heavy chain (smooth muscle myosin heavy chain, hereinafter referred to as "SMMHC") is 20% or more.

Differentiated smooth muscle cells, also called contractile smooth muscle cells, mean mature smooth muscle cells with strong contractile ability, and dedifferentiated smooth muscle cells with weak contractile ability (synthetic Also called smooth muscle cells). It is known that when differentiated smooth muscle cells are cultured in a normal culture environment, they readily undergo dedifferentiation and change into dedifferentiated smooth muscle cells with weak contractility. Differentiated smooth muscle markers include α-smooth muscle actin (hereinafter referred to as “α-SMA”), desmin, calponin, SMMHC and the like.

Mesenchymal stem cells (MSCs) used in the production method of the present invention are a kind of somatic stem cells derived from mesodermal tissues (mesenchyme) such as bone marrow and fat, and are used in bone, cartilage, and fat. Cells that have the ability to differentiate into mesodermal tissues such as. Mesenchymal stem cells can be identified using the expression or non-expression of a specific gene as an index. Genes expressed in mesenchymal stem cells (mesenchymal stem cell markers) include, for example, CD106, CD166, CD29, CD105, CD73, CD44, CD90, CD71, Stro-1 and the like. Examples of genes (negative markers) that are not expressed in mesenchymal stem cells include CD31, CD18, CD56, CD45, CD34, CD14, CD11, CD80, CD86, and CD40.

The mesenchymal stem cells used in the production method of the present invention may be mesenchymal stem cells collected from a living body, or may be mesenchymal stem cells obtained by inducing differentiation from pluripotent stem cells. Mesenchymal stem cells collected from a living body can preferably be mesenchymal stem cells collected from bone marrow or adipose tissue. The mesenchymal stem cells used in the production method of the present invention may be human mesenchymal stem cells or non-human mesenchymal stem cells. Non-human organisms are not particularly limited, and may be, for example, mammals. Examples of mammals include monkeys, chimpanzees, dogs, cats, cows, horses, pigs, rabbits, mice, rats and the like. A method for collecting mesenchymal stem cells from bone marrow or adipose tissue can be appropriately selected from known methods. Examples thereof include the method described in Ishii et al. (Pharmaceutics (2021) 13:1264). A method for inducing differentiation of pluripotent stem cells to obtain mesenchymal stem cells can be appropriately selected from known methods. Examples thereof include the method described in Dupuis V. et al. (World Stem Cells (2021) 13:1094-1111). Pluripotent stem cells that can be used to obtain mesenchymal stem cells include, for example, embryonic stem cells (ES cells), cloned embryo-derived embryonic stem cells (ntES cells) obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), cultured fibroblasts and bone marrow stem cell-derived pluripotent cells (Muse cells). Mesenchymal stem cells collected from a living body may be mesenchymal stem cells obtained by cryopreserving mesenchymal stem cells isolated from a living body, or mesenchymal stem cells deposited in a depository such as a cell bank. good too.

Since the differentiated smooth muscle cells produced by the production method of the present invention can be suitably used as smooth muscle cells for transplantation into smooth muscle injury sites, the mesenchymal stem cells used in the production method of the present invention are Mesenchymal stem cells obtained from a subject in need of smooth muscle cell transplantation are preferred. Subjects requiring transplantation of the present smooth muscle cells include, for example, mammals such as mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, and humans, preferably humans. Since smooth muscle cells obtained by inducing the differentiation of one's own mesenchymal stem cells are used for transplantation, transplantation becomes possible without causing rejection.

In step (1), mesenchymal stem cells are cultured in a medium containing a MEK inhibitor and TGF-β1. The medium used in step (1) is not particularly limited as long as it is suitable for culturing cells in the process of differentiation from mesenchymal stem cells to smooth muscle cells, and a medium generally used as a medium for animal cells may be used. can be done. Examples thereof include minimal essential medium (MEM), Dulbecco's modified minimal essential medium (DMEM), RPMI1640 medium, 199 medium, Ham's F-12 medium, William's E medium, and mixed medium thereof. Serum-free media may be used, and serum-free media may be media containing serum-free supplements. As the serum-free supplement, commercially available serum-free supplements and known serum-free supplements can be suitably used. Specifically, for example, N-2 supplement, B27 supplement, KSR (KnockOut Serum Replacement) and the like. If necessary, other serum substitutes, various amino acids, various inorganic salts, various vitamins, antibiotics, antifungal agents, etc. may be added. When the differentiated smooth muscle cells produced by the production method of the present invention are used for transplantation into a human body, it is preferable to use a Xeno-free medium that does not contain xenogenic components.

The MEK inhibitor used in step (1) is not particularly limited as long as it suppresses MEK (MAPK/ERK kinase), and may be a MEK1 inhibitor and/or a MEK2 inhibitor. For example Trametinib, Cobimetinib, Binimetinib, Selumetinib, Refametinib, Pimasertib, U0126, MEK162/ARRY-162, AZD8330/ARRY-424704, GDC -0973/ RG7420, GDC-0623/RG7421/XL518, CIF/RG7167/RO4987655, CK127/RG7304/RO5126766, E6201, TAK-733, PD-0325901, AS703988/MSC2015103B, WX-554, CI-1040/PD184352, AS703026, PD318088, PD98059, SL327, and pharmaceutically acceptable salts and solvates thereof. Alternatively, the MEK inhibitor may be a nucleic acid (eg, siRNA, shRNA, microRNA, antisense nucleic acid, etc.) that inhibits MEK expression. PD98059 is preferred.

The amount of the MEK inhibitor to be added to the medium is not particularly limited as long as it is an amount suitable for inducing differentiation from mesenchymal stem cells to differentiated smooth muscle cells. Depending on the MEK inhibitor to be used, it is preferable to determine the amount to be added that can achieve the purpose through a preliminary test or the like. For example, when using PD98059 as a MEK inhibitor, it may be 0.1 μg/mL or more, 0.5 μg/mL or more, 1.0 μg/mL or more, 1.5 μg/mL or more, 2.0 μg/mL or more. , 10.0 μg/mL or less, 9.0 μg/mL or less, 8.0 μg/mL or less, 7.0 μg/mL or less, 6.0 μg/mL or less, 5.0 μg/mL or less, 4.5 μg/mL or less, It may be 4.0 μg/mL or less, 3.5 μg/mL or less, or 3.0 μg/mL or less. The amount of PD98059 added to the medium may be 1.0 μg/mL or more and 5.0 μg/mL or less, may be 1.5 μg/mL or more and 4.0 μg/mL or less, or may be 2.0 μg/mL or more. It may be 3.0 μg/mL or less, or may be 2.5 μg/mL.

TGF-β1 used in step (1) includes TGF-β1 derived from humans and other animals, and functional variants thereof. For example, commercially available recombinant human TGF-β1 can be used. The amount of TGF-β1 added to the medium is not particularly limited as long as it is an amount suitable for inducing differentiation from mesenchymal stem cells to differentiated smooth muscle cells. 0.1 ng/mL or more, 0.5 ng/mL or more, 1.0 ng/mL or more, 1.5 ng/mL or more, may be 2.0 ng/mL or more, 10.0 ng/mL or less, 9.0 ng /mL or less, 8.0 ng/mL or less, 7.0 ng/mL or less, 6.0 ng/mL or less, 5.0 ng/mL or less, 4.5 ng/mL or less, 4.0 ng/mL or less, 3.5 ng/mL or less It may be less than mL or less than 3.0 ng/mL. The amount of TGF-β1 added to the medium may be 1.0 ng/mL or more and 5.0 ng/mL or less, may be 1.5 ng/mL or more and 4.0 ng/mL or less, or may be 2.0 ng/mL or more. mL or more and 3.0 ng/mL or less, or 2.5 ng/mL.

The cell culture vessel used in step (1) is not particularly limited as long as it is a culture vessel capable of adherent culture of cultured cells. Examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multidishes, microplates, microwell plates, multiplates, multiwell plates, chamber slides, culture slides, petri dishes, and the like. A culture vessel having a surface that is suitable for cell adhesion may be used.

The number of seeded cells is not particularly limited, and may be appropriately set according to the culture area of the culture vessel used. It is preferable to change the medium every 2 to 3 days and set the number of seeded cells so that the cells will be 80 to 90% confluent at the time of passage. The culture conditions are not particularly limited, and the cells can be cultured under general culture conditions for cultured animal cells. Specifically, for example, it can be cultured at a culture temperature of 37° C. in a 5% CO ₂ atmosphere.

The culture period is not particularly limited, but for example, the time to terminate the culture may be determined based on the ratio of cells expressing markers of mature differentiated smooth muscle cells (positive cell ratio) as an indicator. Mature differentiated smooth muscle cell markers include, for example, desmin, SMMHC, and the like. Cells expressing mature differentiated smooth muscle cell markers can be detected by known immunostaining techniques. The culture period is preferably continued until at least either desmin or SMMHC positive cell rate reaches 20% or more. More preferably, the culture is continued until the percentage of cells positive for either desmin or SMMHC reaches 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more.

The culture period may be 3 weeks or more. By culturing for 3 weeks or more, the rate of positive cells for either desmin or SMMHC becomes 20% or more, and the obtained smooth muscle cells have been confirmed to be differentiated smooth muscle cells with high contractility. there is The culture period may be 6 weeks or less. It is preferably 3 weeks or more and 5 weeks or less, may be 3 weeks or more and 4 weeks or less, may be 4 weeks or more and 5 weeks or less, or may be 4 weeks.

The period of culturing in a medium containing both the MEK inhibitor and TGF-β1 may be the entire culture period, or may be 90% or more of the total culture period from the start of culture, and the entire culture period from the start of culture. may be 80% or more of. Alternatively, the cells may be cultured in a medium containing both the MEK inhibitor and TGF-β1 for a certain period of time from the start of culture, and then cultured in a medium containing TGF-β1 but not the MEK inhibitor. The period of culturing in a medium containing both the MEK inhibitor and TGF-β1 is preferably 4 days or more from the start of culture, 5 days or more, 6 days or more, 7 days (1 week) or more. good. The period of culturing in a medium containing TGF-β1 but not MEK inhibitor may be continued until the end of the culture, or may be continued from one week before the end of the culture to any time after the end of the culture.

In step (2), a cell population in which the percentage of cells expressing desmin and/or SMMHC is 20% or more is collected. Desmin-expressing cells and SMMHC-expressing cells can be detected by known immunostaining techniques. The ratio of expressing cells (positive cell ratio) can be calculated by microscopic observation, image analysis of microscopic images, or the like. A method for collecting the cell population is not particularly limited, and a known method can be used. For example, a method in which adherent cells are peeled off by a known method and collected by centrifugation may be used.

The cell population collected in step (2) may have a positive cell rate of either desmin or SMMHC of 20% or more, and a positive cell rate of one or both of desmin and SMMHC of 25% or more, 30% or more. , 35% or more, 40% or more, or 50% or more.

INDUSTRIAL APPLICABILITY The production method of the present invention can provide a method for producing differentiated smooth muscle cells having strong contractility, which has not been established hitherto. By using the production method of the present invention, it becomes possible to easily obtain a large amount of smooth muscle cells required for regeneration of luminal organs of large animals, and to impart functions such as contraction and peristalsis to the regenerated organs. be able to.

The ability of the cells collected in step (2) to contract can be confirmed by a known method such as a collagen-based cell contraction assay (see Example 3).

[Method for producing smooth muscle cell spheroids]
The present invention provides a method for producing smooth muscle cell spheroids (hereinafter referred to as "the spheroid producing method of the present invention"). The spheroid production method of the present invention includes, in addition to steps (1) and (2) of the production method of the present invention, a step of three-dimensionally culturing the obtained differentiated smooth muscle cells to form spheroids. I wish I had. A three-dimensional culture method for differentiated smooth muscle cells is not particularly limited, and a known three-dimensional culture method can be used. Examples include the method described in Taniguchi et al. (Intractive Cardiovascular and Thoracic Surgery, 26 (2018) 745-752).

Smooth muscle cell spheroids produced by the spheroid production method of the present invention may be spheroids containing cells other than differentiated smooth muscle cells produced from mesenchymal stem cells by the above-described production method of the present invention. Cells other than differentiated smooth muscle cells are not particularly limited, and may be, for example, nerve cells, vascular cells, or fibroblasts. Mixed cells may also be used. By incorporating nerve cells into spheroids, it becomes possible to regenerate smooth muscle tissue that retains nerves, and acquisition of functions closer to those of the living body can be expected.

[Pharmaceutical composition for treatment of smooth muscle injury]
The present invention provides a pharmaceutical composition for treating smooth muscle damage containing differentiated smooth muscle cells obtained by the production method of the present invention or smooth muscle cell spheroids obtained by the spheroid production method of the present invention. , designated “the pharmaceutical composition of the invention”). Pharmaceutical compositions can include pharmaceuticals, medical devices, and regenerative medicine products. Methods of administering the pharmaceutical composition of the present invention include a method of administering (transplanting) differentiated smooth muscle cells or smooth muscle cell spheroids to a smooth muscle injury site. In order to adhere the administered (implanted) differentiated smooth muscle cells or smooth muscle cell spheroids to the injured site, the injured site may be coated with fibrin glue, gelatin gel, collagen gel, hyaluronic acid gel, or the like. The subject of administration of the pharmaceutical composition of the present invention is not particularly limited, and may be mammals, for example. Examples of mammals include mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, humans and the like. Humans are preferred.

Diseases to be treated by the pharmaceutical composition of the present invention include, for example, lung cancer, tracheal cancer, traumatic gastrointestinal injury, gastric cancer, colon cancer, esophageal cancer, gastrointestinal perforation, peptic ulcer (ulcerative colitis, Crohn's disease), Examples include ureteral injury, arteriosclerosis, and uterine fibroids.

The present invention further includes the following inventions.
(i) a smooth muscle cell comprising the step of transplanting the differentiated smooth muscle cells obtained by the production method of the present invention or the smooth muscle cell spheroids obtained by the spheroid production method of the present invention to a site of smooth muscle damage; Muscle injury treatment method.
(ii) Differentiated smooth muscle cells obtained by the production method of the present invention or smooth muscle cell spheroids obtained by the spheroid production method of the present invention, for use in treating smooth muscle damage.
(iii) Differentiated smooth muscle cells obtained by the production method of the present invention or smooth muscle cell spheroids obtained by the spheroid production method of the present invention for producing a pharmaceutical composition for treating smooth muscle damage Use of.

EXAMPLES The present invention will be described in detail below with reference to Examples, but the present invention is not limited to these.

[Example 1: Induction of differentiation of bone marrow-derived mesenchymal stem cells into smooth muscle cells]
<Preparation of rat bone marrow-derived mesenchymal stem cells>
Rat bone marrow-derived mesenchymal stem cells (BM-MSCs) were prepared according to a previous report (Ishii M et al., Pharmaceutics (2021) 13:1264). That is, the femur was removed from the hind leg of a 3-week-old Fischer 344 rat (male), the muscle was removed, and the femur was extruded by vigorously injecting phosphate buffered saline (PBS) from the joint cavity. The harvested cells were cultured in a basal medium having the following composition. Basal medium: 10% FBS (manufactured by Hyclone), 1% penicillin (100 IU/mL)/streptomycin (100 μg/mL) mixed solution (manufactured by Fujifilm Wako) and amphotericin B (0.25 μg/mL, Fujifilm Wako) DMEM/F-12 medium (manufactured by Fujifilm Wako Co., Ltd.). Medium was changed every 3 days. Passaging was performed when 80-90% confluency was reached. After 1-2 passages, it was used for the following experiments.

The cells prepared as described above were divided into the following six groups and cultured. Cultivation was performed at 37° C. under 5% CO ₂ atmosphere, and the medium was changed every 48 hours.
(1) [Group 1] Cultured in basal medium for 1 week.
(2) [Group 2] Cultured for 1 week in a basal medium containing 2.5 μg/mL PD98059 (manufactured by Fujifilm Wako).
(3) [Group 3] Cultured for 1 week in a basal medium containing 2.5 ng/mL TGF-β1 (manufactured by Pepro Tech).
(4) [Group 4] Cultured in basal medium for 4 weeks.
(5) [Group 5] Cultured for 4 weeks in a basal medium containing 2.5 ng/mL TGF-β1.
(6) [Group 6] After one week of culture in a basal medium containing 2.5 μg/mL PD98059 and 2.5 ng/mL TGF-β1, the cells were further cultured in a basal medium containing 2.5 ng/mL TGF-β1 for 3 weeks.

<Immunohistological evaluation of cell differentiation>
Cellular differentiation was assessed by immunofluorescent labeling of differentiated smooth muscle cell markers. Immunofluorescent labeling was performed according to a previous report (Ghionzoli M et al., FASEB Journal (2013) 27:4853-4865). That is, after one week or four weeks of culture, the cells cultured as described above were washed with PBS and then fixed with 4% paraformaldehyde (manufactured by Nacalai Tesque) for 15 to 20 minutes. Fixed cells were permeabilized with 0.5% Triton and incubated in PBS containing 3% BSA for 30 minutes to block non-specific antibody binding sites. After blocking, a primary antibody was added and allowed to react at room temperature for 1 hour. Primary antibodies include monoclonal anti-α smooth muscle actin antibody (α-SMA, sheep polyclonal IgG antibody, 250-fold dilution, manufactured by Abcam), anti-desmin antibody (rabbit polyclonal IgG antibody, 200-fold dilution, Abcam), anti-calponin antibody (rabbit monoclonal IgG antibody, 250-fold dilution, Abcam), anti-smooth muscle myosin heavy chain antibody (SMMHC, mouse monoclonal IgG antibody, 300-fold dilution, Abcam) It was used. After the primary antibody reaction, the cells were washed with PBS containing 3% BSA, and then reacted with a fluorescence-labeled secondary antibody corresponding to each primary antibody at room temperature for 1 hour. After the reaction with the secondary antibody, the cells were mounted with a DAPI-containing mounting medium (manufactured by Vector Laboratories), nuclear stained, and the stained cells were observed under a fluorescence microscope to compare the positive rate for each antibody.

<Results>
FIG. 1 shows stained images for each primary antibody. Cells cultured for 1 week in a medium containing PD98059 (group 2) or TGF-β1 (group 3) showed a slight appearance of calponin-positive cells compared to cells cultured in basal medium (group 1), No desmin and SMMHC positive cells were identified. A slight appearance of calponin-positive and SMMHC-positive cells was confirmed in cells cultured in medium containing only TGF-β1 for 4 weeks (group 5) compared to cells cultured in basal medium (group 4). On the other hand, after culturing in a medium containing PD98059 and TGF-β1 for 1 week, the cells were changed to a medium containing TGF-β1 and cultured for 3 weeks (group 6). Met. That is, the differentiation-inducing method of ``after culturing in a medium containing PD98059 and TGF-β1 for 1 week, replacing the medium with a medium containing TGF-β1 and culturing for 3 weeks'' is an extremely superior method for efficiently inducing smooth muscle cells. method has been identified.

[Example 2: Evaluation of culture time]
Culture time and cell differentiation were evaluated. Rat bone marrow-derived mesenchymal stem cells were cultured in a basal medium containing 2.5 μg/mL PD98059 and 2.5 ng/mL TGF-β1, and harvested after 1 week, 2 weeks, 3 weeks, and 4 weeks, In the same manner as in Example 1, cell differentiation was evaluated immunohistologically. Medium was changed every 48 hours.

<Results>
2 to 6 show immunostained images of each differentiated smooth muscle cell marker. Almost no differentiated smooth muscle cell marker-positive cells were observed in the cells after 1 week of culture (Fig. 2). Although an increase in the desmin-positive rate was observed in the cells after 2 weeks of culture, the number of differentiated smooth muscle cell marker-positive cells was small as a whole (Fig. 3). An increase in the number of positive cells for each differentiated smooth muscle cell marker was observed in the cells after 3 weeks of culture (Fig. 4), and a further increase in the positive rate of each differentiated smooth muscle cell marker was confirmed in the cells after 4 weeks of culture. (Fig. 5).

The cells after 4 weeks of culture were compared with the results of Group 6 in Example 1 (1 week of culture in a medium containing PD98059 and TGF-β1, followed by 3 weeks of culture in a medium containing TGF-β1). A similar positive rate was observed for the type smooth muscle cell marker (Fig. 6). That is, it was shown that smooth muscle cells can be efficiently induced to differentiate not only during the first week but also during the entire culture period (4 weeks).

[Example 3: Collagen-based cell contraction assay]
Induced cell contractility was tested (Fig. 7). Contraction assays were performed and compared for the following five groups.
(1) [Group 1] Medium only (2) [Group 2] Rat skin fibroblasts (DF) cultured in basal medium
(3) [Group 3] Rat smooth muscle cells (SMC) cultured in basal medium
(4) [Group 4] Rat mesenchymal stem cells (MSC) cultured in basal medium
(5) [Group 5] After 1 week of culture in a basal medium containing 2.5 μg/mL PD98059 and 2.5 ng/mL TGF-β1, the cells were further cultured in a basal medium containing 2.5 ng/mL TGF-β1 for 3 weeks. Mesenchymal stem cells derived from rat bone marrow

Each group was adjusted to 5.0×10 ⁵ cells per 1 mL, added to MEM medium containing type I collagen, and incubated at 37° C. in a 5% CO ₂ atmosphere for 2 hours. The collagen gel was peeled off from the culture dish wall, and the diameter (Da) of the gel was measured. After incubation with or without endothelin-1 for an additional 24 hours, the diameter of the gel (Db) was measured. Cell contractility was calculated by the following formula and compared.
Shrinkage = {1-(Db/Da) ² } x 100 (%)

<Results>
FIG. 8 shows gel images at each stage of the contraction assay. The contractility of each cell cultured in normal medium was very weak or absent under the condition of no addition of endothelin-1 (group 2: 10%, group 3: 3%, group 4: 9%). All cells showed weak contractility under the condition of endothelin-1 addition (group 2: 13%, group 3: 28%, group 4: 18%). On the other hand, rat bone marrow-derived mesenchymal stem cells (group 5) cultured in a medium containing PD98059 and TGF-β1 exhibited moderate contractility (50%) in the absence of endothelin-1, and showed moderate contractility (50%) in the absence of endothelin-1. showed a very strong contractility (69%) under the condition of . When comparing the contracted areas, a statistically significant difference was observed between rat MSCs (group 4) and cells differentiated by the induction method of the present invention (group 5) (Fig. 9, p = 0.0006). , t-test). That is, it was revealed that the cells obtained by the method for inducing smooth muscle cell differentiation and the method for producing smooth muscle cells of the present invention are differentiated smooth muscle cells with high contractility. On the other hand, rat SMC (group 3), which are mature smooth muscle cells, were considered to be dedifferentiated into dedifferentiated smooth muscle cells with little contractility.

The present invention is not limited to the above-described embodiments and examples, and can be modified in various ways within the scope of the claims, by appropriately combining technical means disclosed in different embodiments. The resulting embodiment is also included in the technical scope of the present invention. In addition, all scientific and patent documents mentioned in this specification are incorporated herein by reference.

Claims (9)

A method for producing differentiated smooth muscle cells from mesenchymal stem cells, comprising:
(1) culturing mesenchymal stem cells in a medium containing a MEK inhibitor and transforming growth factor β1; A manufacturing method comprising the step of recovering a cell population. 2. The method of claim 1, wherein in step (1), culture is performed in a medium containing a MEK inhibitor and transforming growth factor β1 for at least 4 days, followed by culturing in a medium containing transforming growth factor β1 but not MEK inhibitor. 1. The manufacturing method according to 1. 3. The production method according to claim 1 or 2, wherein the culture period in step (1) is 3 weeks or more and 5 weeks or less. The mesenchymal stem cells according to any one of claims 1 to 3, wherein the mesenchymal stem cells are mesenchymal stem cells obtained by inducing differentiation from mesenchymal stem cells or pluripotent stem cells collected from bone marrow or adipose tissue. manufacturing method. The production method according to any one of claims 1 to 4, wherein the mesenchymal stem cells are mesenchymal stem cells obtained from a subject in need of smooth muscle cell transplantation. The production method according to any one of claims 1 to 5, wherein the MEK inhibitor is PD98059. Smooth muscle comprising a step of producing differentiated smooth muscle cells by the production method according to any one of claims 1 to 6, and a step of three-dimensionally culturing the obtained differentiated smooth muscle cells to form spheroids. A method for producing cell spheroids. 8. The production method according to claim 7, wherein in the three-dimensional culture, the differentiated smooth muscle cells are mixed with one or more cells selected from nerve cells, vascular cells and fibroblasts to form spheroids. Smooth muscle cells obtained by the production method according to any one of claims 1 to 6, or smooth muscle cell spheroids obtained by the production method according to claim 7 or 8 A pharmaceutical composition for treating muscle damage.