JP7540893B2 - Composition for increasing the ratio of CD56 positive cells - Google Patents

Description

The present invention relates to a composition and method for increasing the ratio of CD56-positive cells in a cell population, a cell population having a high ratio of CD56-positive cells, a method for producing a graft using the same, and a graft produced by the production method.

Despite the innovative advances in the treatment of heart disease in recent years, a treatment system for severe heart failure has yet to be established. The development of new regenerative medicine is considered essential as a solution to the treatment of severe heart failure. In severe myocardial infarction, myocardial cells become dysfunctional, and the proliferation of fibroblasts and interstitial fibrosis progress, resulting in heart failure. As heart failure progresses, myocardial cells are damaged and undergo apoptosis, but because myocardial cells hardly undergo cell division, the number of myocardial cells decreases and cardiac function declines further. Cell transplantation is considered useful for restoring cardiac function in patients with such severe heart failure, and clinical application using skeletal myoblasts has already begun.

In recent years, as one example, a three-dimensional cell culture containing skeletal myoblasts that can be transplanted into the heart and a method for producing the same have been provided (Patent Document 1). Skeletal myoblasts used in such cell transplantation are usually obtained by isolating CD56-positive cells such as skeletal myoblasts and muscle satellite cells from the skeletal muscle tissue of the transplant target. As a measure to increase the proportion of CD56-positive cells, for example, a method is known in which skeletal muscle tissue is immersed in a protease solution for a predetermined time to perform an enzyme treatment, the enzyme treatment liquid obtained is discarded, and then the tissue is immersed in the protease solution again for a predetermined time to perform an enzyme treatment to recover the cells contained in the enzyme treatment liquid obtained (Patent Document 2), or a medium containing serum and a basal medium as a culture medium for culturing only muscle cells, the serum concentration being 10 to 40% by volume in the total culture medium, the basal medium being 50 to 90% by volume in the total culture medium, and substantially free of glucose (Patent Document 3).

Special Publication No. 2007-528755 JP 2011-110368 A JP 2018-000194 A

The researchers found that the number of CD56-positive cells, such as skeletal myoblasts and muscle satellite cells, in the cell population obtained from the skeletal muscle tissue obtained as described above was significantly lower than the number of cells originally contained in the tissue, and that the quality of the cells, such as the number of cells and proliferation ability, changed depending on individual differences such as age.

As a result of extensive research into solving the above-mentioned problems, the inventors discovered that adding a TGF-β inhibitor to a cell population prepared from biological tissue could increase the ratio of CD56-positive cells in the cell population. In the course of further research, they discovered that using TGF-β in combination with a TGF-β inhibitor not only improved the ratio but also selectively increased the number of CD56-positive cells, thus completing the present invention.

That is, the present invention relates to the following.
[1] A composition containing a TGF-β inhibitor for increasing the proportion of CD56-positive cells in a cell population.
[2] The composition described in [1], wherein the TGF-β inhibitor is SB-43154.
[3] The composition according to [1] or [2], further comprising TGF-β, for promoting an increase in the number of CD56-positive cells.

[4] The composition according to any one of [1] to [3], wherein the CD56-positive cells are skeletal muscle progenitor cells.
[5] The composition described in [4], wherein the skeletal muscle progenitor cells are skeletal myoblasts or muscle satellite cells.
[6] A therapeutic or preventive agent for a muscular disease, a cardiac disease, or a fibrotic disease, comprising the composition according to any one of [1] to [5].
[7] A method for treating a disease in a subject in need thereof, comprising a step of administering the therapeutic or preventive agent for a muscular disease, a cardiac disease, or a fibrotic disease described in [6] to the subject.

[8] A method for increasing the ratio of CD56-positive cells in a cell population, the method comprising a step of incubating the cell population in the presence of a TGF-β inhibitor.
[9] The method according to [8], wherein the cell population is prepared from skeletal muscle tissue.
[10] The method according to [8] or [9], wherein the TGF-β inhibitor is SB-43154.

[11] The method according to any one of [8] to [10], further comprising a step of incubating in the absence of a TGF-β inhibitor prior to the step of incubating in the presence of a TGF-β inhibitor.
[12] The method according to [11], wherein the incubation is for 192 hours or longer in the absence of a TGF-β inhibitor.
[13] The method according to any one of [8] to [12], wherein TGF-β is further present in the step of incubating in the presence or absence of a TGF-β inhibitor.

[14] A cell population with a high ratio of skeletal myoblasts, obtained by the method according to any one of [8] to [13].
[15] A method for producing a graft using the cell population described in [14].
[16] A graft produced by the method according to [15].
[17] The composition according to any one of [1] to [5], for administration together with a graft produced from a cell population containing CD56-positive cells.

[18] A method for treating a disease in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of the cell population described in [14] or the graft described in [16].
[19] A method for treating a disease in a subject, comprising the step of administering to the subject in need thereof a therapeutically effective amount of a graft produced from a cell population containing CD56-positive cells, together with a therapeutically effective amount of the composition according to any one of [1] to [5].

The ratio of CD56-positive cells in a cell population can be stably increased, thereby providing high-quality grafts, etc. Furthermore, it can provide a new therapeutic strategy to suppress undesirable proliferation of non-CD56-positive cells, such as proliferation of fibroblasts and interstitial fibrosis in the target tissue.

The ratio of CD56 positive cells (hereinafter also referred to as CD56 positivity rate) when TGF-β or a TGF-β inhibitor (SB-43154) was added to a cell population isolated from skeletal muscle tissue is shown. The total cell count and the CD56-positive cell count are shown when TGF-β or a TGF-β inhibitor (SB-43154) was added to a cell population isolated from skeletal muscle tissue. The total cell number when a TGF-β inhibitor was added to primary skeletal myoblasts is shown. The graph shows the CD56 positive cell rate when TGF-β and/or a TGF-β inhibitor (SB-43154) was added to a cell population isolated from skeletal muscle tissue and cultured for 2 hours.

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety. The present invention will now be described based on preferred embodiments thereof.

<TGF-β inhibitor-containing composition>
The present disclosure includes compositions containing a TGF-β inhibitor for increasing the proportion of CD56 positive cells in a cell population.
In the present disclosure, "CD56 positive cells" refers to cells characterized by the cell surface marker CD56. CD56 positive cells may be somatic cells or undifferentiated stem cells (e.g., myoblasts, tissue stem cells such as cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.), and are typically skeletal muscle precursor cells such as skeletal myoblasts and muscle satellite cells. For example, a method for measuring the ratio of CD56 positive cells includes labeling with an anti-CD56 antibody and dividing the number of positive cells bound to the antibody by the total number of cells counted. The counting of CD56 positive cells can be performed by microscopic observation of a specimen stained with a specific antibody, image analysis of the microscopic image, flow cytometry analysis of a cell population stained with a specific antibody, etc.

In the present disclosure, the "proportion of CD56-positive cells" refers to the ratio of the number of CD56-positive cells to the total number of cells constituting a cell population including CD56-positive cells and cells other than CD56-positive cells (non-CD56-positive cells). The cell population may be a living tissue itself, a tissue collected from a living body, a cell suspension prepared from the tissue, or a graft manufactured from the cell, or a cell suspension prepared from cells induced to differentiate from pluripotent stem cells or a graft manufactured from the cell. Typically, the cell population is prepared from tissue collected from a living body, and is typically composed of cells present in the same tissue. For example, a cell population prepared from skeletal muscle tissue is mainly composed of skeletal muscle progenitor cells, such as skeletal myoblasts and muscle satellite cells, and fibroblasts. In this case, the skeletal muscle progenitor cells are CD56-positive cells, and "increasing the proportion of CD56-positive cells" refers to increasing the proportion of skeletal muscle progenitor cells. Therefore, in one aspect of the present invention, the cell population is a cell population prepared from skeletal muscle tissue. In one embodiment of the invention, the CD56 positive cells are preferably skeletal myoblasts and/or muscle satellite cells, and the non-CD56 positive cells are fibroblasts.
If the CD56-positive cells are, for example, skeletal myoblasts, they can be identified by markers such as, but not limited to, Pax7, GATA4, MyoD, α7 integrin, myosin heavy chain IIa, myosin heavy chain IIb, myosin heavy chain IId (IIx), Myf5, myogenin, etc. If the CD56-positive cells are muscle satellite cells, they can be identified by markers such as, but not limited to, Pax7, α7 integrin, CD34, CXCR4, CD29, etc.

The biological tissue or cells from which the cell population is derived may be derived from, but are not limited to, mammals such as humans, non-human primates, rodents (e.g., mice, rats, hamsters, guinea pigs), rabbits, dogs, cats, pigs, horses, cows, goats, and sheep.

A composition containing a TGF-β inhibitor according to the present disclosure can increase the proportion of CD56-positive cells in a cell population.
TGF-β is generally a type of growth factor, and is produced by almost all cells that compose a wide range of tissues and organs. There are five subtypes of TGF-β (β1 to β5). TGF-β is known to promote the proliferation of osteoblasts, as well as the synthesis and proliferation of connective tissues such as collagen, and to have an inhibitory effect on the proliferation of epithelial cells and osteoclasts.
TGF-β inhibitors in the present disclosure are agents that block or inhibit the binding of TGF-β to the TGF-β receptor. In one embodiment, the TGF-β inhibitor is an agent that acts as a TGF-β receptor antagonist or inverse agonist. In one embodiment, the TGF-β inhibitor is an agent that binds to TGF-β forming a complex that neutralizes TGF-β activity.

The TGF-β inhibitors disclosed herein are not limited as long as they block or inhibit the binding of TGF-β to the TGF-β receptor, and include SB-43154 (4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide), A83-01 (3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole), ALK5 Inhibitor, I (3-(pyridin-2-yl)-4-(4-quinonyl)-1H-pyrazole), LDN193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate), SD-208 (2-(5-chloro-2-fluorophenyl)pteridin-4-yl)pyridine Lysin-4-yl-amine), SB-525334 (6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), LY-364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), LY2157299 (4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]-quinoline-6-carboxylic acid amide), TGF-β RI Kinase Inhibitor II 616452 (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), TGF-β RI Kinase Inhibitor III 616453 (2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, HCl), TGF-β RI Kinase Inhibitor IX 616463 (4-((4-((2,6-dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide), TGF-β RI Kinase Inhibitor VII 616458 (1-(2-((6,7-dimethoxy-4-quinolyl)oxy)-(4,5-dimethylphenyl)-1-ethanone), TGF-β RI Kinase Inhibitor VIII 616459 (6-(2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl)-quinoxaline), AP12009, Belagenpumatucel-L, CAT-152, CAT-192, GC-1008 (anti-TGF-β monoclonal antibody), etc. can be used. In the present invention, SB-43154 can be preferably used.

The concentration of the TGF-β inhibitor in the present disclosure is not limited as long as it has TGF-β inhibitory activity, and may be, for example, 5%, 10%, 20%, 30%, 40% or more inhibitory activity compared to the TGF-β activity level in the absence of the TGF-β inhibitor. TGF-β inhibitory activity can be evaluated by methods known to those skilled in the art, such as an assay using cells containing a PAI-1 promoter operably linked to a luciferase reporter gene or a reporter vector containing a Smad binding site.

When SB-43154 is used as a TGF-β inhibitor, it is not limited as long as it can increase the ratio of CD56 positive cells in the cell population, and for example, it is 0.01 ng/mL to 100 ng/ml, 0.05 ng/mL to 50 ng/ml, 0.1 to 10 ng/ml, 0.2 to 5 ng/mL, and 0.5 to 5 ng/mL is preferable. When used in combination with TGF-β, it is also 0.01 ng/mL to 100 ng/ml, 0.05 ng/mL to 50 ng/ml, 0.1 to 10 ng/ml, 0.2 to 5 ng/mL, and 0.5 to 5 ng/mL is preferable.

In one embodiment of the present invention, a TGF-β inhibitor can be used in combination with TGF-β, and in this embodiment, it is possible to promote an increase in the proportion and number of CD56-positive cells in a cell population.
The TGF-β may be any TGF-β classified in the art, and may be derived from a living organism or a recombinant organism. For example, recombinant TGF-β1 (Transforming Growth Factor-beta1, Peprotech, Cat No: 100-21) may be used.

In this embodiment, the concentration of TGF-β is not limited as long as it can increase the ratio of CD56-positive cells and the number of CD56-positive cells in the cell population when combined with a TGF-β inhibitor, and the ratio of TGF-β to TGF-β inhibitor is about 1:1 to 100, 1:1 to 50, about 1:1 to 25, about 1:1 to 10, about 1:1 to 5, about 1:1 to 2.5, about 1:1.25, and preferably about 1:1. When used in combination with SB-43154 as a TGF-β inhibitor, the concentration of TGF-β is, for example, about 0.01 ng/mL to about 100 ng/mL, about 0.05 ng/mL to about 50 ng/mL, about 0.1 to about 10 ng/mL, about 0.2 to about 5 ng/mL, and about 0.5 to about 5 ng/mL is preferred. It is preferable to use about 1-5 ng/mL of TGF-β for 1-5 ng/mL of SB-43154.

The composition of the present invention containing a TGF-β inhibitor can increase the ratio of CD56 positive cells in a cell population by about 1.03 to 4 times, 1.06 to 3 times, about 1.09 to 2.5 times, about 1.10 to 2 times, or about 1.12 to 1.9 times, compared to when the composition does not contain a TGF-β inhibitor.
The composition of the present invention containing a TGF-β inhibitor and TGF-β can increase the ratio of CD56 positive cells in a cell population by about 1.3 to 7 times, about 2 to 6 times, about 3 to 4.5 times, or about 2 to 2.3 times, compared to when a TGF-β inhibitor is not contained.
The composition of the present invention containing a TGF-β inhibitor and TGF-β can increase the number of CD56-positive cells in a cell population by about 1.1 to 4 times, about 1.2 to 3 times, about 1.3 to 2.5 times, or about 1.4 to 1.8 times, compared to a composition containing a TGF-β inhibitor at the same concentration.
The composition of the present invention containing a TGF-β inhibitor and TGF-β can increase the number of CD56-positive cells in a cell population by about 1.1 to 5 times, about 1.4 to 4.5 times, about 1.7 to 4 times, about 2 to 4 times, or about 2.5 to 3.5 times compared to a composition containing a TGF-β inhibitor at the same concentration.

An increase in the ratio and/or number of CD56-positive cells in a cell population is also effective for myoblastic diseases such as various muscular dystrophies, various myopathies, mitochondrial diseases, and glycogen storage diseases; age-related muscle atrophy (sarcopenia); muscle weakness (cachexia) due to chronic disease or cancer cachexia; muscle atrophy due to decreased muscle activity associated with being bedridden or having a fracture; and muscle diseases such as infantile spinal muscular atrophy, Charcot-Marie-Tooth disease, congenital hypomyelination, and amyotrophic lateral sclerosis (ALS); and heart diseases such as heart failure associated with these diseases.
Furthermore, an increase in the ratio of CD56-positive cells in a cell population means a decrease in the ratio of non-CD56-positive cells. The decrease in the ratio of non-CD56-positive cells may be brought about by proliferation of CD56-positive cells, or by inhibition of proliferation or death of non-CD56-positive cells, etc.
For example, an increase in the ratio of CD56-positive cells, such as skeletal myoblasts and muscle satellite cells, in skeletal muscle tissue results in a decrease in the ratio of fibroblasts. Therefore, a composition containing a TGF-β inhibitor and/or TGF-β can be used as a preventive or therapeutic agent for various fibrotic diseases associated with the proliferation of fibroblasts in tissue.
Fibrotic diseases include, but are not limited to, heart failure, arteriosclerosis, coronary artery restenosis after percutaneous transluminal coronary angioplasty, pulmonary fibrosis, liver cirrhosis, scleroderma, interstitial pneumonia, interstitial myocarditis, interstitial cystitis, glomerulonephritis, vasculitis, diabetic nephropathy, hypertensive nephrosclerosis, HIV nephropathy, IgA nephropathy, lupus nephropathy, interstitial nephritis, obstructed kidney due to ureteral obstruction, and skin scarring.

The composition of the present invention is not particularly limited as long as it contains a TGF-β inhibitor and/or TGF-β. The composition of the present invention can be used in combination with a composition containing a compound other than the TGF-β inhibitor and/or TGF-β of the present invention that exhibits the effect of increasing the ratio of CD56-positive cells. In addition, the composition of the present invention may contain any component other than the TGF-β inhibitor and/or TGF-β of the present invention, so long as it does not interfere with the effect of the present invention. Examples of such optional components include pharmacologically acceptable carriers, excipients, liquid media (diluents, buffer solutions, culture media, etc.), etc.

The dosage form of the composition of the present invention is not particularly limited, and examples thereof include liquid or solid preparations containing the TGF-β inhibitor and/or TGF-β of the present invention. Liquid preparations can be produced by adding the TGF-β inhibitor and/or TGF-β of the present invention to an appropriate physiological saline solution, replacement fluid, or pharmaceutical additive and filling the mixture into an ampoule or vial. Solid preparations can be produced by adding an appropriate protective agent to the liquid preparation, filling the mixture into an ampoule or vial, and then lyophilizing or L-drying, or by adding an appropriate protective agent to the liquid preparation, lyophilizing or L-drying, and then filling the mixture into an ampoule or vial.
In one embodiment of the present invention, when a TGF-β inhibitor and TGF-β are used in combination, the TGF-β inhibitor and TGF-β may be contained together in a formulation, or the TGF-β inhibitor and TGF-β may be contained separately in a formulation, and each may be administered during treatment.

Methods for Treating Diseases with TGF-β Inhibitors and/or TGF-β-Containing Compositions
Another aspect of the present disclosure relates to a method for treating a disease in a subject, comprising administering to said subject in need thereof an effective amount of a TGF-β inhibitor and/or a TGF-β-containing composition obtained by the method of the present disclosure. The disease to be treated is as described above.
In the present disclosure, the term "treatment" is intended to include all types of medically acceptable prophylactic and/or therapeutic interventions aimed at curing, temporarily ameliorating, or preventing a disease, etc. For example, the term "treatment" includes medically acceptable interventions for various purposes, including delaying or halting the progression of a disease associated with tissue abnormality, regressing or eliminating a lesion, preventing the onset of the disease or preventing recurrence, etc.
In the treatment method of the present disclosure, other active ingredients useful for treating the target disease can be used in combination with the TGF-β inhibitor and/or TGF-β-containing composition of the present disclosure. The treatment method of the present disclosure may further include cell populations, grafts, and the like.

In the present disclosure, an effective amount is, for example, an amount that can suppress the onset or recurrence of a disease, alleviate symptoms, or delay or stop progression, and is preferably an amount that prevents the onset or recurrence of the disease, or cures the disease. In addition, an amount that does not cause adverse effects that exceed the benefits of administration is preferable. Such an amount can be appropriately determined, for example, by testing in experimental animals such as mice, rats, dogs, or pigs, or disease model animals, and such testing methods are well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important indicator for determining the effective amount.

Methods of administration include, for example, intravenous administration, intramuscular administration, intraosseous administration, intrathecal administration, and direct application to tissue. The frequency of administration is typically once per treatment, but multiple administrations are possible if the desired effect is not achieved.

<Method for increasing the ratio of CD56-positive cells in a cell population>
Another aspect of the invention is a method of increasing the proportion of CD56 positive cells in a cell population, comprising incubating the cell population in the presence of a TGF-β inhibitor.
The cell population and CD56-positive cells of the present disclosure are as described above.
In this aspect, the cell population is preferably cells prepared from biological tissue, more preferably cells prepared from skeletal muscle tissue.
Incubation in the presence of a TGF-β inhibitor may involve adding a composition containing the above-mentioned TGF-β inhibitor, for example any liquid medium containing a TGF-β inhibitor, to a cell population and incubating.
In one embodiment of the present invention, the incubation step may further include the presence of TGF-β, i.e., incubation may be performed in the presence of a TGF-β inhibitor and TGF-β. Thus, one embodiment of the present invention may involve adding a composition containing a TGF-β inhibitor and TGF-β as described above, for example, any liquid medium containing a TGF-β inhibitor and TGF-β, to a cell population and incubating the cell population.
The concentrations of the TGF-β inhibitor and/or TGF-β in the composition are as described above.
The liquid medium can be used without any particular limitation as long as it can maintain the survival of cells, for example, physiological saline, various physiological buffer solutions (e.g., PBS, HBSS, etc.), and those based on various basal media for cell culture. Such basal media include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM/F12, etc. Many of these basal media are commercially available, and their compositions are also publicly known. The basal medium may be used as is with a standard composition (e.g., as it is commercially available), or the composition may be appropriately changed depending on the cell type and cell conditions. The medium may contain additives such as normal serum (eg, bovine serum such as fetal bovine serum (FBS), horse serum, human serum, etc.), various growth factors (eg, FGF, EGF, VEGF, HGF, etc.).

Incubation can be performed under conditions commonly used in the art. For example, typical incubation conditions include incubation at 35° C. and 5% CO ₂ .
The incubation time is not particularly limited as long as it can increase the ratio of CD56-positive cells of interest, and is preferably about 1 hour to 10 days, about 1 hour to 5 days, about 1.5 hours to 3 days (72 hours), about 2 hours to about 48 hours, or about 2 hours to about 24 hours.
The incubation temperature is not particularly limited as long as it can increase the ratio of CD56-positive cells, and may be any temperature commonly used in the art.
After the incubating step, the method may further comprise the step of washing the cell population.

In one embodiment of the present invention, the method may further include a step of incubating in the absence of a TGF-β inhibitor prior to the step of incubating in the presence of a TGF-β inhibitor. Depending on the type of cell or the growth stage, inhibition of TGF signaling may significantly inhibit proliferation, and in such a case, it is preferable to incubate in the presence of a TGF-β inhibitor after incubating in the absence of a TGF-β inhibitor. The type of cell used in this embodiment may be a primary culture cell.
The incubation time in the absence of a TGF-β inhibitor, for example, when primary cultured cells are used, is not particularly limited as long as the cell population is stabilized, and may be, but is not limited to, 8 hours or more, 12 hours or more, 24 hours or more, 48 hours or more, 96 hours or more, 168 hours or more, 192 hours or more, or 240 hours or more. During such incubation time, one or more passages may be included.
Incubation in the absence of a TGF-β inhibitor may be performed in a manner that does not contain a TGF-β inhibitor, and may contain any component such as TGF-β, or may be performed using the same components as those used in incubation in the presence of a TGF-β inhibitor, except that the component does not contain a TGF-β inhibitor. Thus, the step of incubating in the presence of a TGF-β inhibitor can also be initiated by adding any liquid medium containing the above-mentioned TGF-β inhibitor to the cell population incubated in the absence of a TGF-β inhibitor.

By including a step of incubating in the presence of a TGF-β inhibitor, a cell population with a high ratio of CD56 positive cells can be obtained. Furthermore, by including a step of incubating in the presence of a TGF-β inhibitor and TGF-β, a cell population with a high ratio and number of CD56 positive cells can be obtained.

<Method for increasing the ratio of CD56-positive cells in a cell population>
Another aspect of the present invention relates to a method for producing a graft using a cell population with a high ratio of CD56 positive cells obtained by the method of the present invention.
In the present invention, the term "graft" refers to a structure for transplantation into a living body, and particularly refers to a transplantation structure containing cells as a component. In a preferred embodiment, the graft is a transplantation structure that does not contain any structure (e.g., a scaffold, etc.) other than cells and cell-derived substances. Examples of the graft in the present disclosure include, but are not limited to, sheet-shaped cell cultures, spheroids, cell aggregates, etc., and are preferably sheet-shaped cell cultures or spheroids, and more preferably sheet-shaped cell cultures.

In the present disclosure, the term "sheet-shaped cell culture" refers to cells that are connected to each other to form a sheet. In the present disclosure, the term "spheroid" refers to cells that are connected to each other to form a roughly spherical shape. The cells may be connected to each other directly (including through cellular elements such as adhesion molecules) and/or through an intervening substance. The intervening substance is not particularly limited as long as it is a substance that can at least physically (mechanically) connect the cells to each other, and examples thereof include extracellular matrix. The intervening substance is preferably derived from cells, particularly from the cells that constitute the sheet-shaped cell culture. The cells are at least physically (mechanically) connected, but may also be functionally connected, for example, chemically or electrically. The sheet-shaped cell culture may be composed of one cell layer (single layer) or two or more cell layers (laminated (multilayer), for example, two layers, three layers, four layers, five layers, six layers, etc.). The sheet-shaped cell culture may also have a three-dimensional structure with a thickness greater than the thickness of one cell, without the cells showing a clear layer structure. For example, in the vertical cross section of a sheet-shaped cell culture, the cells may not be uniformly aligned in the horizontal direction, but may be arranged unevenly (e.g., in a mosaic pattern).

The graft of the present disclosure, particularly the sheet-shaped cell culture, preferably does not include a scaffold (support). A scaffold may be used in the art to attach cells to its surface and/or inside and maintain the physical integrity of a graft such as a sheet-shaped cell culture; for example, a membrane made of polyvinylidene difluoride (PVDF) is known, but the graft of the present disclosure can maintain its physical integrity even without such a scaffold. In addition, the graft of the present disclosure preferably consists only of substances derived from the cells that constitute the graft, and does not include any other substances.

The culture substrate is not particularly limited as long as the cells can form a cell culture thereon, and includes, for example, containers of various materials and/or shapes, solid or semi-solid surfaces in the container, and the like. The container is preferably made of a structure or material that is impermeable to liquids such as culture fluid. Examples of such materials include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethylacrylamide, metals (e.g., iron, stainless steel, aluminum, copper, brass), and the like. In addition, the container preferably has at least one flat surface. Examples of such containers include, but are not limited to, a culture vessel having a bottom surface made of a culture substrate on which a cell culture can be formed and liquid-impermeable sides. Specific examples of such culture vessels include, but are not limited to, cell culture dishes, cell culture bottles, and the like. The bottom surface of the vessel may be transparent or opaque. If the bottom surface of the vessel is transparent, cells can be observed, counted, and the like from the back side of the vessel. The container may have a solid or semi-solid surface inside. Solid surfaces include plates and containers made of the various materials described above, and semi-solid surfaces include gels and soft polymer matrices. The culture substrate may be made from the materials described above, or a commercially available product may be used.

Preferred culture substrates include, but are not limited to, substrates with an adhesive surface suitable for forming sheet-shaped cell cultures, substrates with a low adhesive surface suitable for forming spheroids, and/or substrates with a uniform well-like structure. Specifically, in the case of forming sheet-shaped cell cultures, substrates with a corona discharge-treated polystyrene, a hydrophilic compound such as collagen gel or a hydrophilic polymer, and substrates with an extracellular matrix such as collagen, fibronectin, laminin, vitronectin, proteoglycan, or glycosaminoglycan, or a cell adhesion factor such as the cadherin family, selectin family, or integrin family are coated on the surface. Such substrates are commercially available (e.g., Corning(R) TC-Treated Culture Dish, Corning, etc.). In the case of forming spheroids, examples of suitable substrates include soft agar, a temperature-responsive gel (trade name: Mebiol Gel) in which poly(N-isopropylacrylamide) (PIPAAm) is crosslinked with polyethylene glycol (PEG), a substrate coated with a non-cell-adhesive compound such as hydrogels such as polyhydroxyethyl methacrylate (polyHEMA) and 2-methacryloyloxyethyl phosphoriccholine (MPC) polymer, and/or a substrate having a uniform uneven surface. Such substrates are also commercially available (e.g., EZSPHERE(R)). The culture substrate may be entirely or partially transparent or opaque.

The surface of the culture substrate may be coated with a material whose physical properties change in response to a stimulus, such as temperature or light. Such materials are not limited to, but include, for example, (meth)acrylamide compounds, N-alkyl-substituted (meth)acrylamide derivatives (e.g., N-ethylacrylamide, N-n-propylacrylamide, N-n-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide, etc.), N,N-dialkyl-substituted (meth)acrylamide derivatives (e.g., N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, N,N-diethylacrylamide, etc.), (meth)acrylamide derivatives having a cyclic group (e.g., 1-(1 Examples of materials that can be used include known materials such as temperature-responsive materials made of homopolymers or copolymers of 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, 4-(1-oxo-2-methyl-2-propenyl)-morpholine, or vinyl ether derivatives (for example, methyl vinyl ether), light-absorbing polymers having an azobenzene group, copolymers of vinyl derivatives of triphenylmethane leucohydroxide and acrylamide monomers, and light-responsive materials such as N-isopropylacrylamide gel containing spirobenzopyran (for example, see JP-A-2-211865 and JP-A-2003-33177). By applying a specific stimulus to these materials, the physical properties, such as hydrophilicity or hydrophobicity, can be changed, and the detachment of cell cultures attached to the materials can be promoted. Culture dishes coated with temperature-responsive materials are commercially available (e.g., UpCell(R) from CellSeed Inc.), and these can be used in the manufacturing method of the present disclosure.

The culture substrate may have various shapes. Its area is not particularly limited, and may be, for example, about 1 cm ² to about 200 cm ² , about 2 cm ² to about 100 cm ² , about 3 cm ² to about 50 cm ² , etc. For example, the culture substrate may be a circular culture dish with a diameter of 10 cm. In this case, the area is 56.7 cm ^2. The culture surface may be flat or may have an uneven structure. When the culture substrate has an uneven structure, it is preferably a uniform uneven structure.

The culture substrate may be coated with serum. By using a culture substrate coated with serum, a sheet-shaped cell culture of higher density can be formed. "Coated with serum" means a state in which serum components are attached to the surface of the culture substrate. Such a state is not limited, and can be obtained, for example, by treating the culture substrate with serum. Treatment with serum includes contacting the culture substrate with serum and, if necessary, incubating for a predetermined period of time.

As the serum, xenogeneic serum and/or allogeneic serum can be used. When a graft, particularly a sheet-shaped cell culture, is used for transplantation, xenogeneic serum means serum derived from an organism of a different species from the recipient. For example, when the recipient is a human, serum derived from a cow or horse, such as fetal bovine serum (FBS, FCS), calf serum (CS), and horse serum (HS), corresponds to xenogeneic serum. In addition, "allogeneic serum" means serum derived from an organism of the same species as the recipient. For example, when the recipient is a human, human serum corresponds to allogeneic serum. Allogeneic serum includes autologous serum (also called autologous serum), i.e., serum derived from the recipient, and allogeneic serum derived from an individual of the same species other than the recipient. In this specification, serum other than autologous serum, i.e., xenogeneic serum and allogeneic serum, are sometimes collectively referred to as non-autologous serum.

Serum for coating the culture substrate is commercially available, or can be prepared by standard methods from blood collected from the desired organism. Specifically, for example, the collected blood is left at room temperature for about 20 to 60 minutes to coagulate, and then centrifuged at about 1000 x g to about 1200 x g to collect the supernatant.

When incubating on a culture substrate, serum may be used in its original form or diluted. Dilution can be performed with any medium, including, but not limited to, water, physiological saline, various buffer solutions (e.g., PBS, HBSS, etc.), various liquid media (e.g., DMEM, MEM, F12, DMEM/F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc.). The dilution concentration is not particularly limited as long as the serum components can adhere to the culture substrate, and is, for example, about 0.5% to about 100% (v/v), preferably about 1% to about 60% (v/v), more preferably about 5% to about 40% (v/v).

The incubation time is not particularly limited as long as the serum components can adhere to the culture substrate, and is, for example, about 1 hour to about 72 hours, preferably about 2 hours to about 48 hours, more preferably about 2 hours to about 24 hours, and even more preferably about 2 hours to about 12 hours. The incubation temperature is also not particularly limited as long as the serum components can adhere to the culture substrate, and is, for example, about 0°C to about 60°C, preferably about 4°C to about 45°C, and more preferably room temperature to about 40°C.

After incubation, the serum may be discarded. Conventional liquid disposal methods such as suction with a pipette or decantation can be used as a method for discarding serum. In a preferred embodiment of the present disclosure, after serum disposal, the culture substrate may be washed with a serum-free washing solution. The serum-free washing solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect the serum components attached to the culture substrate, and can be, for example, water, physiological saline, various buffer solutions (e.g., PBS, HBSS, etc.), various liquid media (e.g., DMEM, MEM, F12, DMEM/F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc.), etc. The washing method can be a conventional culture substrate washing method, such as, but not limited to, a method in which a serum-free washing solution is added to the culture substrate, stirred for a predetermined time (e.g., about 5 to about 60 seconds), and then discarded.

In the present disclosure, the culture substrate may be coated with a growth factor. Here, "growth factor" means any substance that promotes cell proliferation compared to the absence of the growth factor, and includes, for example, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and the like. The method of coating, discarding, and washing the culture substrate with the growth factor is basically the same as that of serum, except that the dilution concentration during incubation is, for example, about 0.0001 μg/mL to about 1 μg/mL, preferably about 0.0005 μg/mL to about 0.05 μg/mL, and more preferably about 0.001 μg/mL to about 0.01 μg/mL.

In the present disclosure, the culture substrate may be coated with a steroid agent. Here, "steroid agent" refers to a compound having a steroid nucleus that may have adverse effects on the living body, such as adrenal insufficiency and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone, etc. The method of coating, disposing, and washing the culture substrate with the steroid agent is basically the same as that of serum, except that the dilution concentration during incubation is, for example, about 0.1 μg/mL to about 100 μg/mL, preferably about 0.4 μg/mL to about 40 μg/mL, and more preferably about 1 μg/mL to about 10 μg/mL, as dexamethasone.

The culture substrate may be coated with serum, growth factors, and steroids, or any combination of these, i.e., serum and growth factors, serum and steroids, serum, growth factors and steroids, or growth factors and steroids. When coated with multiple components, these components may be mixed and coated simultaneously or in separate steps.

The seeding of the cell population into the culture substrate can be carried out by any known method and under any known conditions. For example, the seeding of the cell population into the culture substrate can be carried out by injecting a cell suspension in which the cell population is suspended in a culture medium into the culture substrate (culture vessel). The cell suspension can be injected using a tool suitable for injecting a cell suspension, such as a dropper or pipette.

The culture medium used in the production method of the present invention is not particularly limited as long as it can maintain cell viability, but typically, a medium containing amino acids, vitamins, and electrolytes as its main components can be used. In one embodiment of the present invention, the culture medium is based on a basal medium for cell culture. Such basal media include, but are not limited to, DMEM, MEM, F12, DMEM/F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc. Many of these basal media are commercially available, and their compositions are also publicly known. However, when used in the production method of the present invention, the composition may be appropriately changed depending on the cell type and cell conditions.

The seeding density of cells is not particularly limited as long as it is a density at which a sheet-shaped cell culture can be formed, but in a preferred embodiment, the cell population is seeded at a density at which the cell population reaches confluence or a density higher than that. Therefore, the step of culturing the cell population in the presence of a TGF-β inhibitor and/or TGF-β may be included at any timing before seeding.
In the present disclosure, the term "density at which confluence is achieved" refers to a density at which the cells are expected to completely cover the adhesive surface of the culture vessel when seeded with the cells, such as a density at which the cells are expected to come into contact with each other when seeded, a density at which contact inhibition occurs, or a density at which cell proliferation is substantially stopped due to contact inhibition.

Non-limiting examples of the seeding density of the cell population include about 7.1×10 ⁵ cells/cm ² to about 3.0×10 ⁶ cells/cm ² , about 7.3×10 ⁵ cells/cm ² to about 2.8×10 ⁶ cells/cm ² , about 7.5×10 ⁵ cells/cm ² to about 2.5×10 ⁶ cells/cm ² , about 7.5×10 ⁵ cells/cm ² to about 3.0×10 ⁶ cells/cm ² , about 7.8×10 ⁵ cells/cm ² to about 2.3×10 ⁶ cells/cm ² , about 8.0×10 ⁵ cells/cm ² to about 2.0×10 ⁶ cells/cm ² , about 8.5×10 ⁵ cells/cm ² to about 1.8×10 ⁶ cells/cm ² , and about 9.0×10 ⁵ cells/cm ^{2 .} To about 1.6×10 ⁶ cells/cm ² and the like. Note that these densities refer to the densities of all cells contained in the cell population, unless otherwise specified.

In yet another embodiment, seeding can be performed in a cell culture medium substantially free of growth factors at a density at which at least one type of graft-forming cell that may be included in the cell population does not substantially proliferate. In such an embodiment, other cells that may be included in the cell population can be at a density at which they can proliferate while being growth-suppressed. The culture substrate used in the method of the present disclosure is as described above. In a preferred embodiment, the culture substrate may be coated with serum. In another preferred embodiment, the culture substrate may be coated with a temperature-responsive material. In a further preferred embodiment, the culture substrate may be coated with a temperature-responsive material and serum.

The cell population to be seeded includes at least one type of CD56-positive cell, but may include two or more types of CD56-positive cells, or may include cells other than CD56-positive cells. In one embodiment of the present disclosure, the at least one type of CD56-positive cell contained in the cell population is preferably a cell derived from skeletal muscle, more preferably a skeletal myoblast, and more preferably a skeletal myoblast and a muscle satellite cell. In such an embodiment, the cell population may further include fibroblasts. That is, for example, a graft containing myoblasts and fibroblasts as graft-forming cells is included. In yet another embodiment of the present disclosure, at least one type of sheet-forming cell contained in the cell population is a mesenchymal stem cell. In such an embodiment, the cells may further include vascular endothelial cells.
The high ratio is as described above. The graft of the present invention may be one produced by the sheet-shaped cell culture production method of the present invention. The sheet-shaped cell culture of the present invention is preferably sterile. The graft of the present invention contains a high ratio of CD56-positive cells, and therefore has a higher therapeutic effect, etc., than a sheet-shaped cell culture not produced by the method of the present invention.

Another aspect of the present disclosure relates to the above-mentioned TGF-β inhibitor-containing composition for administration together with a graft produced from a cell population containing CD56-positive cells. Here, the "graft produced from a cell population containing CD56-positive cells" may be any graft produced from a cell population containing CD56-positive cells, and may be, for example, a graft produced using a cell population obtained by the method of the present disclosure for increasing the ratio of CD56-positive cells in a cell population.
Without being bound by any particular theory, it is believed that by administering to a subject a pharmaceutical composition comprising a graft produced from a cell population containing CD56-positive cells and a composition containing a TGF-β inhibitor, it is possible to maintain a high proportion of CD56-positive cells even after administration.

<Kit>
In another aspect, the present invention relates to a method for increasing the ratio of CD56-positive cells in a cell population, a method for producing a graft, and a kit for producing a sheet-shaped cell culture, which includes some or all of the elements of the production.
The kit of the present invention may include, but is not limited to, for example, SB-43154 or TGF-β as a TGF-β inhibitor, as well as cells constituting the sheet-shaped cell culture (a cell population containing CD56-positive cells), culture medium, culture dishes, tools (e.g., pipettes, droppers, tweezers, etc.), instructions regarding the production method of the sheet-shaped cell culture (e.g., instructions for use, media recording information regarding the production method and the method for recovering the cryopreserved cells of the present invention, such as a flexible disk, CD, DVD, Blu-ray disk, memory card, USB memory, etc.), and the like.

Methods of Treating Disease
Another aspect of the present disclosure relates to a method of treating a disease in a subject in need thereof, comprising administering to said subject an effective amount of a TGF-β inhibitor-containing composition. In this disclosure, the term "treatment" is intended to include all kinds of medically acceptable prophylactic and/or therapeutic interventions aimed at curing, temporarily ameliorating or preventing a disease, etc. For example, the term "treatment" includes medically acceptable interventions for various purposes, including delaying or halting the progression of a disease associated with tissue abnormality, regressing or eliminating a lesion, preventing the onset of the disease or preventing recurrence, etc.

In the treatment method of the present disclosure, the graft of the present disclosure can be used in combination with components that enhance the survival, engraftment and/or function of the graft, or other active ingredients that are useful for treating the target disease.

The treatment method of the present disclosure may include the cell population and graft of the present disclosure. The treatment method of the present disclosure may further include a step of collecting cells (e.g., skin cells, blood cells, etc., in the case of using iPS cells) for producing the cell population from a subject or a biological tissue (e.g., skin tissue, blood, etc., in the case of using iPS cells) that serves as a source of the cell population, before enriching the cell population rich in CD56-positive cells.
In one embodiment, the subject from which the cells or tissue source of the cells are harvested is the same individual as the subject receiving the cell population or graft, etc.
In another embodiment, the subject from which the cells or tissue that serves as a source of the cells is harvested is a different individual of the same species as the subject receiving the cell population or graft, etc. In another embodiment, the subject from which the tissue that serves as a source of the cell population or graft, etc. is harvested is a different individual of the same species as the subject receiving the sheet cell population or graft, etc.

In the present disclosure, an effective amount is, for example, an amount that can suppress the onset or recurrence of a disease, alleviate symptoms, or delay or stop progression (e.g., size, weight, number of sheets, etc. of the sheet-like cell culture), and is preferably an amount that can prevent the onset or recurrence of the disease or cure the disease. In addition, an amount that does not cause adverse effects that exceed the benefits of administration is preferable. Such an amount can be appropriately determined, for example, by tests on experimental animals such as mice, rats, dogs, or pigs or disease model animals, and such test methods are well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important indicator for determining the effective amount.

Methods of administration include, for example, intravenous administration, intramuscular administration, intraosseous administration, intrathecal administration, and direct application to tissue. The frequency of administration is typically once per treatment, but multiple administrations are possible if the desired effect is not obtained. When applied to tissue, the cell culture, composition, or sheet-shaped cell culture of the present invention may be fixed to the target tissue by a fastening means such as sutures or staples.

Yet another aspect of the present disclosure relates to a method for treating a disease in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a graft produced by a cell population comprising CD56-positive cells together with a therapeutically effective amount of a composition comprising a TGF-β inhibitor as described above. Here, the "graft produced by a cell population comprising CD56-positive cells" may be any graft produced from a cell population comprising CD56-positive cells, and may be, for example, a graft produced using a cell population obtained by the method of increasing the ratio of CD56-positive cells in a cell population disclosed herein.
In one embodiment of the present invention, the administration may be simultaneous administration of the graft and the TGF-β inhibitor-containing composition. In another embodiment, the administration may be administration of the graft to which the TGF-β-containing composition has been added in advance. In yet another embodiment, the administration may be administration of a TGF-β-containing composition to the administered graft.
Without being bound by any particular theory, it is believed that by administering to a subject both a graft produced from a cell population containing CD56-positive cells and a TGF-β inhibitor, it is possible to maintain a high proportion of CD56-positive cells even after administration.

The present invention has been described above in terms of preferred embodiments, but the present invention is not limited to these. In the present invention, each component can be replaced with any component that can perform a similar function, or any component can be added.

Example 1: Effect of TGF-β and TGF-β inhibitor on CD56 positive cells Six samples of cell suspensions of two human skeletal myoblasts (passage number: 3) were seeded in a 12-well multiwell (Corning, Costar®) at 400 cells/ ^cm2 and pre-cultured overnight at 37°C under 5% _CO2 . Culture media (0, 0.1, 0.5, 2, 5, 10 ng/mL) containing TGF-β (PEPROTECH, Cat#100-21) and culture media (0, 0.1, 0.5, 2, 5, 10 ng/mL) containing a TGF-β inhibitor (SB-43154 (Cayman, Cat#13031)) were prepared. After removing the pre-culture medium, culture medium containing TGF-β at various concentrations and culture medium containing SB-43154 were added to the wells containing the cells derived from each individual, and cultured for 10 days at 37° C. under 5% CO ₂ .
The cells from each well were collected, and the cell populations were reacted with CD56 antibodies, and the CD56 positivity rate was measured using a flow cytometer. The CD56 positivity rates are shown in Table 1 and Figure 1. Figure 2 shows the total cell count and the number of CD56-positive cells at each concentration.

It was found that in the group to which TGF-β was added, the total cell count and CD56 positivity rate decreased in a dose-dependent manner. On the other hand, in the group to which a TGF-β inhibitor (SB-43154) was added, except for the group to which 0.1 μM was added, the total cell count decreased in a dose-dependent manner, but the CD56 positivity rate was maintained at a high level at all concentrations of the TGF-β inhibitor. From these results, it was found that the TGF-β inhibitor has the effect of increasing the ratio of CD56-positive cells such as skeletal myoblasts and muscle satellite cells, and decreasing the ratio of non-CD56-positive cells such as fibroblasts. Furthermore, an increase in the total cell count was confirmed when 0.1 μM of the TGF-β inhibitor was added, suggesting that excessive TGF signals suppress proliferation in cell populations of passaged skeletal myoblasts.

Example 2: Effect of TGF-β and TGF-β inhibitors on cell proliferation Cell suspensions of human skeletal myoblasts (passage number 3) were precultured in the same manner as in Example 1. Culture solutions containing TGF-β (0, 0.5, 5 ng/mL) and culture solutions containing TGF-β and SB-43154 (TGF-β: 5 ng/mL and SB-43154: 5 μM) were prepared. After removing the preculture medium, the prepared TGF-β-containing culture solutions and TGF-β and SB-43154-containing culture solutions of each concentration were added to each well, and 10 μl of Cell Counting Kit-8 (Dojindo Chemical Industries) was added at 1 and 2 hours after each addition, and the cells were incubated at 37° C. for 1 hour, after which the absorbance at 450 nm was measured using a microplate reader. The results are shown in Table 2.

Addition of TGF-β alone reduces the total cell count, as in Example 1, but regardless of the concentration added, the cell count increases over the culture time, indicating that addition of TGF-β alone reduces the cell count immediately after addition. Furthermore, when both TGF-β and SB-43154 are added (combined use), there is no reduction in cell count, but rather it has been revealed to have the effect of promoting cell growth.

Example 3: Effect of TGF-β inhibitor on cell proliferation in primary skeletal myoblasts A cell suspension of primary cells isolated from porcine skeletal muscle was cultured for 8 days in a culture medium (5 μM) containing a TGF-β inhibitor (SB-43154). The cells in each well were harvested and the cell numbers were compared. The results are shown in FIG. 3.
It can be seen that proliferation is strongly inhibited in primary cultured cells compared to the subculture in Example 2. This suggests that TGF-β signaling contributes greatly to proliferation in young cell populations such as primary cultures. It was found that the timing of adding a TGF-β inhibitor depending on the cell population affects the proliferation of the cell population.

Example 4: Effect on CD56-positive cells when TGF-β and TGF-β inhibitor are used together Preculture was performed in the same manner as in Example 2. Culture solutions containing TGF-β (0, 0.5, 5 ng/mL) and culture solutions containing TGF-β and SB-43154 (TGF-β: 5 ng/mL and SB-43154: 5 μM) were prepared. After removing the preculture medium, the prepared TGF-β-containing culture solutions and TGF-β and SB-43154-containing culture solutions of each concentration were added to each well and cultured for 8 days at 37° C. under 5% CO _2. The CD56 positivity rate was measured in the same manner as in Example 1. The results are shown in Table 3 and FIG. 4.

As in Example 1, the addition of TGF-β caused a dose-dependent decrease in the CD56 positivity rate in the cell population. On the other hand, it was revealed that the combined use of TGF-β and SB-43154 significantly increased the CD56 positivity rate. This shows that the cells that increased with the combined use of TGF-β and SB-43154 in Example 2 were CD56 positive cells. It was therefore revealed that the combined use of TGF-β and SB-43154 has the effect of significantly increasing the CD56 positivity rate and CD56 positive cells.

Claims (9)

A composition containing a TGF-β inhibitor for increasing the ratio of CD56-positive cells in skeletal myoblasts isolated from biological tissue, the composition being added to a culture medium when the skeletal myoblasts have been passaged three times, the TGF-β inhibitor being SB-431542. The composition according to claim 1, further comprising TGF-β, for promoting an increase in the number of CD56-positive cells. The composition according to claim 1 or 2, wherein the CD56-positive cells are skeletal muscle progenitor cells. The composition according to claim 3, wherein the skeletal muscle progenitor cells are skeletal myoblasts or muscle satellite cells. A composition containing a TGF-β inhibitor for suppressing cell proliferation in skeletal myoblasts isolated from biological tissue, the TGF-β inhibitor being SB-431542, the composition being added to a culture medium when the skeletal myoblasts are primary skeletal myoblasts. A method for increasing the ratio of CD56-positive cells in skeletal myoblasts isolated from biological tissue, comprising a step of incubating skeletal myoblasts that have been passaged three times in vitro in the presence of a TGF-β inhibitor, the TGF-β inhibitor being SB-431542. 7. The method of claim 6 , further comprising the step of incubating in vitro in the absence of a TGF-β inhibitor prior to the step of incubating in vitro in the presence of a TGF-β inhibitor. The method of claim 7 , wherein the in vitro incubation in the absence of a TGF-β inhibitor is for 192 hours or more. The method according to any one of claims 6 to 8 , wherein the step of incubating in vitro in the presence or absence of a TGF-β inhibitor further comprises the presence of TGF-β.