JP2017023008A - Method for culturing stem cells using incubator having polyrotaxane block copolymer surface - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique for culturing new pluripotent stem cells, which is highly safe, easy to handle, and/or inexpensive.SOLUTION: The above problems are solved by culturing stem cells using an incubator coated with a polyrotaxane (PRX) block copolymer. The inventors of the present invention have found that culturing the stem cells using an incubator coated with a polyrotaxane (PRX) block copolymer allows the undifferentiated state of pluripotent stem cells to be maintained and allows pluripotent stem cells to be differentiated into cardiomyocytes.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for culturing stem cells. Specifically, the present invention relates to a method for culturing stem cells using a culture vessel whose surface is coated with a polyrotaxane block copolymer. More specifically, the present invention relates to a method for culturing pluripotent stem cells using a culture vessel having a polyrotaxane block copolymer surface and maintaining the undifferentiation property, or a method for inducing differentiation into specific cells.

  Human pluripotent stem cells (hPSC) such as human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) are expected to be applied to drug discovery and regenerative medicine. Cultivating and proliferating pluripotent stem cells safely and reproducibly is an essential fundamental technology for medical application of these cells. In particular, in the industrial use of regenerative medicine, it is necessary to handle stem cells in large quantities in an undifferentiated state. Therefore, natural and synthetic polymers are used to support the proliferation of pluripotent stem cells while maintaining pluripotency. Extensive research has been conducted on the technology to be performed (Non-Patent Document 1).

  Currently the most commonly used techniques for maintaining hPSCs in an undifferentiated state are on feeder cells (eg mouse embryonic fibroblasts (MEF), human foreskin fibroblasts, human adipose derived cells, etc.) or The culture is carried out on Matrigel or Geltrex. However, culturing hPSCs using feeder cells is time consuming, has a problem that it varies from lot to lot of feeder cells, and is labor intensive. Matrigel and Gertrex, on the other hand, are composed of substrates isolated from mouse EHS sarcoma, including laminin, type IV collagen, heparan sulfate proteoglycan, enactin, growth factors, etc., and support the multipotency of many hESC strains It has been reported to do. However, since the exact composition is not clear and contains components derived from different organisms, the clinical application of hPSC is hindered when such culture conditions are used. There are also concerns regarding immunogenic epitopes such as sialic acid N-glycolylneuraminic acid (Neu5Gc), which are not found in humans, and transmission of heterologous infectious viruses to humans (Non-patent Document 2).

  The production of cells such as hESC and hiPSC is currently required to be carried out on a large scale under conditions that do not contain components derived from feeder cells and heterologous organisms in facilities that conform to the Good Manufacturing Practice (cGMP). The presence in particular limits the use of hPSC. The basic FGF (bFGF), Wnt, and activin / Nodal pathways are thought to be important for maintaining hPSC pluripotency, and various studies have shown that alternative hPSCs that do not involve feeder cells Although culture conditions have been tried, the main component responsible for maintaining the self-renewal ability of hPSC has not been clarified (Non-patent Document 3).

  In order to create a storable hPSC population required for future research toward regenerative medicine, it is important to control proliferation so that the pluripotency of hPSC is maintained. In recent years, in order to maintain the pluripotency while culturing hPSC, a cell culture matrix has been developed together with a medium having a clear chemical composition. These matrices have a clear chemical composition or are free of xenogeneic components, but require specific media combinations. Also, only specific hPSCs can maintain pluripotency on these cell matrices. It is currently not easy to determine the optimal matrix for hPSC culture (Non-patent Document 4). In order to utilize human pluripotent stem cells (hPSC) for medical treatment, it cannot be said that a culture technique for proliferating these cells safely, simply and inexpensively has already been sufficiently established. There are also various needs for techniques for controlling (promoting) differentiation of pluripotent stem cells.

  On the other hand, in cells that adhere to culture equipment, it is known that a physiological activity signal is transmitted into the cells reflecting the physicochemical characteristics of the equipment, thereby regulating metabolic activities such as cell proliferation, differentiation, and death. ing. For this reason, research on physicochemical factors on the surface of materials and cell function control according to them has been actively conducted for the past 10 years (Non-Patent Documents 5 to 7). For example, the differentiation characteristics of mesenchymal stem cells on elastomeric surfaces with various elasticity are enhanced by the differentiation of nerves and adipocytes being promoted by lowering the Young's modulus of the adhered elastomer. It is known that differentiation into osteoblasts is promoted (Non-patent Document 8). In addition, hESC is sensitive to physical stimuli, and it is known that when the matrix stiffness of the micropost array on the incubator increases, the contractility of the cytoskeleton increases and the maintenance of pluripotency of hESC is promoted by a hard substrate. (Non-Patent Documents 9 and 10). However, a technique for controlling the differentiation or undifferentiation maintenance of stem cells by adjusting the physicochemical characteristics of such equipment has not been sufficiently established to satisfy various needs in this field.

International Publication No. 02/02159 International Publication No. 03/074099 JP 2005-143920 A

Higuchi, A., Ling, Q., Kumar, S., Munusamy, M., Alarfajj, A., Umezawa, A., Wu, G., 2014.Design of polymeric materials for culturing humanpluripotent stem cells: Progress toward feeder -free andxeno-free culturing.Progress in Polymer Science 39, 1348-1374. Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat Med 2005; 11: 228-32. Abraham S, Sheridan SD, Miller B, Rao RR.Stable propagation of human embryonic and induced pluripotent stem cells on decellularized human substrates. Biotechnol Prog 2010; 26: 1126-34. Meng GL, Liu SY, Rancourt DE. Synergistic effect of medium, matrix, and exogenous factors on the adhesion and growth of humanpluripotent stem cells under defined, xeno-free conditions.Stem Cells Dev 2012; 21: 2036-48. Ji-Hun Seo, Nobuhiko Yui.The effect of molecular mobility of supramolecular polymer surfaces on fibroblast adhesion.Biomaterials Volume 34, Issue 1, January 2013, Pages 55-63. Ji-Hun Seo, Sachiro Kakinoki, Yuuki Inoue, Kwangwoo Nama, Tetsuji Yamaoka, Kazuhiko Ishihara, Akio Kishida, Nobuhiko Yui.The significance of hydrated surface molecular mobility in the control of the morphology of adhering fibroblasts.Biomaterials Volume 34, Issue 13, April 2013, Pages 3206-3214. Ji-Hun Seo, Sachiro Kakinoki, Tetsuji Yamaoka, and Nobuhiko Yui. Directing Stem Cell Differentiation by Changing the Molecular Mobility of Supramolecular Surfaces. Adv Healthc Mater. 2015 Jan 28; 4 (2): 215-22. Adam J. Engler, Shamik Sen, H. Lee Sweeney, Dennis E. Discher.Matrix Elasticity Directs Stem Cell Lineage Specification.Cell Volume 126, Issue 4, 25 August 2006, Pages 677-689 Higuchi A, Ling QD, Chang Y, Hsu ST, Umezawa A. Physical cuesof biomaterials guide stem cell differentiation fate.Chem Rev 2013; 113: 3297-328. Sun Y, Villa-Diaz LG, Lam RH, Chen W, Krebsbach PH, Fu J. Mechanics regulates fate decisions of human embryonic stem cells.PLoSONE 2012; 7, e37178 / 1-7.

  An object of the present invention is to provide a new pluripotent stem cell culture technique. Another object of the present invention is to provide a highly safe culture technique for pluripotent stem cells, and to provide a culture technique for pluripotent stem cells that is easy to handle. Furthermore, another object is to provide a pluripotent stem cell culture technique at low cost.

  In one embodiment, the present invention provides a method of culturing stem cells using a culture vessel whose surface is coated with a polyrotaxane (PRX) block copolymer. The present inventors can maintain the undifferentiation of pluripotent stem cells by culturing stem cells using an incubator whose surface is coated with a polyrotaxane (PRX) block copolymer, They found that it is possible to promote differentiation of pluripotent stem cells into cardiomyocytes.

  In one embodiment, the present invention provides a cell culture method for maintaining the undifferentiated nature of pluripotent stem cells, the pluripotent stem cells on a culture vessel having a surface coated with a polyrotaxane block copolymer. A method comprising the step of culturing

  In one embodiment of the method, the pluripotent stem cells are ES cells (embryonic stem cells) or iPS cells (artificial pluripotent stem cells), in particular skin fibroblast-derived iPS cells, fetal lung cell-derived iPS cells, foreskin Either a fibroblast-derived iPS cell or a cardiac fibroblast-derived iPS cell. In one embodiment of the method, the pluripotent stem cell is a primate or rodent cell, more specifically either a human, mouse or rat cell. Alternatively, porcine, rabbit, monkey pluripotent stem cells can also be used.

  In one embodiment of the method, the polyrotaxane block copolymer comprises α-, β-, or γ-cyclodextrin as a cyclic molecule. Cyclodextrin (hereinafter also referred to as CD) may be modified with a methoxy group. In one embodiment of the method, the polyrotaxane block copolymer comprises polyethylene glycol (PEG) or polypropylene glycol (PPG) as the linear molecule. When the polyrotaxane block copolymer includes polyethylene glycol (PEG) as a linear molecule, the molecular weight of PEG can be 15000-25000. The ratio of the number of molecules of cyclic molecules and linear molecules can be 20: 1 to 10: 1. In one embodiment of the method, the polyrotaxane block copolymer comprises MPC (2-methacryloyloxyethyl phosphorylcholine) and / or BMA (n-butyl methacrylate) as the block molecule. When a combination of MPC (2-methacryloyloxyethyl phosphorylcholine) and BMA (n-butyl methacrylate) is used as the block molecule of the polyrotaxane block copolymer, the BMA molar fraction can be 0.3 to 0.4. .

  In one embodiment of this method, the polyrotaxane block copolymer has a molecular mobility factor Mf value of 0.3 or more on the incubator.

  In one embodiment of the method, the incubator material comprises glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, or acrylic block copolymer (BCF).

  In one embodiment, the method further comprises the step of adding a cell culture medium for maintaining the undifferentiation of pluripotent stem cells to a culture vessel having a surface coated with a polyrotaxane block copolymer. In one embodiment, the method further comprises seeding pluripotent stem cells in a culture vessel having a surface coated with a polyrotaxane block copolymer.

  In one embodiment of this method, a culture medium prepared to maintain the undifferentiated nature of pluripotent stem cells is used as the culture medium.

  In one embodiment, the present invention provides a cell culture kit, a culture vessel having a surface coated with a polyrotaxane block copolymer, and a cell culture medium for maintaining the undifferentiation of pluripotent stem cells And a kit comprising:

  In one embodiment of this kit, the pluripotent stem cells are ES cells (embryonic stem cells) or iPS cells (artificial pluripotent stem cells), in particular skin fibroblast-derived iPS cells, fetal lung cell-derived iPS cells, foreskin Either a fibroblast-derived iPS cell or a cardiac fibroblast-derived iPS cell. In one embodiment of the kit, the pluripotent stem cell is a primate or rodent cell, more specifically either a human, mouse or rat cell.

  In one embodiment of the kit, the polyrotaxane block copolymer comprises cyclodextrin (α-, β-, or γ-cyclodextrin) as a cyclic molecule. The cyclodextrin may be modified with a methoxy group. In one embodiment of the kit, the polyrotaxane block copolymer comprises polyethylene glycol (PEG) or polypropylene glycol (PPG) as a linear molecule. When the polyrotaxane block copolymer includes polyethylene glycol (PEG) as a linear molecule, the molecular weight of PEG can be 15000-25000. The ratio of the number of molecules of cyclic molecules and linear molecules can be 20: 1 to 10: 1. In one embodiment of the kit, the polyrotaxane block copolymer comprises MPC (2-methacryloyloxyethyl phosphorylcholine) and / or BMA (n-butyl methacrylate) as a block molecule. When a combination of MPC (2-methacryloyloxyethyl phosphorylcholine) and BMA (n-butyl methacrylate) is used as the block molecule of the polyrotaxane block copolymer, the BMA molar fraction can be 0.3 to 0.4. .

  In one embodiment of this kit, the polyrotaxane block copolymer has a molecular mobility factor Mf value of 0.3 or more on the incubator.

  In one embodiment of the kit, the incubator material comprises glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, or acrylic block copolymer (BCF).

  In one embodiment of the kit, the culture medium includes a medium formulated to maintain the undifferentiated nature of pluripotent stem cells. The culture medium may be provided in liquid form or in the form of a dry powder. The culture medium may contain all the components in one container, or may be divided into a plurality of containers for each component.

  In one embodiment, the present invention provides an incubator having a surface coated with a polyrotaxane block copolymer comprising a cell culture medium for maintaining the undifferentiated nature of pluripotent stem cells.

  In one embodiment of the incubator, the pluripotent stem cells are ES cells (embryonic stem cells) or iPS cells (artificial pluripotent stem cells), in particular skin fibroblast-derived iPS cells, fetal lung cell-derived iPS cells, Either foreskin fibroblast-derived iPS cells or cardiac fibroblast-derived iPS cells. In one embodiment of the incubator, the pluripotent stem cells are primate or rodent cells, more specifically either human, mouse, or rat cells.

  In one embodiment of the incubator, the polyrotaxane block copolymer contains α-, β-, or γ-cyclodextrin as a cyclic molecule. The cyclodextrin may be modified with a methoxy group. In one embodiment of the incubator, the polyrotaxane block copolymer contains polyethylene glycol (PEG) or polypropylene glycol (PPG) as a linear molecule. When the polyrotaxane block copolymer includes polyethylene glycol (PEG) as a linear molecule, the molecular weight of PEG can be 15000-25000. The ratio of the number of molecules of cyclic molecules and linear molecules can be 20: 1 to 10: 1. In one embodiment of the incubator, the polyrotaxane block copolymer contains MPC (2-methacryloyloxyethyl phosphorylcholine) and / or BMA (n-butyl methacrylate) as a block molecule. When a combination of MPC (2-methacryloyloxyethyl phosphorylcholine) and BMA (n-butyl methacrylate) is used as the block molecule of the polyrotaxane block copolymer, the BMA molar fraction can be 0.3 to 0.4. .

  In one embodiment of the present incubator, the polyrotaxane block copolymer has a molecular mobility factor Mf value of 0.3 or more on the incubator.

  In one embodiment of the incubator, the incubator material includes glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, or acrylic block copolymer (BCF).

  In one embodiment of the incubator, a culture medium prepared to maintain the undifferentiated nature of pluripotent stem cells is included as the culture medium.

  In one embodiment, the present invention provides a cell culture method for promoting differentiation of pluripotent stem cells into cardiomyocytes, wherein the pluripotent stem cell has a surface coated with a polyrotaxane block copolymer. A method comprising the step of culturing sex stem cells is provided.

  In one embodiment of the method, the polyrotaxane block copolymer comprises α-, β-, or γ-cyclodextrin as a cyclic molecule. The cyclodextrin may be modified with a methoxy group. In one embodiment of the method, the polyrotaxane block copolymer comprises polyethylene glycol (PEG) or polypropylene glycol (PPG) as the linear molecule. When the polyrotaxane block copolymer includes polyethylene glycol (PEG) as a linear molecule, the molecular weight of PEG can be 15000-25000. The ratio of the number of molecules of cyclic molecules and linear molecules can be 20: 1 to 10: 1. In one embodiment of the method, the polyrotaxane block copolymer comprises MPC (2-methacryloyloxyethyl phosphorylcholine) and / or BMA (n-butyl methacrylate) as the block molecule. When a combination of MPC (2-methacryloyloxyethyl phosphorylcholine) and BMA (n-butyl methacrylate) is used as the block molecule of the polyrotaxane block copolymer, the BMA molar fraction can be 0.3 to 0.4. .

  In one embodiment of this method, the polyrotaxane block copolymer has a molecular mobility factor Mf value of 0.3 or more on the incubator.

  In one embodiment of the method, the incubator material comprises glass, polystyrene, polypropylene, polyethylene, polyolefin, or polycarbonate.

  In one embodiment, the method further comprises the step of adding a cell culture medium for promoting differentiation of pluripotent stem cells into cardiomyocytes into a culture vessel having a surface coated with a polyrotaxane block copolymer. . In one embodiment, the method further comprises seeding pluripotent stem cells in a culture vessel having a surface coated with a polyrotaxane block copolymer.

  In one embodiment of this method, a culture medium prepared to promote differentiation of pluripotent stem cells into cardiomyocytes is used as the culture medium.

  In one embodiment, the method further comprises the step of providing a pluripotent stem cell with a stimulus that promotes differentiation into cardiomyocytes.

  Furthermore, the present invention, in a further aspect, a cell culture comprising a culture vessel having a surface coated with a polyrotaxane block copolymer, and a cell culture medium for promoting differentiation of pluripotent stem cells into cardiomyocytes. And a culture vessel having a surface coated with a polyrotaxane block copolymer, including a cell culture medium for promoting the differentiation of pluripotent stem cells into cardiomyocytes.

It is drawing which shows typically the synthesis | combination of the polyrotaxane block copolymer by RAFT polymerization. A summary of an exemplary reaction is as follows. 0.018 g of dimethylaminopyridine (5 mL dichloromethane) was dissolved in 1 g of a both-end aminated PEG having a molecular weight of 20 kDaltons (5 mL dichloromethane), and then 0.14 g of a reversible addition-cleavage chain transfer polymerization agent (RAFT agent) and 0.082 g of carbodiimide Then, after reacting at room temperature for 12 hours, terminal RAFT agent functionalized PEG (RAFT-PEG) is recovered by reprecipitation and dialysis. 0.35 g of RAFT-PEG is stirred with 3.5 g of α-CD and 25 mL of pure water, and an inclusion complex of pseudopolyrotaxane is obtained by centrifugation and lyophilization. 0.6 g of the obtained pseudopolyrotaxane was dissolved in 7 mL of ethanol-toluene (1: 1) together with 0.354 g of MPC and 0.633 g of BMA, and 0.82 mg of azobisisobutyronitrile (AIBN) Is added and polymerized at 60 ° C. for one day. Thereafter, the PRX block copolymer (PRX-PMB) is recovered by reprecipitation, dialysis, and lyophilization. 200 mg of PRX-PMB is dispersed in 7 mL of dehydrated DMSO, 0.155 g of sodium hydride is added and stirred for 30 minutes, and then 0.102 g of iodomethane is gradually added and reacted at room temperature for 3 hours. The reaction solution is neutralized with a 6N hydrochloric acid solution, and methylated PRX-PMB is obtained by dialysis and lyophilization. It is a figure which shows the phase-contrast image and GFP-Nanog fluorescence expression (Bar = 100micrometer) of an adhesion | attachment iPS cell (the undifferentiation 8th day). The first and third columns from the left show phase contrast images of cultured cells, and the second and fourth from the left show fluorescence expression of GFP-Nanog. The upper two rows on the left are cells cultured on glass, the middle is cells cultured on PRX-B, the lower are cells cultured on gelatin, and the upper two rows on the right are cells cultured on PRX-A, The middle row shows cells cultured on the immobile Random surface, and the lower row shows cells cultured on SNL feeder cells. It is drawing which shows expression of Oct-4 and actin of an iPS cell (the undifferentiated 8th day). From left to right, protein expression of cells cultured on glass, PRX-A, PRX-B, Random surface, gelatin surface, and SNL feeder cells was examined by Western blot. The expression and the lower part show the expression of actin, respectively. It is a figure which shows the phase-contrast image and GFP-Nanog fluorescence expression (Bar = 100micrometer) of an adhesion | attachment iPS cell (myocardial differentiation 8th day). The upper left two photographs show the phase contrast image (left) and GFP-Nanog fluorescence expression (right) of cells cultured on glass. Similarly, the upper right two cells are cells cultured on gelatin, the middle is PRX-A, the lower left is PRX-B, the lower right is a phase contrast image (left) and GFP-Nanog of the cells cultured on the Random surface. Fluorescence expression (right) is shown. It is drawing which shows the number of beating colonies in a myocardial differentiation iPS cell. From the left, the number of beating colonies of cells cultured on glass, PRX-A, PRX-B, Random surface, and gelatin surface after 8 days, 12 days, or 17 days after myocardial differentiation, respectively. It is drawing which shows the expression level (RT-PCR) of the differentiation marker gene in a cardiac myocyte. The upper left graph shows the α-MHC gene, the upper right graph shows the HCN gene, and the lower left graph shows the expression of the TnT2 gene (8 days, 12 days and 17 days after myocardial differentiation). The lower right is an electrophoresis band. It is drawing which shows the expression of Rac1, RhoA, and ROCK of iPS cells (myocardial differentiation day 17). From the left, protein expression of cells cultured on glass, PRX-A, PRX-B, Random surface, and gelatin surface was examined by Western blotting. The top row shows active Rac1 (Rac1-GTP). ), The second row from the top shows the expression of active ROCK (ROCK-GTP), the third row shows the expression of active RhoA (RhoA-GTP), and the bottom row shows the expression of all RhoA. It is drawing which shows the expression level (ELISA) of E-cadherin and N-cadherin of an iPS cell (Undifferentiation day 8, differentiation day 8, differentiation day 17). The upper graph shows cadherin expression on day 8 of undifferentiation. The lower left graph shows the expression level of E-cadherin on differentiation day 8 and differentiation day 17, and the lower right graph shows the expression level of N-cadherin on differentiation day 8 and differentiation day 17, respectively. It is a figure which shows N-cadherin expression of the iPS cell (the myocardial differentiation 17th day) on PRX-A (left) and gelatin (right).

  The present invention is described in detail below. The present inventors have found that when pluripotent stem cells are cultured on the surface of a PRX block copolymer having high surface molecular mobility, the stem cells can be maintained in an undifferentiated state. The behavior of mouse fibroblasts or rat mesenchymal stem cells cultured on an incubator whose surface has been processed with polyrotaxane has been previously investigated (see Non-Patent Documents 5 to 7 above). In contrast to the literature showing accelerated cell changes. In addition, the present inventors have unexpectedly found that the differentiation of pluripotent stem cells into cardiomyocytes can be promoted by culturing on the surface of the PRX block copolymer.

Polyrotaxane (PRX)
A rotaxane is one in which a linear molecule penetrates a macrocyclic molecule and bonds bulky sites to both ends of the linear molecule, so that the ring cannot be removed from the axis due to steric hindrance. In polyrotaxane, one linear molecule penetrates the ring of a plurality of macrocyclic molecules. In one embodiment, the present invention provides a method of culturing stem cells using a culture vessel whose surface is coated with a polyrotaxane (PRX) block copolymer. The linear molecule and cyclic molecule used in the present invention are not particularly limited, but the linear molecule is selected from the group consisting of polyethylene glycol, polypropylene glycol, a copolymer of polyethylene glycol and polypropylene glycol, and polymethyl vinyl ether. It is preferable that it is 1 type, or 2 or more types. The average molecular weight of the linear molecules is preferably 200 to 100,000, particularly 10,000 to 40,000 or 15,000 to 25,000. For example, polyethylene glycol having a molecular weight of 20,000 is used. The cyclic molecule is preferably α, β or γ-cyclodextrin, but may have a similar cyclic structure, such as cyclic polyether, cyclic polyester, cyclic Examples include polyether amines and cyclic polyamines. As a combination of a linear molecule and a cyclic molecule, a combination of α-cyclodextrin and polyethylene glycol is preferable. In addition, the synthesis example of the polyrotaxane by the combination of (alpha) -cyclodextrin and polyethyleneglycol is also disclosed by said patent document 1-3, The content is taken in in this specification by reference. The ratio of the number of molecules of cyclic molecules to the number of molecules of linear molecules is 50: 1 to 4: 1, more preferably 20: 1 to 10: 1, for example, a ratio of 12: 1 is used. . That is, 4 to 50 cyclic molecules are preferably included in one molecule of the linear molecule, more preferably 10 to 20 cyclic molecules are included, for example, 12 cyclic molecules are included. When penetrating α-cyclodextrin into polyethylene glycol, the stoichiometric number of the ratio of repeating units (ethylene oxide units) of α-cyclodextrin and polyethylene glycol is said to be 1: 2.

  The bulky substituent used in the present invention may be any group as long as the cyclic molecule can be prevented from falling off from the linear molecule, for example, a group having one or more benzene rings, and one or more third groups. A group having butyl, MPC (2-methacryloyloxyethyl phosphorylcholine), BMA (n-butyl methacrylate), or a combination of MPC and BMA is preferred. Examples of the group having one or more benzene rings include a benzyloxycarbonyl (Z) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, a benzyl ester (OBz) group, and the like. Examples of the group having tributyl include a tertiary butylcarbonyl (Boc) group and an amino acid tertiary butyl ester (OBu group). Among the above, a combination of MPC (2-methacryloyloxyethyl phosphorylcholine) and BMA (n-butyl methacrylate) is particularly preferable.

  In the present invention, when a combination of MPC (2-methacryloyloxyethyl phosphorylcholine) and BMA (n-butyl methacrylate) is used as a block molecule of the polyrotaxane block copolymer, the BMA molar fraction is 0.2 to 0.00. 5 may be sufficient, Preferably it is 0.3-0.4. Here, the molar fraction refers to the ratio of the polymerization degree of each monomer when the total polymerization degree of MPC and BMA (PBM) is 1.

  As the polyrotaxane of the present invention, the linear molecule is polyethylene glycol, the cyclic molecule is α-cyclodextrin, and the portion having the bulky substituent is MPC (2-methacryloyloxyethyl phosphorylcholine) and BMA (n-butyl methacrylate). It is particularly preferable to use a combination. In this case, the molecular weight of PEG is about 20000, and the number of cyclodextrins contained in one PEG molecule can be about 12, for example, 9-15. FIG. 1 schematically shows an outline of synthesis of a polyrotaxane block copolymer by RAFT polymerization.

Preparation of the PRX block copolymer PRX part requires the steric complementarity of the cyclodextrin (CD), which is the cyclic molecule used, and the cross section of the linear polymer chain. PEG is used for CD, and polypropylene glycol (PPG) or a copolymer thereof is used for the next largest β-CD (the internal diameter of cyclodextrin pores is 0.45-0.6 nm for α-form and 0 for β-form. .6 to 0.8 nm, about 0.8 to 0.95 nm for γ-form). Stoichiometrically, α-CD can include 2 units of EG, which is a repeating unit of PEG, so the number of cyclic molecules has an upper limit depending on the molecular weight of the linear polymer used. . For example, when synthesized by the method of Example 1, the penetration rate of CD is approximately several to 60%. The high molecular mobility PRX block copolymer preferably comprises 4-50 α-CD molecules, more preferably 8-30 α-CD molecules, even more preferably, per PEG molecule having a molecular weight of 20000. Contains 10-20 α-CD molecules.

  The chain introduced to form a block copolymer with PRX can be any monomer that can undergo a polymerization reaction, but for example, it can be immobilized on an existing material and the influence on biological components is minimized. For this purpose, a random copolymer of MPC (2-methacryloyloxyethyl phosphorylcholine) and BMA (n-butyl methacrylate) can be introduced. The composition of MPC and BMA aimed at biocompatibility has been studied previously and it has been shown that a BMA molar fraction of 0.3 shows the best biocompatibility. From the viewpoint of minimizing the influence on biological components while aiming at immobilization, the BMA molar fraction of 0.4 can also be used as a guide.

  The CD according to the present invention is preferably methoxy-modified. The methoxy modification (OMe) of PRX is originally intended to impart water solubility, and CD non-modified PRX is poor in water solubility, so CD motility in a living environment cannot be expected. The degree of OMe conversion ranges from a few to over 90%. When a methoxy group is introduced into cyclodextrin, the introduction rate (number of introduced methoxy groups / number of cyclodextrins) is preferably 50% or more, and may be 60% or more, 70% or more, or 80% or more. 90% or more is more preferable. As a range, the introduction rate is preferably 50 to 100%, and may be 60 to 100%, 70 to 100%, or 80 to 100%, but 90 to 100% is more preferable.

  In addition to methoxy, cyclic molecules such as cyclodextrin in PRX may contain a modifying group such as methyl, hydroxypropyl, or monoacetyl, and are modified with a peptide such as a peptide containing an RGD motif. Also good. Furthermore, proteins such as collagen may be bound to the cyclodextrin.

  For the CD motility that has the most influence on the cell response, the amount of energy dissipation in water of the PRX copolymer obtained by QCM-D (Quartz Crystal Microbalance-Dissipation) measurement was standardized by the frequency change corresponding to the coating mass. The numerical value (Mf) can be used as a measure of hydration molecular mobility. The ratio of the change in energy dissipation (ΔD) when the air-water environment changes to the change in resonance frequency (Δf) due to mass increase when the polymer is cast is the molecular mobility factor in water (ΔD / Δ f = Mf) (Inoue Y, Ye L, Ishihara K, Yui N: Preparation and surface properties of polyrotaxane-containing tri-block copolymers as a design for dynamic biomaterials surfaces. Colloid Surf. B: Biointerface 2012, 89: 223-227). Mf shows a value of about 0.1 to 0.2 as the polymer becomes hydrophobic from the hydrophobicity to the general purpose polymer. However, in the PRX block copolymer, 0.1% is maintained while maintaining the hydrophilicity by adjusting the CD number. It can be changed over a wide range up to about 1.0. The Mf of the PRX block copolymer having a high molecular mobility is preferably 0.1 or more, more preferably 0.2 or more, and still more preferably 0.3 or more. As a range, Mf is preferably 0.1 to 1.0, more preferably 0.2 to 1.0, and still more preferably 0.3 to 1.0.

Stem cells As pluripotent stem cells that can be used in the present invention, ES cells (embryonic stem cells) or iPS cells (artificial pluripotent stem cells), particularly skin fibroblast-derived iPS cells, fetal lung cell-derived iPS cells, foreskin Examples include fibroblast-derived iPS cells or cardiac fibroblast-derived iPS cells. Also, GS cells (pluripotent germ stem cells), fusion cells of ES cells and somatic cells can be used. The pluripotent stem cells that can be preferably used in the present invention are primate or rodent cells, and more specifically human, mouse, or rat cells. Alternatively, porcine, rabbit, monkey pluripotent stem cells can also be used.

  Human skin fibroblast-derived iPS cells, human fetal lung cell-derived iPS cells, human foreskin fibroblast-derived iPS cells, and human cardiac fibroblast-derived iPS cells, which are iPS cells, are, for example, RIKEN BioResource Center and ATCC, JCRB Available from cell banks. As human iPSC strains, 253G1, IMR90-1, IMR90-4 and the like can be used. Human embryonic stem cells that are ES cells are available from, for example, the RIKEN BioResource Center. Examples of human ESC strains that can be used include KhES-1, KhES-3, H1, and H9.

The culture medium for culture media undifferentiated maintained, for example, Primate ES Cell Medium + 4 ng / ml human basic FGF (ReproCELL Co.) and human ES medium (ReproCell Co.), Pluripotent Stem Cell SFM XF / FF (ATCC Corp.), DMEM / HamF12 (2 mM L-Glutamine) + 20% KSR + 0.1 mM NEAA + 0.1 mM 2-Mercaptoethanol + 5 ng / ml human bFGF can be used. Examples also include mTeSR1 (StemCell Technologies), TeSR2 (StemCell Technologies), StemPro hESC SFM (Invitrogen), hESF-GRO (Cell Science Laboratory). In the examples of the present application, Gibco's DMEM, EQUITECH-BIO's bovine serum 15%, Gibco's 2-mercaptoethanol 0.1 mM, Gibco's NEAA 0.1 mM, Millipore's LIF are used as undifferentiated media. A 0.1% mixed medium is used. In addition, the media described in the following literature can also be used:
(1) Liu, Y. et al., Biochem. Biophys. Res. Commun., 346: 131-139, 2006.
(2) Vallier, L. et al., J. Cell Sci. 118: 4495-4509, 2005.
(3) Li, Y. et al., Biotechnol. Bioeng., 91: 688-698, 2005.
(4) Yao, S. et al., Proc. Natl. Acad. Sci. USA, 103: 6907-6912, 2006.
(5) Lu, J. et al., Proc. Natl. Acad. Sci. USA, 103: 5688-5693, 2006.

  As a medium for myocardial differentiation, for example, myocardial differentiation medium based on IMDM medium (for example, 20% FBS (Gibco), 1% MEM non-essential amino acid solution (Sigma), 1% penicillin-streptomycin (GIBCO), 2 mM Contains L-glutamine (Sigma), 0.001% 2-mercaptoethanol (GIBCO), and 0.005N NaOH, and 10 ng / ml BMP4 (R & D Systems), or 1% MEM non-essential amino acid solution (Sigma), 1 % Penicillin-streptomycin (GIBCO), 2 mM L-glutamine (Sigma), 0.5 mM L-carnitine (Sigma), 0.001% 2-mercaptoethanol (GIBCO), and 1-2% bovine serum albumin (Wako), Or 0.4% human serum albumin (Sigma), and 5 μM CHIR99021 (Axon) and μM BIO (containing Calbiochem), DMEM-based myocardial differentiation medium (for example, DMEM / F12 medium (Sigma) 200 ml, fetal calf serum (GIBCO) 50 ml, MEM non-essential amino acid solution (Sigma) 2.5 ml), Examples of the medium include penicillin-streptomycin (GIBCO) 2.5 ml, 200 mM L-glutamine 2.5 ml, 2-mercaptoethanol), or StemPro-34SFM (GIBCO) + BMP4 (10 ng / ml). In the examples of the present application, a mixed medium of αMEM manufactured by GIBCO and 0.05 mM 2-mercaptoethanol and 10% bovine serum is used as the differentiation induction medium.

In another aspect, the present invention provides an incubator for undifferentiated maintenance culture of pluripotent stem cells, and in another aspect, an incubator for promoting cardiomyocyte differentiation of pluripotent stem cells. To do. As a material of the incubator, plastic materials such as polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, and glass materials can be used, but are not limited thereto. In recent years, acrylic block copolymers (BCF) and the like have also been developed. Also in the shape, there is no limited shape as long as the container shape has a large bottom area without causing contamination or cross-contamination. So far, a thin columnar culture dish or a cube-like culture flask has been mainly used. In addition, cell culture inserts processed into a membrane shape are also used, and the shape is not limited. As the incubator, for example, a glass bottom dish (manufactured by IWAKI) having a diameter of 27φ can be used.

Coating Method The polyrotaxane can be coated on the incubator by, for example, casting a polymer solution (for example, 0.05 wt%, distilled water: ethanol = 1: 1 mixed solvent) and drying it overnight. In addition, dip coating in which a plate-like surface is immersed in a polymer solution, free standing in a clean bench and dried, or coating by spin coating is also possible. The culture vessel coated with polyrotaxane can be used as a culture vessel for undifferentiated maintenance culture of pluripotent stem cells and a culture vessel for promoting cardiomyocyte differentiation of pluripotent stem cells.

The kit according to the present invention is a kit for cell culture, comprising a culture vessel having a surface coated with a polyrotaxane block copolymer, and a cell culture medium for maintaining the undifferentiated nature of pluripotent stem cells or A cell culture medium for promoting differentiation of pluripotent stem cells into cardiomyocytes is included. As a cell culture medium for maintaining the undifferentiation of pluripotent stem cells contained in the kit according to the present invention or a cell culture medium for promoting differentiation of pluripotent stem cells into cardiomyocytes, Although what is described in a specification is mentioned, it is not limited to them. The culture medium may be in any form such as liquid, solid, gel, powder, etc., and may be thawed or dissolved in water. The culture medium may contain all the components in one container, or may be divided into a plurality of containers for each component, and there is no limitation on the type, number, etc. of the culture medium included in the kit. Further, the medium may be used after adding any supplement. Further, the kit according to the present invention may include an instruction manual and other accessories in addition to the incubator and the culture medium.

PRX surface-coated incubator containing culture medium Cell culture medium for maintaining undifferentiated pluripotent stem cells or cells for promoting differentiation of pluripotent stem cells into cardiomyocytes according to one embodiment of the present invention An incubator having a surface coated with a polyrotaxane block copolymer comprising a culture medium is provided. This incubator can also be produced by adding the medium contained in the kit according to the present invention to the incubator included in the kit according to the present invention. Desired cells can be seeded in this incubator and used for culture. That is, the cell culture medium in this incubator may contain cells to be cultured.

Cell culture conditions are not particularly limited as long as the cells can be maintained or amplified, but the culture temperature is, for example, in the range of 30 to 40 ° C, preferably in the range of 36 to 38 ° C. Preferably 37 degreeC can be illustrated suitably. In addition, from the viewpoint of more suitably maintaining or amplifying the cells, it is preferable to perform medium exchange or passage at an appropriate time. The frequency of medium exchange is not particularly limited as long as the cells can be maintained or amplified, but may be, for example, every 1 to 5 days, preferably every 3 days. The frequency of passage is not particularly limited as long as the cells can be maintained or amplified, and can be appropriately performed at the timing when the population of cells becomes large. For example, every 4 to 12 days, preferably 6 to 10 It can be every day.

  Pluripotent stem cells cultured on the incubator according to the present invention may be induced to differentiate into various tissues and cells. For example, pluripotent stem cells can be sent to cells of the central and sensory systems, cornea, retina, neural stem cells, cardiomyocytes, insulin-producing cells, hepatocytes, bone, cartilage, hematopoietic stem cells, muscle, blood vessels, or kidney cells And can be differentiated. In particular, differentiation from pluripotent stem cells to cardiomyocytes can be efficiently carried out by culturing on a culture vessel coated with polyrotaxane according to the present invention.

  Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.

Example 1: Synthesis of PRX block copolymer Preparation of PEG macro CTA (4- (benzodithioyl) -4-cyanopentanoic acid) Dissolve PEG (molecular weight 20000, 1 g, 0.05 mmol) and dimethylaminopyridine (0.0018 g, 0.15 mmol) in dichloromethane (5 mL). CTA (0.14 g, 0.5 mmol) and water-soluble carbodiimide (0.082 g, 0.5 mmol) were added and stirred for 12 hours. Further dichloromethane was added and recovered by precipitation in cold diethyl ether. The obtained crude product was dissolved in water, dialyzed and lyophilized.

B. Preparation of CD inclusion complex (pseudo-PRX) The obtained PEG macro CTA (0.35 g) was added to an α-CD (3.5 g) aqueous solution (25 ml) at room temperature, and the resulting precipitate was collected by centrifugation. Water (25 mL) was added, and the mixture was centrifuged again and lyophilized.

C. Synthesis of PRX block copolymer CD inclusion complex (0.6 g) in MPC (0.354 g, 1.2 mmol) and BMA (0.633 g, 4.45 mmol) and ethanol / toluene (1/1) mixed solvent Then, AIBN (0.82 mg, 5 μmol) was reacted as an initiator in an argon atmosphere for 15 minutes, and further reacted at 60 ° C. for 24 hours. 15 mL of solvent was added, and the resulting precipitate was collected by centrifugation. The crude product was subsequently washed with ethanol, acetone, dimethyl sulfoxide to remove unreacted monomer and CD. The resulting precipitate was further vacuum dried at 40 ° C. to obtain the final product.

D. Synthesis of OMe-PRX block copolymer (OMe conversion of PRX block copolymer)
The synthesized PRX block copolymer (200 mg) was dissolved in dehydrated dimethyl sulfoxide (7 mL), sodium hydride (0.155 g, 6.3 mmol) was added, and the mixture was reacted at room temperature for 30 minutes in an argon atmosphere. Next, iodomethane (0.102 g, 0.719 mmol) was gradually added and reacted at room temperature for 3 hours. After neutralizing the pH, the reaction solution was dialyzed for 3 days and freeze-dried to obtain the desired product.

E. Synthesis of Random Copolymer (Control) A random copolymer having OMe and OH groups similar to the PRX block copolymer is BMA (0.633 g, 4.45 mmol), MPC (0.354 g, 1.2 mmol). , HEMA (0.637 g, 4.9 mmol), MEA (1.16 g, 8.51 mmol) in toluene / ethanol mixed solvent (1/1) (8 mL) with AIBN (0.4 mg) as an initiator in an argon atmosphere The reaction was carried out at 60 ° C. for 24 hours under synthesis. The reaction solution was precipitated in cold diethyl ether, dialyzed in water for 2 days, and lyophilized to obtain the desired product.

Example 2: Evaluation of undifferentiated characteristics of artificial multifunctional stem cells (iPS cells) using PRX treatment equipment In a glass bottom dish (manufactured by IWAKI) having a diameter of 27φ, 150 μL of a polymer solution (0.05 wt%, distilled water) : Ethanol = 1: 1 mixed solvent), and dried overnight on a clean bench. The polymer used was PRX-A (a PRX block copolymer having the highest molecular mobility, 12 α-CD penetrations per PEG molecule having a molecular weight of 20000, and 90% introduction rate of methoxy group into α-CD). , PRX-B (PRX block copolymer with low molecular mobility, α-CD penetration number 104, methoxy group introduction rate 61% to α-CD), and Random (universal methacrylate with non-PRX structure) Polymers having a methoxy group introduction rate of 11 mol% and having the same methoxy group as PRX-2). For comparison, an unmodified glass bottom dish and SNL (manufactured by European Collection of Cell Cultures), which is a feeder cell for maintaining undifferentiation, were added to the sample group for experiments. Mouse-derived iPS cells (iPS-MEF-Ng-178B-5, Kyoto University iPS Cell Research Institute CiRA establishment) at a concentration of 3.0 × 10 ⁴ cells / mL, undifferentiated medium (Dib manufactured by Gibco, cattle manufactured by EQUITECH-BIO) The mixture was dispersed in serum 15%, Gibco 2-mercaptoethanol 0.1 mM, Gibco NEAA 0.1 mM, Millipore LIF 0.1% mixed medium), and each 1 mL was seeded on a sample dish. The state of adhesion was observed for 8 days while changing the medium every day after seeding. The expression analysis of undifferentiated marker protein (Oct-4) by Western blot was performed according to the following procedure. First, the total protein of iPS cells (8 days) cultured in an undifferentiated medium was extracted using Minute Detergent-Free protein extraction kit manufactured by Invent Biotechnology. Thereafter, the extracted protein concentration was quantified using a Micro-BCA kit manufactured by Pierce. The calibration curve used was prepared from a bovine serum albumin (Pierce Standard) solution with a known concentration. The extracted protein solution was adjusted to a concentration of 900 μg / mL, 10 μL of the protein solution was mixed with 10 μL of Lamiel Buffer (manufactured by BioRad), and each was electrically charged at 200 V in a 10-20% acrylamide gradient gel (manufactured by BioRad) for 30 minutes. Electrophoresed. Thereafter, the protein on the electrophoresis gel was transferred to a PVDF membrane using a Turbo Blotting System manufactured by Biorad. Thereafter, blocking was performed with skim milk (5%) manufactured by Wako for 1 hour, and a 1% skim milk solution diluted with anti-Oct4 antibody (derived from rabbit, Cell Signaling Technology) and anti-actin antibody (derived from rabbit, manufactured by Sigma-Aldrich) was diluted. For 1 hour and a half at room temperature. Thereafter, the plate was washed three times with PBS, and reacted with a 1% skim milk solution diluted with Cell Signaling Technology HRP-labeled anti-rabbit IgG for 1 hour at room temperature. After washing with PBS three times and processing with GE Healthcare Blotting Substrate, the images were taken with an ImageQuant LAS500 system manufactured by GE Healthcare.

  FIG. 2 shows a state 8 days after seeding of iPS cells. It was confirmed that iPS cells seeded on all surfaces adhered stably and proliferated almost confluently. Even in the quantitative results, the same level of cell adhesion was found on most surfaces (excluding the number of adhesions on feeder cells that grow three-dimensionally), and the constructed PRX surface was also sufficiently effective for iPS cell adhesion and proliferation. It was shown that In addition, when the fluorescence expression of GFP-Nanog was observed, the strongest Nanog expression was seen in the iPS cell cultured on the SNL surface which is a feeder cell. Weak Nanog expression was observed on the untreated glass surface, the non-movable Random surface, and the gelatin surface, and it was confirmed that the undifferentiated ability was greatly reduced. However, a slightly strong Nanog expression was observed on the PRX-A surface having high molecular mobility, and it was confirmed that the morphology of the embryoid-like cell mass was more maintained. This is thought to be because iPS cells adhered to a highly molecularly motile surface have a weak interaction with the material surface and a strong cell-cell interaction. It is effective in maintaining undifferentiated characteristics.

  Furthermore, in order to confirm the undifferentiation maintenance property of iPS cells adhered to each surface at the protein level, the expression of Oct-4, which is a kind of undifferentiated marker protein, was examined by Western blot. FIG. 3 shows the expression results of Oct-4 and actin in undifferentiated day 8 iPS cells. On the SNL surface, since the interaction with the substrate is weak, the expression level of actin is detected to be relatively low with respect to the same total protein amount, while the expression of Oct-4 is detected to be high with respect to the expression of actin. It was confirmed that the environment suitable for maintaining the undifferentiated characteristics was given while weakening the interaction. On the other hand, on the PRX-A surface having high molecular mobility, Oct-4 expression was detected high while the expression level of actin relative to the total amount of protein was significantly reduced. In addition, the degree was remarkable even when compared with a surface having strong interaction with a substrate such as gelatin. This indicates that the undifferentiated nature of iPS cells is maintained by the high molecular mobility of the PRX surface coated on the culture equipment.

Example 3: Differentiation of iPS cells into cardiomyocytes using PRX treatment equipment and gene analysis by RT-PCR The above three types of polymer surfaces, unmodified glass surfaces, and gelatin-modified dishes (in 0.1% gelatin solution) In this way, 1 mL each of 2.0 × 10 ⁴ cells / mL of iPS cell solution was seeded on Life Technology Co., Ltd.), and differentiation characteristics with different culture devices were examined while differentiating into cardiomyocytes. The differentiation induction medium was a mixed medium of Gibco αMEM and 0.05 mM 2-mercaptoethanol, 10% bovine serum, and the medium was changed every two days after seeding. On day 7 of sowing, the medium was replaced with a differentiation-inducing medium containing 10 ng / mL trichostatin A (manufactured by Wako), and after one day of incubation, the state of differentiation was replaced again with a differentiation-inducing medium containing no trichostatin A. Observed.

  FIG. 4 shows the adhesion form 8 days after differentiation of iPS cells into cardiomyocytes (one day after treatment with trichostatin A). IPS cells stably adhered and proliferated in the differentiation medium on all surfaces. In addition, almost no expression of GFP-Nanog was observed on all surfaces, and it was confirmed that the pluripotency of adherent iPS cells was lost due to the influence of the differentiation medium. Compared to the gelatin (positive control) surface, which is generally known to be advantageous for differentiation into cardiomyocytes, the PRX-A surface with the highest molecular mobility has a pulsation on the 8th day of differentiation. Colonies began to be seen. Even in the state of adhesion, it is characteristically that dense cell colonies are spreading on the surface of gelatin and PRX-A where pulsation is observed, differentiation of iPS cells into cardiomyocytes and pulsatile tissue It was shown that the PRX block copolymer surface having high surface molecular mobility is excellent in the formation of.

  FIG. 5 shows the number of beating colonies in iPS cells on the 8th, 12th and 17th days after myocardial differentiation. On the 8th day, which was the early stage of differentiation, the largest number of beating colonies were observed on the gelatin surface, but the number decreased as the differentiation time increased. On the other hand, on the PRX-A surface, the number of colonies at the initial stage of differentiation was about 20% of that of gelatin. However, the number of colonies increased with time, and the largest number of colonies was shown on the 17th day of differentiation. This result shows that the PRX-A surface with high surface molecular mobility provides the most advantageous environment for increasing the number of beating colonies.

Example 4: Analysis of expression level of cardiomyocyte differentiation marker mRNA by RT-PCR In order to examine the expression level of a differentiation marker gene in each iPS cell differentiated in a differentiation-inducing medium, quantification of mRNA expression level by RT-PCR Went. The total RNA in the differentiated cells was extracted using the PureLink RNA Mini kit manufactured by Ambion, and then the RNA concentration was quantified using Nanodrop 2000 manufactured by Thermo. The total RNA concentration was adjusted to 100 ng / μL, and cDNA was synthesized using a cDNA reverse transcription kit manufactured by Applied Biosystems. 50 ng of cDNA was mixed with 25 μL of Toyobo SYBR-RT-PCR Master mix, mixed with 2 μL of primer solution (25 μM), and then PCR analysis was performed with an Applied Biosystems Step-One Puls RT-PCR machine. It was. PCR primers and PCR reaction conditions are as follows.

Primer (Sigma-Aldrich):
αMHC:
F-ACGGTGACCATAAAGAGGA
R-TGTCCTCGATCTTGTCGAAC
HCN4:
F-CGACAGCGCATCCATGAACTA
R-GCTGGAAGACCTCGAAACGC
TnT2:
F-GAAACAGGATCAACGACAACCA
R-CGCCCCGGTGACTTTGG
GAPDH:
F-TGCGACTTCAACAGCAACTC
R-CTTGCTCAGTGTCCTTCTGTG
Reaction conditions:
After 2 minutes at 95 ° C, 40 cycles of 95 ° C for 20 seconds, 55 ° C for 30 seconds, 68 ° C for 30 seconds

  FIG. 6 shows the results of RT-PCR analysis of mRNA expression levels in iPS cells differentiated in myocardium on each surface. In any of the results, the expression of the differentiation marker gene on the PRX-A surface where the number of beating colonies was the largest was seen highest on the 17th day of differentiation, and the high differentiation ability to cardiomyocytes was also confirmed at the gene level. It was.

Example 5: Expression quantification of intracellular skeletal system signal by Western blot To analyze the state of adhesion and differentiated iPS cells at the protein level, iPS cells at 17 days of differentiation were first analyzed by the method described in Example 2. Total protein extraction and concentration quantification were performed. In order to examine the protein expression of active RhoA and ROCK, 100 μg of total protein was reacted with the AbA RhoA Activation Assay kit and ROCK Assay kit, respectively. Mixed and electrophoresed in a 12% acrylamide gel at 150V for 30 minutes. Thereafter, the protein on the electrophoresis gel was transferred to a PVDF membrane using a Turbo Blotting System manufactured by Biorad. Thereafter, blocking was performed with skim milk (5%) manufactured by Wako for 1 hour, and the mixture was reacted at room temperature for 1 hour and a half with a 1% skim milk solution in which the anti-active RhoA antibody and the anti-active ROCK antibody in the kit were diluted. Thereafter, the plate was washed three times with PBS, and reacted with a 1% skim milk solution diluted with Cell Signaling Technology HRP-labeled anti-rabbit IgG for 1 hour at room temperature. After washing with PBS three times and processing with GE Healthcare Blotting Substrate, the images were taken with an ImageQuant LAS500 system manufactured by GE Healthcare.

  FIG. 7 shows protein expression of active Rac1, active RhoA (RhoA-GTP) and ROCK (ROCK-GTP) on day 17 of differentiation by Western blot. Highly active RhoA expression was confirmed on the gelatin surface, which is expected to have a high interaction with cells, and it was confirmed that the skeletal system signal transduction pathway composed of RhoA is dominant on the gelatin surface.

Example 6: Quantification of cadherin expression by ELISA The amount of cadherin expression in 10 μg of total protein was quantified with an N-Cadherin ELISA kit manufactured by Boster Biological Technology and an E-Cadherin ELISA kit manufactured by RayBio, respectively. The calibration curve used was prepared from mouse-derived N-cadherin and E-cadherin standard with known concentrations in the kit, and the protein expression level was quantified from the absorption intensity at 450 nm. It is considered that an increase in the expression level of cadherin is necessary for promoting differentiation into cardiomyocytes, which is essential for maintaining undifferentiated iPS cells and forming cell-cell interactions. In particular, multiple expression of N-cadherin is required for efficient differentiation into cardiomyocytes. As shown in FIG. 8, the expression of N-cadherin is most frequently observed from the undifferentiated stage on the surface of PRX-A having high molecular mobility, and it is found that an environment favorable for differentiation into cardiomyocytes is constructed. It was. Furthermore, on day 17 of differentiation into cardiomyocytes, high N-cadherin expression was observed together with gelatin, and the results consistent with the results of forming the most pulsatile colonies shown in FIG. 5 were obtained. On the other hand, gelatin showed high N-cadherin expression like PRX-A, but also high E-cadherin expression, and a tendency different from typical cadherin expression of differentiated myocardial cells was observed. In PRX-A, the expression level of E-cadherin, which was highly detected in an undifferentiated state, was significantly reduced during differentiation, and at the same time, only N-cadherin maintained a high expression level.

Example 7: Distribution state of N-cadherin by immunostaining In order to examine the distribution state of N-cadherin expression detected highly after myocardial differentiation, N-cadherin expression in cardiomyocyte differentiated cells was observed by immunostaining. The myocardial differentiated cells were washed with PBS, and then fixed with a 4% formaldehyde (Merck) solution for 15 minutes at room temperature. After washing 3 times with PBS, it was contacted for 1 hour with a blocking solution containing 5% goat serum and 0.3% Triton-X100. Thereafter, a rabbit anti-N-cadherin antibody (manufactured by Abcam) was brought into contact with the diluted solution and reacted overnight at 4 ° C. Thereafter, the plate was washed 3 times with PBS, and reacted with Alexa488-labeled anti-rabbit IgG (Abcam) solution at room temperature for 1 hour. The plate was washed three times with PBS, sealed with a DAPI-containing ProLong gold antifade solution (manufactured by Life Technology), and observed with a confocal laser microscope.

  FIG. 9 shows a fluorescent immunostaining image of N-cadherin of iPS cells on day 17 of myocardial differentiation. It can be seen that even with a similar expression level of N-cadherin, the PRX-A surface has a small and dense structure of nuclei, and further, a cluster of cells is formed by N-cadherin. On the other hand, on the gelatin surface, it was confirmed that the cell aggregate formation by N-cadherin was not as dense as PRX-A. This result suggests that the adhesion of stem cells is controlled on the surface simply coated with the PRX block copolymer with high molecular mobility and affects the regulation of cell-cell interaction. ing. That is, a PRX block copolymer having high molecular mobility is useful for producing an undifferentiated maintenance surface of stem cells and a differentiation-inducing surface into cardiomyocytes, in which control of cell-cell interaction is essential.

  From the above results, it has been clarified that a simple coating of the PRX block copolymer realizes a surface capable of controlling cell-cell interaction and adjusting characteristics including stem cell differentiation ability. From the analysis of undifferentiated marker protein of adherent iPS cells, it was found that the PRX block copolymer surface having high surface molecular mobility can maintain stem cells in an undifferentiated state. This is probably because the intracellular signal transduction system is regulated by a decrease in RhoA-ROCK activity. This can be said to be extremely important for industrial use of regenerative medicine in which stem cells need to be handled in a large amount in an undifferentiated state.

In addition, the present inventors have found that the surface of a PRX block copolymer having high surface molecular mobility is excellent in differentiation from stem cells such as iPS cells into cardiomyocytes. Furthermore, it has also been found that N-cadherin expression that mediates cell-cell interaction can be induced in constructing mesenchymal differentiated tissue from cardiomyocytes. This suggests that it is excellent not only for induction of cell differentiation but also for tissue regeneration by differentiated cells. Compared with the strong E-cadherin expression and weak N-cadherin expression seen on the collagen surface, the PRX block copolymer surface with high surface molecular mobility shows higher N-cadherin expression than E-cadherin expression. It has been suggested that it excels in epithelial-mesenchymal transition.

Claims (20)

A cell culture method for maintaining the undifferentiated nature of pluripotent stem cells,
A method comprising culturing the pluripotent stem cell on an incubator having a surface coated with a polyrotaxane block copolymer.   The method according to claim 1, wherein the polyrotaxane block copolymer contains cyclodextrin as a cyclic molecule.   The method according to claim 2, wherein the cyclodextrin is modified with a methoxy group.   The method according to claim 1, wherein the polyrotaxane block copolymer contains polyethylene glycol (PEG) or polypropylene glycol (PPG) as a linear molecule.   The method according to claim 4, wherein the polyrotaxane block copolymer contains polyethylene glycol (PEG) as a linear molecule, and the molecular weight of the PEG is 15000 to 25000.   The method according to claim 1, wherein the polyrotaxane block copolymer has a ratio of the number of molecules of cyclic molecules to linear molecules of 20: 1 to 10: 1.   The said polyrotaxane block copolymer contains MPC (2-methacryloyloxyethyl phosphorylcholine) and / or BMA (n-butylmethacrylate) as a block molecule, The any one of Claims 1-6 characterized by the above-mentioned. Method.   The polyrotaxane block copolymer contains MPC (2-methacryloyloxyethyl phosphorylcholine) and BMA (n-butyl methacrylate) as block molecules, and has a BMA molar fraction of 0.3 to 0.4. The method of claim 7.   The method according to claim 1, wherein the polyrotaxane block copolymer has a molecular mobility factor Mf value of 0.3 or more on an incubator.   The method according to any one of claims 1 to 9, wherein the incubator material comprises glass, polystyrene, polypropylene, polyethylene, polyolefin, polycarbonate, or acrylic block copolymer (BCF). .   The pluripotent stem cell is a group consisting of embryonic stem cell (ES cell) or dermal fibroblast-derived iPS cell, fetal lung cell-derived iPS cell, foreskin fibroblast-derived iPS cell, and cardiac fibroblast-derived iPS cell. The method according to claim 1, wherein the method is an artificial pluripotent stem cell (iPS cell) selected more.   The method according to claim 1, wherein the pluripotent stem cell is a human, mouse, or rat cell.   The method according to any one of claims 1 to 12, further comprising a step of adding a cell culture medium for maintaining the undifferentiation of pluripotent stem cells to a culture vessel having a surface coated with a polyrotaxane block copolymer. the method of.   The method according to any one of claims 1 to 13, further comprising the step of seeding pluripotent stem cells in a culture vessel having a surface coated with a polyrotaxane block copolymer. A cell culture kit comprising:
An incubator having a surface coated with a polyrotaxane block copolymer;
A cell culture medium for maintaining the undifferentiated nature of pluripotent stem cells.   16. A kit according to claim 15, wherein the cell culture medium is provided in the form of a liquid or a dry powder.   The kit according to claim 15 or 16, wherein the cell culture medium is divided into a plurality of containers for each component, or all components are contained in one container.   An incubator having a surface coated with a polyrotaxane block copolymer, comprising a cell culture medium for maintaining the undifferentiated nature of pluripotent stem cells. A cell culture method for promoting differentiation of pluripotent stem cells into cardiomyocytes,
A method comprising culturing the pluripotent stem cell on an incubator having a surface coated with a polyrotaxane block copolymer.   20. The method of claim 19, further comprising the step of providing a pluripotent stem cell with a stimulus that promotes differentiation into a cardiomyocyte.