WO2010090177A1

WO2010090177A1 - Method for inducing differentiation of mesenchymal stem cells

Info

Publication number: WO2010090177A1
Application number: PCT/JP2010/051405
Authority: WO
Inventors: 紀ノ岡正博; 金美海; 田谷正仁
Original assignee: 国立大学法人大阪大学; 財団法人大阪産業振興機構
Priority date: 2009-02-03
Filing date: 2010-02-02
Publication date: 2010-08-12
Also published as: JP5641468B2; JP2010200745A

Abstract

Disclosed is a novel technique relating to the induction of the differentiation of mesenchymal stem cells (MSCs). Specifically disclosed is a method for inducing the differentiation of mesenchymal stem cells, which comprises culturing the mesenchymal stem cells in a cell culture vessel having a culture surface modified with a dendrimer compound.

Description

Method for inducing differentiation of mesenchymal stem cells

The present invention relates to a method for inducing differentiation of mesenchymal stem cells.

One of the major trends in recent regenerative medicine research is to artificially induce undifferentiated stem cells to reconstruct target tissues and organs outside or inside the body. Among them, mesenchymal stem cells (mesenchymal stem cells: MSCs) are expected as a powerful cell source applicable to regenerative medicine because of their various differentiation potentials. On the other hand, methods for inducing differentiation of mesenchymal stem cells into the desired differentiation state are currently in the research and development stage. Much of the current research on methods for inducing differentiation of mesenchymal stem cells relates to differentiation control by soluble factors (medium components) in the liquid phase.

In addition, there is a technique for modifying the culture surface with a dendrimer compound as one of the techniques for modifying the culture surface (bottom surface with which the culture medium comes into contact) of the cell culture vessel. Patent Documents 1 and 2 below show that a culture surface modified with a dendrimer compound having a terminal substance (D-glucose) that can be taken up by a cell transporter, and promoting cell immobilization by the culture surface. Disclose. Non-Patent Documents 1 and 2 below report the promotion of focal adhesion and morphological changes in human epithelial cells cultured on dendrimer-modified culture surfaces presenting D-glucose. Non-Patent Document 3 below reports the effects on morphological changes and migration in rabbit articular chondrocytes cultured on dendrimer-modified culture surfaces presenting D-glucose. Further, Non-Patent Document 4 described below reports that the undifferentiated state of mouse ES cells (embryonic stem cells) is maintained in culture using a dendrimer-modified culture surface presenting D-glucose.

JP-A-2005-192406 JP 2005-245224 A

The present invention provides a new technique relating to differentiation induction of mesenchymal stem cells.

The present invention is a method for inducing differentiation of a mesenchymal stem cell, which comprises culturing the mesenchymal stem cell in a cell culture vessel having a culture surface modified with a dendrimer compound. Regarding the method.

According to the present invention, a dendrimer-modified culture surface that is a solid phase factor can be provided as a factor for inducing differentiation of mesenchymal stem cells. Further, according to the present invention, differentiation induction of mesenchymal stem cells including use of a dendrimer-modified culture surface as a solid phase factor for inducing differentiation of mesenchymal stem cells can be achieved. Furthermore, according to the present invention, it is possible to preferably induce differentiation of mesenchymal stem cells accompanied by cell proliferation.

FIG. 1 shows a control surface (PS surface) (a), a generation 1 dendrimer-modified surface (G1 surface) (b), a generation 3 dendrimer-modified surface (G3 surface) (c), and a generation 5 dendrimer. It is a figure which shows the microscope observation photograph of the mesenchymal stem cell cultured by the culture | cultivation surface of the modification surface (G5 surface) (d) 1 day after culture, 3 days later, and 7 days after. FIG. 2 shows staining of desmin, a striated / smooth muscle cell differentiation marker, after culturing mesenchymal stem cells on the PS, G1, G3, and G5 culture surfaces for 7 days. And a microscopic observation photograph in which nuclear staining (red) was performed. FIG. 3 shows mesenchymal stem cells cultured on the PS, G1, G3, and G5 culture surfaces for 7 days, and then stained for type II collagen, a differentiation marker for chondrocytes (red). It is a figure which shows the microscope observation photograph. FIG. 4 shows mesenchyme cultured on dendrimer-modified culture surfaces with generation numbers 3, 4, and 5 (G3, G4, and G5) presenting 0%, 50%, and 100% D-glucose. It is a figure which shows the microscopic observation photograph of a stem cell after 4 days and 7 days of culture | cultivation. FIG. 5 shows that a cell clump obtained by culturing mesenchymal stem cells on the G5 culture surface for 7 days is fluorescent immunostaining for each of the markers of desmin, MHC Fast Skeletal, cTnT and type II collagen (green). ) And a nuclear micrograph (Topro-3, blue). FIG. 6 shows fluorescence of a cell cluster of mesenchymal stem cells prepared without using a dendrimer-modified culture surface according to the present invention for each marker of desmin, MHCＭFast Skeletal, cTnT, type II collagen and CD105. An example of a microscopic observation photograph obtained by immunostaining (green) and nuclear staining (Topro-3, blue) is shown. FIG. 7 shows fluorescence immunostaining (green) for the respective markers of desmin, cTnT and myosin heavy chain after culturing mesenchymal stem cells on the PS, G1, G3 and G5 culture surfaces for 7 days. In addition, an example of a microscopic photograph obtained by nuclear staining (Topro, blue) is shown. FIG. 8 is a graph showing the relationship between the culture time and the cell density when mesenchymal stem cells are cultured on the PS, G1, G3, and G5 culture surfaces. FIG. 9 shows an example of a photograph obtained by culturing cells on the PS surface or G5 surface for 7 days, then treating cells with trypsin, re-culturing on the PS surface, and observing the cells on the fifth day under a microscope. FIG. 10 shows that after culturing on the PS surface or G5 surface for 7 days, the cells were trypsinized and re-cultured on the PS surface, and the cells on the 5th day were desmin, cTnT, MHC Fast Skeletal and α-smooth muscle actin. An example of a microscopic observation photograph after fluorescent immunostaining (green) and nuclear staining (Topro, blue) for each of the markers is shown.

The present invention relates to a culture condition in which differentiation is not induced by culture on a general plane culture surface when a mesenchymal stem cell is cultured on a culture surface modified with a dendrimer compound, for example, a culture medium component for differentiation induction or differentiation induction. Based on the knowledge that differentiation of mesenchymal stem cells can be induced even under culture conditions in which no medium is added or used. The present invention is preferably further based on the finding that differentiation induction of the mesenchymal stem cells can be performed with cell proliferation.

That is, the present invention includes:
[1] A method for inducing differentiation of mesenchymal stem cells (hereinafter also referred to as “differentiation induction method of the present invention”), wherein the mesenchymal stem cells are cultured in a cell culture vessel having a culture surface modified with a dendrimer compound. A method of inducing differentiation of mesenchymal stem cells, comprising culturing
[2] The method for inducing differentiation of mesenchymal stem cells according to [1], which comprises directing differentiation of mesenchymal stem cells into different cells by adjusting the number of generations of the dendrimer compound;
[3] The method for inducing differentiation of mesenchymal stem cells according to [1], wherein the differentiation is differentiation into cells of striated muscle / smooth muscle system, and the generation number of the dendrimer compound is 1 or more;
[4] The differentiation induction method of mesenchymal stem cells according to [1], wherein the differentiation is differentiation into cartilage cells, and the generation number of the dendrimer compound is 4 or more;
[5] The differentiation is differentiation into cardiomyocytes,
The method for inducing differentiation of mesenchymal stem cells according to [1], wherein the generation number of the dendrimer compound is 4 or more.
[6] The substance according to any one of [1] to [5], wherein a substance that can be taken up by the transporter of the cell is presented on the culture surface, and the compound is bound to an end of the dendrimer compound. A method for inducing differentiation of mesenchymal stem cells;
[7] The method for inducing differentiation of mesenchymal stem cells according to any one of [1] to [6], wherein the dendrimer compound includes a structural unit having a cationic group;
[8] The substance that can be taken up by the transporter of the cell is at least one sugar selected from the group consisting of D-glucose, D-fructose, and D-galactose, [6] or [7] Method for inducing differentiation of mesenchymal stem cells;
[9] A mesenchymal system in which differentiation is directed, comprising directing differentiation of the mesenchymal stem cell by the method for inducing differentiation of a mesenchymal stem cell according to any one of [1] to [8] A method for producing stem cell-derived cells;
[10] The method for producing a cell according to [9], wherein the direction of differentiation is accompanied by cell proliferation;
[11] A cell derived from a mesenchymal stem cell with a direction of differentiation, which can be produced by the method for producing a cell according to [9] or [10];
[12] A mesenchymal system having a direction of differentiation, comprising directing differentiation of the mesenchymal stem cell by the method for inducing differentiation of a mesenchymal stem cell according to any one of [1] to [8] A method for producing a biomaterial containing cells derived from stem cells;
[13] The method for producing a biomaterial according to [12], wherein the direction of differentiation involves cell proliferation;
[14] A biomaterial containing cells derived from mesenchymal stem cells with a direction of differentiation, which can be produced by the method for producing a biomaterial according to [12] or [13];
[15] A kit for inducing differentiation of mesenchymal stem cells, a cell culture container having a culture surface modified with a dendrimer compound, and the mesenchyme according to any one of [1] to [8] A differentiation induction kit for mesenchymal stem cells, comprising an instruction manual describing a method for inducing differentiation of stem cell stem cells;
[16] A method for producing a cell cluster having a round shape derived from a mesenchymal stem cell, comprising culturing the mesenchymal stem cell in a cell culture vessel having a culture surface modified with a dendrimer compound;
[17] The production method according to [16], wherein the generation number of the dendrimer compound is 4 or more;
[18] The production according to [16] or [17], wherein a substance that can be taken up by a transporter of a mesenchymal stem cell is presented on the culture surface, and the compound is bound to an end of the dendrimer compound. Method;
[19] The production method of [18], wherein the substance that can be taken up by the transporter is at least one sugar selected from the group consisting of D-glucose, D-fructose, and D-galactose.

[Mesenchymal stem cells]
In the present specification, a mesenchymal stem cell preferably refers to a somatic stem cell that can be differentiated into cells constituting many types of mesenchymal tissues. As mesenchymal stem cells, mammalian mesenchymal stem cells are preferable, primate mesenchymal stem cells are more preferable, and human mesenchymal stem cells are more preferable from the viewpoint of clinical application to humans. In addition, the mesenchymal stem cells can be present in bone marrow, fat, muscle, etc., but the mesenchymal stem cells in the present invention are not particularly limited, and mesenchyme obtained from any tissue of bone marrow, fat, muscle. It may be a stem cell. Accordingly, using mesenchymal stem cells herein may include using a cell population that includes mesenchymal stem cells. In addition, the mesenchymal stem cells in the present invention may be obtained from commercially available cells or cell banks.

The mesenchymal stem cells in the present invention may be those subcultured. The number of passages is not particularly limited, but is preferably 1 to 50 times, more preferably 1 to 30 times, and even more preferably 1 to 10 times from the viewpoint of maintaining differentiation ability. The mesenchymal stem cells in the present invention preferably have the ability to differentiate into cartilage cells and striated / smooth muscle cells.

In the present invention, the mesenchymal stem cells are preferably positive for the mesenchymal stem cell marker. A known marker can be used as a marker for mesenchymal stem cells. In the case of human mesenchymal stem cells, for example, at least one cell surface marker of CD105, Stro-1, CD106, and CD271 is preferably positive.

[Induction of differentiation of mesenchymal stem cells]
In the present specification, the induction of differentiation of mesenchymal stem cells includes starting differentiation of mesenchymal stem cells from an undifferentiated state, and is preferably mesenchymal tissue-specific not detected from an undifferentiated state to an undifferentiated state. A state in which a specific differentiation marker can be detected. A person skilled in the art can appropriately select a mesenchymal tissue-specific differentiation marker and a detection method thereof, and determine whether or not the differentiation marker is detected.

Differentiation markers specific to mesenchymal tissues include cell surface markers including sugars and proteins present in cell membranes; intracellular markers including mRNAs, proteins and enzymes present in cells; peptides secreted outside the cells , Extracellular markers including proteins, enzymes, compounds, and the like. The method for detecting the differentiation marker is not particularly limited, and examples thereof include a method using a labeled antibody (staining method, flow cytometry, ELISA, etc.), a staining method utilizing enzyme activity, and an RT-PCR method.

As a method for detecting a differentiation marker specific to a mesenchymal tissue, the following method may be mentioned. Examples of the method for confirming differentiation induction into cartilage cells include type II collagen staining. Examples of the method for confirming induction of differentiation into adipocytes include an oil red O staining method. Examples of the method for confirming differentiation induction into bone cells include ALP staining, and examples of the method for confirming differentiation induction into striated muscle / smooth muscle cells include desmin staining. . As a method for confirming differentiation induction into myocardial cells, for example, cTnT (cardiac troponic T) staining method can be mentioned. Examples of the method for confirming differentiation induction into skeletal muscle cells include myosin heavy chain staining. Examples of the method for confirming differentiation induction into vascular cells include the CD31 staining method. However, the method for detecting a differentiation marker is not limited to these.

In the present specification, “chondrocyte cells” include chondrocytes, chondroblasts, and cells directed to differentiation into chondrocytes, and preferably include cells that express a chondrocyte-specific differentiation marker. More preferably, cells that can be stained by a type II collagen staining method are included. In the present specification, “striated muscle / smooth muscle cell” includes myoblasts, striated muscles and / or smooth muscle myocytes, and cells in which differentiation into myoblasts is directed. Preferably cells that express a myoblast-specific differentiation marker, more preferably cells that can be stained by the desmin staining method. In the present specification, “cardiomyocytes” include cardiomyocytes, myocardial blasts, cells that can constitute myocardial tissue, and cells that have been differentiated into cardiomyocytes or myocardial blasts. A cell or a cell expressing a cardiomyocyte-specific differentiation marker, more preferably a cell that can be stained by the cTnT staining method. In the present specification, “bone system cells” include osteoblasts, bone cells, and cells in which differentiation into osteoblasts is directed.

In the present specification, the term “differentiated mesenchymal stem cell-derived cell” means a property specific to a cell of a specific mesenchymal tissue rather than an undifferentiated mesenchymal stem cell. An altered cell, preferably a cell that expresses or can be detected a differentiation marker that is not expressed or detected in undifferentiated mesenchymal stem cells.

[Dendrimer compounds]
In the present specification, the dendrimer compound is used for modifying the culture surface. By modifying the dendrimer compound, irregularities in the order of nanometers can be formed on a flat culture surface. The dendrimer compound is not particularly limited, and those that can form nanometer-order irregularities on the culture surface as described above can be used. For example, a known polyamidoamine (PAMAM) dendrimer or JP-A-2005-192406 can be used. And polyiminoamine dendrimers represented by the following formula (I) or (II) disclosed in the above. Further, from the viewpoint of promoting the fixation of cells to the culture surface, the dendrimer compound used in the present invention is preferably such that the molecule (dendron) constituting the branched portion of the dendrimer compound is cationic, and particularly the terminal group. Is more preferably an amino group.

In the above formulas (I) and (II), X represents a culture surface or a binding group bonded to the culture surface. In the above formula (I), n is an integer of 1 or more.

As a method of forming a dendrimer compound that modifies the culture surface, a method of forming a molecule (dendron) that constitutes a branched portion of the dendrimer compound directly or through a compound that serves as a linker (Devergent method) ). Specifically, the method disclosed in Non-Patent Document 1 (Kim et al., J. Biosci. Bioeng., Vol. 103 (2007) 192-199) can be mentioned. In the present specification, the number of generations of the dendrimer compound refers to the number of branches of the branched portion. For example, when n = 1 in the dendrimer compound represented by the above formula (I), the number of generations is 2, It may be expressed as G2. Moreover, the generation number of the dendrimer compound represented by the above formula (II) is 1, and may be represented as G1. A person skilled in the art can appropriately design the number of generations of the dendrimer compound to a desired number of generations, and specifically, it can be designed as in the examples described later.

If necessary, various substances may be bound to the terminal in the branching direction of the dendrimer compound that modifies the culture surface (for example, the amino group in the above formulas (I) and (II)). By binding a desired substance to the terminal, it can be presented to mesenchymal stem cells in culture. From the viewpoint of promoting the immobilization of cells on the culture surface, the substance to be bound to the end of the dendrimer compound is preferably a substance that can be taken up by a cell transporter. As a substance that can be taken up by a cell transporter, L-amino acids and sugars such as D-glucose, D-fructose, and D-galactose are preferable from the viewpoint of promoting morphological change and / or differentiation induction of mesenchymal stem cells. . On the other hand, in a less preferred embodiment, a substance (eg, L-glucose) in which an uptakeable transporter is not present in the cell may be bound to the end of the dendrimer compound.

[Culture surface]
As described above, the culture surface used in the present invention is a culture surface modified with a dendrimer compound, and is preferably a culture surface on which unevenness of nanometer order is formed on the culture surface. The irregularities preferably have a surface roughness (Ra) measured by an atomic force microscope (AFM) of 1 to 10 nm, and more preferably 2 to 7 nm. As described above, from the viewpoint of promoting morphological change and / or differentiation induction of mesenchymal stem cells, it is preferable that a substance that can be taken up by the transporter of the cells is presented on the culture surface.

[Method of inducing differentiation of mesenchymal stem cells]
The method for inducing differentiation of mesenchymal stem cells of the present invention is a method for inducing differentiation comprising culturing mesenchymal stem cells in a cell culture vessel having a culture surface modified with a dendrimer compound as described above. As the cell culture vessel, a cell culture vessel having the same material, shape and size as those used in normal cell culture can be used except that it has a modified culture surface. As the culture conditions, normal conditions such as 4 to 6% CO ₂ , preferably 5% CO ₂ , such as 35 to 38 ° C., preferably 37 ° C. can be employed. As the culture medium, a medium for culturing mesenchymal stem cells in an undifferentiated state or a medium for growing mesenchymal stem cells can be used. That is, even if the culture conditions do not induce differentiation of mesenchymal stem cells (for example, culture conditions that do not use a culture medium for differentiation induction or a culture medium component for differentiation induction), the planar culture surface is a dendrimer-modified culture surface. It is possible to induce differentiation of mesenchymal stem cells, and preferably to induce differentiation accompanied by cell proliferation. However, the differentiation induction method of the present invention may include the use of a culture medium and / or a culture medium for differentiation induction in addition to the use of a dendrimer-modified culture surface.

Therefore, according to the present invention, a dendrimer-modified culture surface that is a solid phase factor can be provided as a factor for inducing differentiation of mesenchymal stem cells. According to the present invention, it is possible to induce differentiation of mesenchymal stem cells, including the use of a dendrimer-modified culture surface that is a solid phase factor for inducing differentiation of mesenchymal stem cells. Furthermore, according to the present invention, preferably, differentiation induction of mesenchymal stem cells accompanied by cell proliferation becomes possible. From the viewpoint of promoting cell proliferation, the dendrimer compound generation is preferably as young as possible.

Furthermore, according to the present invention, as described later, the mesenchymal stem cells can be differentiated into different cells by adjusting the number of generations of dendrimer compounds. For example, if the number of generations is 1 or more, it is possible to direct differentiation into striated muscle / smooth muscle cell, and if the number of generations is 4 or more, it is possible to further direct differentiation into chondrocytes and cardiomyocytes. It becomes possible.

[Induction of differentiation into striated / smooth muscle cells]
Therefore, one embodiment of the present invention is a method for inducing differentiation of mesenchymal stem cells into striated muscle / smooth muscle cells, which has a culture surface modified with a dendrimer compound having a generation number of 1 or more. A differentiation induction method comprising culturing the mesenchymal stem cells in a container. According to the present invention in this embodiment, preferably, it is possible to direct differentiation into mesenchymal stem cells and differentiation of mesenchymal stem cells into striated muscle / smooth muscle cells, more preferably cells. It is possible to direct the differentiation of mesenchymal stem cells into striated / smooth muscle cells with proliferation. The generation number of the dendrimer compound is preferably 1 to 10, 1 to 8, or 1 to 6. From the viewpoint of obtaining a cell population in which the cells extend in the plane direction of the culture surface, the generation number is preferably 1 to 3. Also, from the viewpoint of obtaining a cell clump in which the cells are round, the generation number is preferably 4 or more, more preferably 4 to 7, and further preferably 4 to 6.

From the viewpoint of promoting differentiation into striated muscle / smooth muscle cells, a substance that can be taken up by the transporter of mesenchymal stem cells is presented on the modified culture surface by binding to the end of the dendrimer compound. In terms of promoting differentiation into striated / smooth muscle cells, the substance is preferably an L-amino acid and a sugar such as D-glucose, D-fructose, or D-galactose. -Glucose, D-fructose, and D-galactose are more preferred, and D-glucose is even more preferred. As described above, differentiation into striated muscle / smooth muscle cell can be determined based on, for example, a positive differentiation marker (for example, desmin) specific to striated muscle / smooth muscle cell.

[Induction of differentiation into chondrocytes]
Another embodiment of the present invention is a method for inducing differentiation of mesenchymal stem cells into cartilage cells, wherein the cell culture vessel has a culture surface modified with a dendrimer compound having a generation number of 4 or more. A differentiation induction method comprising culturing leaf stem cells. According to the present invention in this embodiment, it is possible to direct differentiation of mesenchymal stem cells into chondrocytes, and more preferably, mesenchymal stem cells accompanied by cell proliferation. The direction of differentiation into chondrocytes can be achieved. From the viewpoint of promoting differentiation into cartilage cells, the number of generations is preferably 4 to 10 or 4 to 8, more preferably 4 to 6, and even more preferably 5.

From the point of promoting differentiation into chondrocyte cells, it is preferable that a substance that can be taken up by the transporter of mesenchymal stem cells is presented on the modified culture surface by binding to the end of the dendrimer compound, From the viewpoint of promoting differentiation into cartilage cells, the substance is preferably L-amino acid and sugars such as D-glucose, D-fructose, D-galactose, and D-glucose, D-fructose, and D- Galactose is more preferred and D-glucose is even more preferred. As described above, differentiation into cartilage cells can be determined based on, for example, that a differentiation marker specific for chondrocytes (eg, type II collagen) is positive.

[Induction of differentiation into myocardial cells]
Another embodiment of the present invention is a method for inducing differentiation of mesenchymal stem cells into myocardial cells, wherein the cell culture vessel has a culture surface modified with a dendrimer compound having a generation number of 4 or more. A differentiation induction method comprising culturing leaf stem cells. According to the present invention in this embodiment, differentiation of mesenchymal stem cells can be induced, and differentiation of mesenchymal stem cells into myocardial cells can be directed. From the viewpoint of promoting differentiation into cardiomyocytes, the number of generations is preferably 4 to 10 or 4 to 8, more preferably 4 to 6, and further preferably 5 or 6.

From the viewpoint of promoting differentiation into cardiomyocytes, it is preferable that a substance that can be taken up by the transporter of mesenchymal stem cells is presented on the modified culture surface by binding to the end of the dendrimer compound. From the viewpoint of promoting differentiation into myocardial cells, the substance is preferably L-amino acid and sugars such as D-glucose, D-fructose, D-galactose, and D-glucose, D-fructose, and D- Galactose is more preferred and D-glucose is even more preferred. As described above, differentiation into cardiomyocytes can be determined based on, for example, that a differentiation marker specific to cardiomyocytes (eg, cTnT) is positive.

When the mesenchymal stem cells are cultured on a culture surface modified with a dendrimer compound having a generation number of 4 or more, as described above, a rounded cell aggregate can be formed. As shown in Examples described later, cells located on the surface portion (surface layer portion) of this cell cluster tend to be cells that are directed to differentiation into type II collagen-positive chondrocytes. On the other hand, cells located in the interior corresponding to portions other than the surface portion (surface layer portion) of the cell cluster tend to be cells that are directed to differentiation into cardiomyocytes. In addition, the surface part (surface layer part) and the inside in the cell agglomeration can be determined with reference to marker-stained data as shown in Examples described later.

Therefore, in one embodiment of the method for inducing differentiation of mesenchymal stem cells into cartilage cells, the cells are obtained by further culturing in a cell culture vessel having a culture surface modified with a dendrimer compound having a generation number of 4 or more. Separating cells that are located on the surface portion (surface layer portion) of the cell aggregate from the obtained cell aggregate, and preferably directed to differentiation into chondrocytes.

Similarly, in one embodiment of the method for inducing differentiation of mesenchymal stem cells into cardiomyocytes, the cells are further cultured in a cell culture vessel having a culture surface modified with a dendrimer compound having a generation number of 4 or more. The method may include separating the cells located in the cell aggregate from the obtained cell aggregate, and preferably directed to differentiation into cardiomyocytes.

In another embodiment of the method for inducing differentiation of mesenchymal stem cells into cardiomyocytes, the cells are located inside the isolated cell cluster, preferably directed to differentiation into cardiomyocytes. The method may include culturing the cells in a cell culture vessel on a flat culture surface that is not dendrimer-modified. Cells that have been directed to differentiation into cardiomyocytes isolated from the cell clumps are preferably specific for cardiomyocytes such as cTnT even if cultured on a flat culture surface that is not dendrimer-modified. Can maintain positive differentiation markers.

As described above, according to the differentiation inducing method of the present invention, it is possible to produce preferably mesenchymal stem cell-derived cells in which differentiation is directed.

[Method for producing cells]
Therefore, the present invention provides, as another aspect, a method for directing differentiation of mesenchymal stem cells derived from mesenchymal stem cells, comprising directing differentiation to mesenchymal stem cells by the differentiation induction method of the present invention. The present invention relates to a production method (hereinafter also referred to as “the production method of the cell of the present invention”). According to the method for producing a cell of the present invention, it is possible to produce a cell derived from a mesenchymal stem cell that is preferably directed to differentiation while accompanying cell proliferation. Moreover, according to the method for producing cells of the present invention, a mesenchymal stem cell with a direction of differentiation that can be used as a biomaterial or a cell supply source (cell source) in tissue engineering, regenerative medicine, regenerative medicine engineering, and the like. Derived cells can be produced.

As described above, the cells that can be produced by the cell production method of the present invention include mesenchymal stem cell-derived cells that have been directed to differentiation into striated muscle / smooth muscle cells, and the direction of differentiation into chondrocytes. And mesenchymal stem cell-derived cells that have been subjected to differentiation, and mesenchymal stem cell-derived cells that have been directed to differentiation into cardiomyocytes.

As an embodiment of the method for producing cells of the present invention, there is provided a method for producing mesenchymal stem cell-derived cells in which differentiation has been directed to striated muscle / smooth muscle cells, which is the striated muscle / smooth muscle described above. Examples include a production method including culturing mesenchymal stem cells by a method for inducing differentiation into a systemic cell. Further, as another embodiment of the method for producing cells of the present invention, a method for producing mesenchymal stem cell-derived cells that have been directed to differentiation into chondrocytes, the method for inducing differentiation into chondrocytes described above A production method including culturing a mesenchymal stem cell is mentioned. Furthermore, as another embodiment of the method for producing a cell of the present invention, a method for producing a mesenchymal stem cell-derived cell that has been directed to differentiation into a myocardial cell, the differentiation induction into the cardiomyocyte described above The production method includes culturing mesenchymal stem cells by the method.

In yet another aspect, the present invention provides a method for producing a biomaterial or cell source containing cells derived from mesenchymal stem cells that have been subjected to differentiation, and the mesenchymal stem cells are produced by the differentiation induction method of the present invention. The present invention relates to a production method comprising directing differentiation to a cell or producing a cell derived from a mesenchymal stem cell whose differentiation is directed by the cell production method of the present invention. According to the method for producing a biomaterial or cell source of the present invention, it is possible to produce a biomaterial or cell source containing cells derived from mesenchymal stem cells that are preferably differentiated with cell proliferation. .

Further, as described above, the differentiation induction method of the present invention can be performed without using a medium for differentiation induction and / or a medium component for differentiation induction conventionally used for induction of differentiation of mesenchymal stem cells. Therefore, according to the method for producing a cell of the present invention, it is preferable that the mesenchymal stem cell which is not contacted with these differentiation-inducing medium and / or medium components (has no history of contact) and is directed to differentiation. The cell can be produced, and more preferably, the cell can be produced with cell proliferation.

The cells, biomaterials or cell sources that can be produced according to the present invention can preferably be used in the field of regenerative medicine. For example, cells directed to differentiation into cardiomyocytes are preferably applied to repair myocardium, and cells directed to differentiation into chondrocytes are preferably applied to repair articular cartilage. Cells that have been directed to differentiation into striated / smooth muscle cells can preferably be applied to repair muscle tissue. Alternatively, cells, biomaterials, or cell sources that can be produced according to the present invention can preferably be used for cell assay applications in drug discovery screening. However, the use of cells, biomaterials, or cell sources that can be produced according to the present invention is not limited thereto.

[kit]
As yet another aspect, the present invention provides a kit for inducing differentiation of mesenchymal stem cells, a cell culture container having a culture surface modified with a dendrimer compound, and the differentiation induction method of the present invention. The present invention relates to a differentiation induction kit for mesenchymal stem cells (hereinafter also referred to as “differentiation induction kit of the present invention”) including an instruction manual including the description described above. The differentiation induction kit of the present invention may further contain mesenchymal stem cells. In addition, the form provided on the web may be sufficient as the instruction manual in the differentiation-inducing kit of this invention, without being bundled with the differentiation-inducing kit of this invention. The culture surface and cell culture vessel in the differentiation induction kit of the present invention can be those described above. From the viewpoint of efficient differentiation induction, as described above, a substance that can be taken up by the transporter of mesenchymal stem cells is preferably presented on the modified culture surface by binding to the end of the dendrimer compound. As the substance, L-amino acids and sugars such as D-glucose, D-fructose and D-galactose are preferable, D-glucose, D-fructose and D-galactose are more preferable, and D-glucose is further preferable.

In one embodiment of the differentiation induction kit of the present invention, the cell culture container is modified with a dendrimer compound having a generation number of 1 or more, preferably 1 to 10, 1 to 8, 1 to 6, 1 to 3, or 4 to 6 This kit has a culture surface and induces differentiation of mesenchymal stem cells into striated muscle / smooth muscle cells. According to the differentiation-inducing kit of the present invention in this embodiment, it is possible to direct differentiation into mesenchymal stem cells and differentiation into striated muscle / smooth muscle cells with respect to mesenchymal stem cells. It is possible to produce cells derived from mesenchymal stem cells that have been directed to differentiation into striated muscle / smooth muscle cells, and more preferably, the direction of differentiation into striated muscle / smooth muscle cells with cell proliferation. Cells with attached mesenchymal stem cells can be produced.

In another embodiment of the differentiation induction kit of the present invention, the cell culture container is modified with a dendrimer compound having a generation number of 4 or more, preferably 4 to 10 or 4 to 8, more preferably 4 to 6, and further preferably 5. This kit has a culture surface and induces differentiation of mesenchymal stem cells into cartilage cells and / or cardiomyocytes. According to the differentiation-inducing kit of the present invention in this embodiment, it is possible to direct differentiation into mesenchymal stem cells and cartilage cells and / or cardiomyocytes, as well as to induce differentiation into mesenchymal stem cells, It is possible to produce cells derived from mesenchymal stem cells that have been directed to differentiation into cartilage cells and / or cardiomyocytes, and more preferably, to cells of cartilage and / or cardiomyocytes while accompanying cell proliferation Cells derived from mesenchymal stem cells with a direction of differentiation can be produced.

[Method of treatment]
In still another aspect, the present invention relates to a therapeutic method and / or a regenerative medicine method including the use of cells that can be produced by the method for producing cells of the present invention. For example, a treatment in which a mesenchymal stem cell that is deficient in cartilage is used to prepare a cell in which differentiation into a cartilage cell is induced by the method for producing a cell of the present invention, and the cell is transplanted to a target cartilage defect site Or the method of regenerative medicine is mentioned. The transplantation can be performed, for example, by seeding the cells on a collagen gel sponge or the like and placing the sponge in the defect. In addition, for example, by using a mesenchymal stem cell of a subject with dilated cardiomyopathy, a cell in which differentiation into striated muscle / smooth muscle cell or cardiomyocyte is induced by the method for producing a cell of the present invention, Examples thereof include a method for treating dilated cardiomyopathy or regenerative medicine in which a myoblast sheet (cardiac function restoring material) is produced from these cells. That is, the treatment method and / or regenerative medicine method of the present invention aims to restore cardiac function by covering the outside of the heart of the subject with the cardiac function recovery material, inducing blood vessels to the heart wall, repairing cardiomyocytes, A method for treating dilated cardiomyopathy or regenerative medicine. In addition, the treatment method and / or regenerative medicine method in this invention are not limited to these.

Hereinafter, the present invention will be further described using examples.

Human mesenchymal stem cells were cultured on a culture surface modified with dentrimers of different generations with D-glucose bonded to the terminal. The production method of the culture surface and the cell culture conditions are as follows.

[Preparation of culture surface]
For the culture surface modified with dendrimer, a commercially available square 8-well plate (made of polystyrene, surface area of 9.6 cm ² / well, manufactured by Nunc) was used as a starting material equipped with a flat surface. It produced as follows. The planar culture surface of this 8-well plate was used as a control surface (hereinafter also referred to as “PS surface”).

[Production of generation 1 (G1) D-glucose-presenting dendrimer surface]
The D-glucose-presenting dendrimer surface (G1 surface) of G1 was prepared in the following four steps under aseptic conditions.
Step 1: A 50 μmol / ml aqueous solution of potassium tert-butoxide (t-BuOK) was added to the well and allowed to stand at room temperature for 1 hour in order to present hydroxyl groups on the planar culture surface as the starting material. The wells were then washed 3 times with sterile water.
Step 2: A 360 μmol / ml aqueous solution of glutaraldehyde was added to the well, allowed to stand at room temperature for 1 hour, and then washed with a large amount of sterile water. A 360 μmol / ml tris (2-aminoethyl) amine aqueous solution (pH 9.0) was added to the well and left to stand for 1 hour to form a dendron structure, which was washed with sterile water.
Step 3: In order to present D-glucose at the end of the dendrimer, 0.5 μmol / ml D-glucose aqueous solution was added to the well and allowed to stand for 2 hours.
Step 4: 0.5 μmol / ml sodium borohydride (NaBH ₄ ) was added to the well and allowed to stand for 24 hours. The well was washed with sterilized water to obtain a culture surface modified with a dendrimer on which D-glucose was presented.

[Preparation of D-glucose-presenting dendrimer surfaces of generations 3 and 5 (G3 and G5)]
For the D-glucose-presenting dendrimer surfaces (G3 surface and G5 surface) of G3 and G5, after step 1, the above step 2 is repeated 3 and 5 times, respectively, so that the number of generations of dendrimers is 3 and 5, It was produced by performing Step 3 and Step 4.

[Used cells]
Human bone marrow mesenchymal stem cells (manufactured by Lonza) were used as human mesenchymal stem cells.

[Medium used]
A DMEM medium (Dulbecco's modified Eagle medium) containing 10% FBS (fetal bovine serum) (manufactured by Sigma) was used as a culture medium on the dendrimer-modified culture surface. In addition, a human bone marrow mesenchymal stem cell culture medium (manufactured by Lonza) was used as the medium for proliferation of the mesenchymal stem cells.

[Culture conditions]
Cell culture was performed at 37 ° C. in a 5% CO ₂ atmosphere. In addition, the inoculation density | concentration to the well of a cell was 5 * 10 < ³ > cell / cm < ² >.

[Evaluation of cell differentiation]
The differentiation of mesenchymal stem cells was confirmed using the differentiation marker staining method shown in Table 1 below. The human mesenchymal stem cells cultured on the PS surface using DMEM medium containing 10% FBS (fetal bovine serum) were all negative for the differentiation marker staining shown in Table 1 below.

[Cell shape change and its result]
The mesenchymal stem cells were cultured on the PS, G1, G3, and G5 planes under the above culture conditions, and the morphology of the cells after 1, 3, and 7 days was observed with a microscope. An example of the result is shown in FIG. FIG. 1 shows mesenchymal stem cells cultured on the culture planes of the PS plane (a), G1 plane (b), G3 plane (c), and G5 plane (d) after 1 day, 3 days, and 7 It is a figure which shows the microscope observation photograph after a day.

As shown in FIG. 1, the mesenchymal stem cells cultured on the culture planes of the PS, G1, and G3 planes tended to show a shape that expanded in the plane direction. On the other hand, when the cells were cultured on the G5 surface, the cell shape was rounded, and some cells became clumps to form spherical cell clumps. In addition, the cell clumps tended to increase with the number of culture days.

[Confirmation of differentiation direction: differentiation into striated muscle / smooth muscle system]
After culturing mesenchymal stem cells on the PS, G1, G3, and G5 culture surfaces for 7 days, desmin staining was performed to confirm the presence or absence of the differentiation marker. An example of the result is shown in FIG. FIG. 2 is a view showing a microscopic observation photograph obtained by staining (green) and nuclear staining (red) of desmin, which is a differentiation marker of striated / smooth muscle cells and smooth muscle cells. In addition, about the culture result in G5 surface, in addition to the photograph of a plane (upper surface), the confocal microscope observation photograph of a side surface is shown.

As shown in FIG. 2, the mesenchymal stem cells cultured on the PS culture surface were negative for desmin staining, but the mesenchymal stem cells cultured on the G1 and G3 culture surfaces were positive for desmin staining. It was. Therefore, it can be seen that the mesenchymal stem cells cultured on the culture planes of the G1 plane and the G3 plane have been directed to differentiation into striated / smooth muscle cells.

In addition, the mesenchymal stem cells cultured on the culture surface of G5 form a cell clump as described above and desmin stain. As shown in FIG. 2, the cells located on the surface of the cell clump are desmin stained. Positive.

[Confirmation of differentiation direction: differentiation into cartilage system]
After culturing mesenchymal stem cells on the PS, G1, G3, and G5 culture surfaces for 7 days, type II collagen staining was performed to confirm the presence or absence of the differentiation marker. An example of the result is shown in FIG. FIG. 3 is a view showing a microscopic observation photograph obtained by staining (red) type II collagen, which is a differentiation marker for cartilage cells.

As shown in FIG. 3, the mesenchymal stem cells cultured on the PS, G1, and G3 culture surfaces were negative for type II collagen staining, but the mesenchymal stem cells cultured on the G5 culture surface were desmin. Staining was positive. Therefore, it can be seen that the mesenchymal stem cells cultured on the culture surface of G5 have been directed to differentiation into chondrocytes.

As described above, culture conditions that do not induce differentiation of human mesenchymal stem cells in culture on the PS surface (for example, culture conditions that do not use conditioned medium for differentiation induction or medium components for differentiation induction) Even if the culture plane is changed from the PS plane to the G1 or G3 plane, differentiation of human mesenchymal stem cells and differentiation into striated / smooth muscle cells can be directed. It was shown that in addition to inducing differentiation of human mesenchymal stem cells and differentiating into striated / smooth muscle cells, it is possible to direct differentiation into chondrocytes by simply changing from PS to G5. .

Dendrimer-modified culture surfaces in which the amount of D-glucose presented was 0%, 50%, and 100% were prepared, and human mesenchymal stem cells were cultured on these culture surfaces. The production method of the culture surface and the cell culture conditions are as follows. The amount of D-glucose presented was adjusted by the mixing rate of L-glucose. In human cells, there is no transporter that can take up L-glucose.

[Preparation of culture surface]
For the culture surface modified with dendrimer, a commercially available square 8-well plate (made of polystyrene, surface area of 9.6 cm ² / well, manufactured by Nunc) was used as a starting material equipped with a flat surface. It produced as follows.

[Production of 100% D-glucose-presenting dendrimer surface]
Generation numbers 3, 4, and 5 (G3, G4, and G5) 100% D-glucose-presenting dendrimer surfaces were prepared under aseptic conditions in the following 4 steps.
Step 1: A 50 μmol / ml aqueous solution of potassium tert-butoxide (t-BuOK) was added to the well and allowed to stand at room temperature for 1 hour in order to present hydroxyl groups on the planar culture surface as the starting material. The wells were then washed 3 times with sterile water.
Step 2: A 360 μmol / ml aqueous solution of glutaraldehyde was added to the well, allowed to stand at room temperature for 1 hour, and then washed with a large amount of sterile water. A 360 μmol / ml tris (2-aminoethyl) amine aqueous solution (pH 9.0) was added to the well and left to stand for 1 hour to form a dendron structure, which was washed with sterile water. This step 2 was repeated three, four, and five times, respectively, when creating G3, G4, and G5 culture surfaces.
Step 3: In order to present D-glucose at the end of the dendrimer, 0.5 μmol / ml D-glucose aqueous solution was added to the well and allowed to stand for 2 hours.
Step 4: 0.5 μmol / ml sodium borohydride (NaBH ₄ ) was added to the well and allowed to stand for 24 hours. The wells were washed with sterilized water to obtain dendrimer culture surfaces (G3, G4, and G5) on which 100% D-glucose was presented.

[Preparation of 0% and 50% D-glucose-presenting dendrimer surfaces]
The 0% D-glucose-presenting dendrimer surface (G3, G4, and G5) was the same as that described above except that the 0.5 μmol / ml D-glucose aqueous solution in Step 3 was changed to a 0.5 μmol / ml L-glucose aqueous solution. It was prepared in the same manner as the 100% D-glucose-presenting dendrimer surface (G3, G4, and G5). The 50% D-glucose-presenting dendrimer surface (G3, G4, and G5) has a 0.5 μmol / ml D-glucose aqueous solution in Step 3 described above, and has an equivalent concentration of D-glucose and L-glucose. It was prepared in the same manner as the 100% D-glucose-presenting dendrimer surface (G3, G4, and G5) except that the aqueous solution was 0.5 μmol / ml glucose.

[Cell shape change and its result]
The mesenchymal stem cells were cultured under the above culture conditions on a culture surface modified with 0%, 50%, and 100% D-glucose-presenting dendrimers (G3, G4, and G5). The morphology was observed with a microscope. An example of the result is shown in FIG.

As shown in FIG. 4, the mesenchymal stem cells cultured on the G3 culture surface showed a shape that expanded in the plane direction regardless of the amount of glucose presented. On the other hand, when cultured on the culture surfaces of G4 and G5, the shape of the cells was rounded, and some cells became clumps to form spherical cell clumps. The number of cells forming a cell cluster was larger on the G5 surface than on the G4 surface. In addition, the number of cells forming the cell cluster was larger as the presentation of D-glucose was increased. Thus, it has been shown that the formation of cell clumps is promoted as the number of generations of dendrimers increases and as the presentation of D-glucose increases.

[Confirmation of differentiation direction: differentiation into myocardial system]
On the G5 surface, the mesenchymal stem cells were cultured for 7 days to obtain cell clusters. The mesenchymal stem cells and culture conditions used are the same as in Example 1. Next, specific markers for skeletal muscle cells (desmin, MHC fast Skeletal), markers specific for cardiomyocytes (desmin, cTnT), and chondrocytes Fluorescent immunostaining for the marker (type II collagen) was performed. To confirm the degree of staining, nuclear staining was also performed simultaneously with Topro-3. The observation results are shown in FIG.

As shown in FIG. 5, desmin and cTnT were expressed throughout the cell mass. On the other hand, expression of MHC fast Skeletal and type II collagen was confirmed on the surface (surface layer portion). Therefore, in the cell cluster, cells on the surface (surface layer part) are directed to differentiation into cartilage cells, but cells inside the cell cluster are directed to differentiation into cardiomyocytes. Has been confirmed.

[Confirmation of the role of dendrimer-modified surface]
In order to confirm that culture on the dendrimer-modified culture surface is necessary for the induction of differentiation into cardiomyocytes as described above, mesenchymal stem cells using a non-cell-adhesive round bottom 98 well plate Cell clusters were prepared. The cell culture conditions were the same as in Example 1 except that the cell culture vessel was a non-cell-adhesive round bottom 98 well plate. Next, fluorescence immunostaining for each marker of desmin, MHC fast Skeletal, cTnT, type II collagen, and CD105 was performed on the obtained cell cluster. CD105 is one of the markers for undifferentiated mesenchymal stem cells. The observation results are shown in FIG.

As shown in FIG. 6, desmin was observed to be slightly expressed around the cell cluster, but in contrast to FIG. 5, MHC fast Skeletal, cTnT, and type II collagen markers were negative. . Moreover, CD105 which shows the undifferentiated state of a mesenchymal stem cell was positive. Therefore, it was confirmed that merely using a mesenchymal stem cell as a cell clump is insufficient for induction of cardiomyocyte differentiation, and that culture on a dendrimer-modified culture surface is necessary.

[Presence or absence of differentiation into myocardial cells on the PS, G1, and G3 planes]
The presence or absence of differentiation induction into myocardial cells on the PS, G1, and G3 planes was confirmed. Under the same conditions as those described in Example 1, mesenchymal stem cells were cultured on the PS, G1, G3, and G5 culture surfaces for 7 days. Thereafter, fluorescence immunostaining for desmin, MHC fast Skeletal, and cTnT was performed and observed under a microscope. The result is shown in FIG.

As shown in FIG. 7, the cTnT marker was negative on the PS, G1, and G3 surfaces that do not form cell clumps. Therefore, it was suggested that the formation of cell clumps on the dendrimer-modified culture surface is necessary for induction of differentiation into cardiomyocytes.

[Relationship between differentiation induction and cell proliferation]
The relationship between the culture time and cell proliferation when culturing mesenchymal stem cells on the PS, G1, G3, and G5 culture surfaces was confirmed. Specifically, the cells were cultured under the cell culture conditions described in Example 1, and the cell density was measured every predetermined time. The result is shown in FIG.

As shown in FIG. 8, cells derived from mesenchymal stem cells continued to proliferate on the culture surfaces of the PS, G1, and G3 surfaces. On the other hand, in the culture on the G5 surface, the cells derived from the mesenchymal stem cells stopped growing from a certain time. The growth arrest on the G5 plane is suggested to be related to differentiation into cardiomyocytes.

Note that the results of Example 3 were also reproduced with different bone marrow mesenchymal stem cells.

[Cell differentiation plasticity]
The mesenchymal stem cells are cultured for 7 days on the G5 culture surface to obtain cell clumps that are directed to differentiation into cardiomyocytes, and the cells that have been dissociated by trypsinization of the cell clumps are displayed on the PS plane. The culture surface was transferred and re-cultured. As a control, mesenchymal stem cells cultured for 7 days on the PS-cultured surface were similarly trypsinized and then re-cultured on the PS-cultured surface. The cells and culture conditions used are the same as in Example 1. On the fifth day of re-culture, the cell morphology was confirmed by microscopic observation, and the expression of various differentiation markers was further confirmed.

The cell morphology observation results are shown in FIG. Cells that were re-cultured on the PS surface after culturing the G5 surface (bottom of FIG. 9) were slightly flattened compared to normal mesenchymal stem cells and cells that were re-cultured on the PS surface after culturing the PS surface (FIG. 9). The shape was shown.

Next, FIG. 10 shows the results of fluorescent immunostaining for markers of desmin, cTnT, MHC Fast Skeletal and α-smooth muscle actin. In cells re-cultured on the PS surface after culturing the PS surface, all of these markers were negative. On the other hand, many of the cells re-cultured on the PS surface after culturing the G5 surface were positive for desmin and cTnT markers. Therefore, it was suggested that differentiation into myocardial cells induced on the G5 culture surface is superior in cell differentiation stability.

The present invention is useful, for example, in the field of tissue engineering, the field of regenerative medicine, the field of regenerative medical engineering, and the like.

Claims

A method for inducing differentiation of mesenchymal stem cells,
A method for inducing differentiation of mesenchymal stem cells, comprising culturing the mesenchymal stem cells in a cell culture vessel having a culture surface modified with a dendrimer compound.
The method for inducing differentiation of mesenchymal stem cells, comprising directing differentiation of mesenchymal stem cells into different cells by adjusting the number of generations of the dendrimer compound.
The differentiation is differentiation into striated / smooth muscle cell,
The method for inducing differentiation of mesenchymal stem cells according to claim 1 or 2, wherein the generation number of the dendrimer compound is 1 or more.
The differentiation is differentiation into cartilage cells,
The method for inducing differentiation of mesenchymal stem cells according to claim 1 or 2, wherein the generation number of the dendrimer compound is 4 or more.
The differentiation is differentiation into cardiomyocytes,
The method for inducing differentiation of mesenchymal stem cells according to claim 1 or 2, wherein the generation number of the dendrimer compound is 4 or more.
A substance that can be taken up by the transporter of mesenchymal stem cells is presented on the culture surface,
The method for inducing differentiation of mesenchymal stem cells according to any one of claims 1 to 5, wherein the substance is bound to an end of the dendrimer compound.
The method for inducing differentiation of mesenchymal stem cells according to any one of claims 1 to 6, wherein the dendrimer compound has a cationic molecule constituting the branched portion.
The mesenchymal stem cell differentiation according to claim 6 or 7, wherein the substance that can be taken up by the transporter is at least one sugar selected from the group consisting of D-glucose, D-fructose, and D-galactose. Guidance method.
A mesenchymal stem cell-derived cell having a direction of differentiation, comprising directing differentiation of the mesenchymal stem cell by the method for inducing differentiation of a mesenchymal stem cell according to any one of claims 1 to 8. Production method.
The method for producing cells according to claim 9, wherein the direction of differentiation is accompanied by cell proliferation.
A cell derived from a mesenchymal stem cell with a direction of differentiation, which can be produced by the method for producing a cell according to claim 9 or 10.
A mesenchymal stem cell-derived cell with a direction of differentiation, comprising directing differentiation of the mesenchymal stem cell by the method for inducing differentiation of a mesenchymal stem cell according to any one of claims 1 to 8. A method for producing a biomaterial.
The method for producing a biomaterial according to claim 12, wherein the direction of differentiation is accompanied by cell proliferation.
A biomaterial comprising cells derived from mesenchymal stem cells with a direction of differentiation, which can be produced by the method for producing a biomaterial according to claim 12 or 13.
A kit for inducing differentiation of mesenchymal stem cells,
A cell culture vessel having a culture surface modified with a dendrimer compound, and
A mesenchymal stem cell differentiation-inducing kit comprising an instruction manual that describes the method for inducing differentiation of mesenchymal stem cells according to any one of claims 1 to 8.
A method for producing a cell cluster having a round shape derived from a mesenchymal stem cell, comprising culturing the mesenchymal stem cell in a cell culture vessel having a culture surface modified with a dendrimer compound.
The production method according to claim 16, wherein the generation number of the dendrimer compound is 4 or more.
A substance that can be taken up by the transporter of mesenchymal stem cells is presented on the culture surface,
The production method according to claim 16 or 17, wherein the substance is bonded to an end of the dendrimer compound.
The production method according to claim 18, wherein the substance that can be taken up by the transporter is at least one sugar selected from the group consisting of D-glucose, D-fructose, and D-galactose.