JP2013223446A - Cell culture substrate, method for cell culture using the same, and method for differential induction of multipotent stem cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture substrate that can differentiate multipotent stem cells into cardiomyocytes without needing IGFBP of high concentration, and a method for cell culture using the cell culture substrate, and a method for differential induction of multipotent stem cells.SOLUTION: A cell culture substrate is characterized in that polypeptide comprising the whole or partial region of protein that belongs to an insulin-like growth factor binding protein (IGFBP) family, or fusion protein containing the whole or part of the protein belonging to the insulin-like growth factor binding protein family is fixed or coated onto the surface.

Description

  The present invention relates to a cell culture substrate, a cell culture method using the same, and a method for inducing differentiation of pluripotent stem cells. Specifically, pluripotent stem cells can be cultured. The present invention relates to a cell culture substrate that can be differentiated into cells, a cell culture method using the same, and a method for inducing differentiation of pluripotent stem cells.

  Since cardiomyocytes do not proliferate in adults, damaged heart tissue does not regenerate as it is. Regenerative treatment using ES cells or iPS cells is promising for the establishment of treatments for heart diseases. Since ES cells and iPS cells have totipotency, cardiomyocytes obtained by differentiation of ES cells and iPS cells can be a cell source for transplantation. So far, methods of using bone morphogenetic proteins (BMPs, bone morphology proteins), BMP inhibitors, Wnts, and Wnts inhibitors have been proposed for differentiation induction from ES cells and iPS cells into cardiomyocytes (Non-patent Document 1). ~ 3).

  These factors lead to cardiac development by regulating the Wnt / β-catenin signaling pathway. Wnt / β-catenin signaling is believed to be important for promoting differentiation induction of ES cells into cardiomyocytes. Recently, insulin-like growth factor protein 4 (IGFBP4) inhibits Wnt / β-catenin signaling to induce differentiation of ES cells into cardiomyocytes at a later stage after embryoid body formation (EB, embryoid body) Has been reported to be strongly promoted (Non-Patent Document 4).

  IGFBP4 inhibits the binding of Wnt3a to the Wnt receptor Frizzled 8 (Frz8) and the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) through binding to these receptors. It was revealed. Furthermore, in chicken and frog embryos, IGFBP4 was found to be required for heart development through inhibition of Wntβ-Catenin signaling (Non-Patent Document 4). Therefore, promotion of cardiomyocyte differentiation of ES cells using IGFBP4 is promising.

Yuasa S. et al., Nat. Biotechnol. 2005; 23: 607-11. Kami D. et al., Plos One 2008; 3: e2407. Monzen K. et al., Mol. Cell Biol. 1999; 19: 7096-105. Zhu W. et al., Nature 2008; 454: 345-9. Nadanaka et al., J. Biol. Chem. 2008; 283: 27333-43.

  However, since IGFBP4 is unstable in the culture medium, a high concentration of IGFBP4 is required for cardiomyocyte differentiation. In addition, Wnts is controlled autonomously and binds to heparan sulfate proteoglycans on the cell surface (Non-patent Document 5). Since Wnt molecules bind to the cell surface, high concentrations of IGFBP4 are required to inhibit Wnt binding to the receptor.

  Accordingly, an object of the present invention is to provide a cell culture substrate capable of differentiating pluripotent stem cells into cardiomyocytes without requiring a high concentration of IGFBP, a cell culture method using the same, and pluripotent stem cells. It is to provide a differentiation induction method.

  As a result of intensive studies, the present inventors have found that the above problems can be solved and cardiomyocyte differentiation can be induced by using a base layer on which a protein belonging to the IGFBP family or a fusion protein containing a part of the protein is immobilized. I found it.

  That is, the cell culture substrate of the present invention is a polypeptide comprising all or part of a protein belonging to the insulin-like growth factor binding protein (IGFBP) family, or a protein belonging to the insulin-like growth factor binding protein family. It is characterized in that the fusion protein including all or part thereof is fixed or coated on the surface.

  In the cell culture substrate of the present invention, it is preferred that the protein belonging to the insulin-like growth factor binding protein (IGFBP) family is IGFBP4 or a protein having homology with IGFBP4.

  Further, in the cell culture substrate of the present invention, a polypeptide comprising all or part of the protein belonging to the insulin-like growth factor binding protein (IGFBP) family, or a protein belonging to the insulin-like growth factor binding protein family It is preferable that the fusion protein containing all or a part of the protein comprises an IGFBP4 Thy domain or an amino acid sequence homologous to the IGFBP4 Thy domain.

  In the cell culture substrate of the present invention, it is preferable that the fusion protein containing all or part of the protein belonging to the insulin-like growth factor binding protein family contains all or part of the elastin-like polypeptide.

  In the cell culture substrate of the present invention, the fusion protein containing all or part of the protein belonging to the insulin-like growth factor binding protein family is represented by (GVGVP) n (n is a number of 2 or more). It is preferable to include a repetitive sequence.

  In the cell culture substrate of the present invention, it is preferable that the repetitive sequence is represented by (GVGVP) n (n is a number of 65 or more).

  In the cell culture substrate of the present invention, the fusion protein contains (GVGVP) n (n is a number of 65 or more) on the N-terminal side, and contains all or part of IGFBP4 on the C-terminal side. It is preferable that it is a protein containing. .

  In the cell culture substrate of the present invention, the fusion protein contains (GVGVP) n (n is a number of 65 or more) on the N-terminal side, and the IGFBP4 Thy domain or IGFBP4 on the C-terminal side. A protein containing an amino acid sequence homologous to the Thy domain is preferred.

  The cell culture method of the present invention is characterized in that pluripotent stem cells are proliferated while maintaining undifferentiation and differentiation pluripotency using any one of the above cell culture substrates and a liquid medium. It is.

  The method for producing cardiomyocytes of the present invention is characterized in that pluripotent stem cells are differentiated into cardiomyocytes using any one of the above cell culture substrates and a liquid medium.

  The method for producing cardiomyocytes of the present invention preferably differentiates pluripotent stem cells into cardiomyocytes by inhibiting the Wnt / β-catenin signaling pathway.

  According to the present invention, a cell culture substrate capable of differentiating pluripotent stem cells into cardiomyocytes without requiring high concentrations of IGFBP, a cell culture method using the same, and a method for inducing differentiation of pluripotent stem cells Can be provided.

FIG. 1A is a Western blot of IGFBP4 (Thy), (GVGVP) ₁₂ -IGFBP4, (GVGVP) ₂₃ -IGFBP4, (GVGVP) ₄₅ -IGFBP4, (GVGVP) ₆₇ -IGFBP4 protein. The upper part of FIG. 1A is a photograph of a SDS-PAGE gel obtained by CBB staining, and the lower part is a photograph showing the results of Western blotting using an anti-IGFBP4 antibody. FIG. 1B is a graph showing temperature-dependent aggregation profiles of (GVGVP) ₄₅ -IGFBP4, (GVGVP) ₆₇ -IGFBP4 protein. Results are shown as mean ± standard deviation. The vertical axis is turbidity (%), and the horizontal axis is temperature. FIG. 2A shows IGFBP4 (Thy), (GVGVP) ₁₂ -IGFBP4, (GVGVP) ₂₃ -IGFBP4, (GVGVP) ₄₅ -IGFBP4, (GVGVP) at 37 ° C. for 1 day (left) and 4 ° C. for 1 day (right). It is a graph which shows the result of the adsorption stability test (ELISA assay) with respect to polystyrene dish of _67- IGFBP4. The vertical axis represents OD450 nm, and the horizontal axis represents the concentration of each protein. FIG. 2B shows the adsorption stability of IGFBP4 (Thy), (GVGVP) ₁₂ -IGFBP4, (GVGVP) ₂₃ -IGFBP4, (GVGVP) ₄₅ -IGFBP4, (GVGVP) ₆₇ -IGFBP4 to polystyrene dishes at 37 ° C. for 1 week. It is a graph which shows the result of a test (ELISA assay). The vertical axis represents OD450 nm, and the horizontal axis represents the concentration of each protein. FIG. 2C shows one day at 37 ° C. for polystyrene dishes of 10 μg / mL IGFBP4 (Thy), (GVGVP) ₁₂ -IGFBP4, (GVGVP) ₂₃ -IGFBP4, (GVGVP) ₄₅ -IGFBP4, (GVGVP) ₆₇ -IGFBP4. It is a graph which shows the result of a weekly adsorption stability test (ELISA assay). FIG. 3 confirms whether LRP5 / 6 interacts with IGFBP4 (Thy), (GVGVP) ₁₂ -IGFBP4, (GVGVP) ₂₃ -IGFBP4, (GVGVP) ₄₅ -IGFBP4, (GVGVP) ₆₇ -IGFBP4 It is a western blot photograph figure which shows the result of an immunoprecipitation test. P19CL6 cells were cultured for 3 hours on a dish on which each recombinant protein had been fixed, disrupted, and immunoprecipitated. It is a photograph figure which shows the result of the western blotting using the anti- IGFBP4 antibody in the upper part and the anti-LRP5 / 6 antibody in the lower part. FIG. 4A shows a gelatin coated dish (control), a gelatin coated dish to which 1 μg / mL IGFBP4 (Thy) was added, a gelatin coated dish to which 1 μg / mL (GVGVP) ₆₇ -IGFBP4 was added, and (GVGVP) ₆₇ -IGFBP4 was immobilized. It is the microscope picture figure (phase contrast) of the ES cell cultured on the prepared dish. The upper part is a photograph after 0 days, and the lower part is a culture after 24 days. FIG. 4B is a photograph showing the results of RT-PCR using ES cells after 24 days of culture. FIG. 4C is a confocal photomicrograph showing the results of immunostaining using an anti-αMHC antibody (MF20) (left). The right is a graph showing the ratio of the MF20 positive region to the DAPI positive region in the ES cells after 24 days of culture. FIG. 5A is a photomicrograph of embryoid bodies on the first, third, and fifth days of hanging drop culture. FIG. 5B is a photographic diagram showing the results of RT-PCR of embryoid bodies (EB) in each culture day. FIG. 6A is a photomicrograph (phase) of embryoid bodies (EB) cultured on a gelatin-coated dish (control), (GVGVP) ₆₇ -IGFBP4-immobilized dish on day 1, day 7, and day 20. contrast). FIG. 6B is a differential interference micrograph of ES cells cultured for 20 days on a dish coated with gelatin-coated dish (control), (GVGVP) ₆₇ -IGFBP4, and differentiated from cardiomyocytes. The area surrounded by the line represents the area that has autonomously beaten. FIG. 6C is a photograph showing the results of RT-PCR using embryoid bodies after 20 days of culture. FIG. 6D is a confocal photomicrograph showing the results of immunostaining using an anti-αMHC antibody (MF20) (left). The right is a graph showing the ratio of the MF20 positive region to the DAPI positive region in the embryoid body after 20 days of culture. It is a confocal microscope photograph figure of the MF20 positive area | region of the embryoid body (EB) cultured for 20 days on a 10 microgram / mL (GVGVP) n-IGFBP4 fixed dish. It is a schematic diagram showing the prediction mechanism of inhibition of the Wnt / βcatenin signaling pathway when IGFBP4 is added as a soluble factor (left) and when IGFBP4 is immobilized on the base layer (right).

[IGFBP family proteins]
IGFBP (Insulin-like Growth Factor Binding Protein) family proteins include six types of IGFBPs 1-6 as proteins that specifically bind to insulin-like growth factor (IGF) I and II in higher organisms. It has been reported. It has been suggested that it has functions such as activity control of insulin-like growth factor and inhibition of degradation. Among these, IGFBP4 is preferable.

  IGFBP4 consists of an insulin-like growth factor binding domain and a type 1 thyroglobulin (Thy) domain. The Thy domain at the C terminus of IGFBP4 is the Wnt receptor binding domain (Non-patent Document 4). The amino acid sequence of IGFBP4 is registered under the accession number: AAH16041.1 in GenBank for humans, for example. In addition, the mouse is registered in GenBank with an accession number :: AAH19836.1. The peptide sequence of IGFBP4 used in the present invention is not limited to these, and may be a deletion, insertion or substitution of 1 to 9 amino acids, or may be IGFBP4 in other organisms. . Moreover, it has a homology of 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more at the amino acid level with the above protein molecule, and has insulin-like growth factor binding activity. Proteins can also be used.

  The method for producing a protein belonging to the IGFBP family is not particularly limited, but it is desirable to produce and purify a recombinant protein using a molecular biological technique and use it. In addition, any method that exhibits the same effect can be used. For example, a protein belonging to the IGFBP family of pluripotent stem cells can be extracted from a biological tissue / cell, purified, and used. Alternatively, the peptide can be chemically synthesized and used.

Elastin is the main protein of elastic fibers such as the aorta, the ligamentum flavum, the yellow ligament, the lungs, the skin, the uterus and the elastic cartilage. Elastin is present in a large amount in vivo next to collagen, and its most important function is elasticity. Elastin is insoluble in tissues. The major repeating peptide sequences in elastin include the repeating sequences of pentapeptide Gly-Val-Gly-Val-Pro (hereinafter GVGVP) and hexapeptide Val-Gly-Val-Ala-Pro-Gly (hereinafter VGVAPG). These two types of peptide repeat sequences exist in common in mammals such as humans, cows, pigs, etc., but their properties are completely different, and the GVGVP repeat sequence has an elastic function and exhibits coacervation. Not recognized at all. On the other hand, the VGVAPG repeat sequence does not show elasticity and coacervation, but induces migration and proliferation of various cells. Water-soluble elastin obtained by solubilizing insoluble elastin, the precursor protein tropoelastin (soluble), and elastin-derived polypentapeptide exhibit a unique phenomenon called coacervation. The aqueous solution of water-soluble elastin and tropoelastin is a transparent homogeneous solution at low temperatures, but when heated to near body temperature (37 ° C), it causes phase separation and white turbidity to form coacervate droplets. It is separated into two layers, a lower layer made of and an upper layer made of the equilibrium solution. This process is reversible and returns to the original homogeneous solution when the temperature is cooled below room temperature.
In the present invention, the fusion protein to be immobilized on the base layer preferably contains the main repetitive peptide sequence in the elastin-like polypeptide, and preferably contains the repetitive sequence of Gly-Val-Gly-Val-Pro (hereinafter GVGVP). preferable. The number of GVGVP repeats is preferably 65 or more, and more preferably 70 or more.

In the present invention, a preferred embodiment of the protein immobilized on the surface of the culture substrate includes a chimeric protein (fusion protein) composed of a Wnt receptor binding domain of IGFBP4 and an elastin-like polypeptide. IGFBP4 consists of an insulin-like growth factor binding domain and a type 1 thyroglobulin (Thy) domain. The Thy domain at the C terminus of IGFBP4 is the Wnt receptor binding domain (Non-patent Document 4). Elastin-like polypeptide has a repeating sequence of glycine-valine-glycine-valine-proline (GVGVP), and has the property that it changes its hydrophobicity upon receiving heat (McPherson DT et al., Biotechnol. Prog. 1992; 8: 347-52.McPherson DT et al., Protein Expr.Purif.1996; 7: 51-7. Mie M et al., J. Biomed. Mater.Res. B Appl. 86: 283-90.). Above the phase transition temperature (Tg (t)), these polypeptides can be stably adsorbed to a base layer such as a polystyrene dish through hydrophobic interactions.
At low temperatures, these polypeptides are released from the base layer due to increased hydrophilicity. Tg (t) of elastin-like polypeptide depends on the number of GVGVP repeats (Non-patent Document 13). Thus, the elastin-like polypeptide, (GVGVP) n is useful for immobilizing various factors. In the present invention, a fusion protein having (GVGVP) n-IGFBP4 in which the N terminus of Thy and (GVGVP) n are bound to each other is particularly preferred.

  Examples of the culture substrate for cell culture include dishes (also referred to as culture dishes), petri dishes and plates (microtiter plates such as 6 holes, 24 holes, 48 holes, 96 holes, 384 holes, and 9600 holes, microplates, Deep well plates, etc.), flasks, chamber slides, tubes, cell factories, roller bottles, spinner flasks, follower fibers, microcarriers, beads and the like. These culture substrates may be made of either inorganic materials such as glass or organic materials such as polystyrene, but materials such as polystyrene, polyethylene, and polypropylene that have high adsorptivity to proteins, peptides, etc., or adsorptivity It is preferable to use a material that has been subjected to a treatment for increasing the viscosity, for example, a hydrophilic treatment or a hydrophobic treatment. Moreover, it is preferable to consist of the material which has the heat resistance and water resistance which can be sterilized. As an example of a suitable device for that purpose, a polystyrene dish and / or plate (hereinafter referred to as an untreated polystyrene plate) that is frequently used mainly for culturing Escherichia coli, etc. and not specifically treated for cell culture is used. The culture substrate is generally commercially available.

  In the implementation of the method disclosed in the present invention, as a method for fixing or coating a protein or fusion protein belonging to the IGFBP family on the culture substrate solid phase surface, a physical method such as adsorption, a covalent bond, etc. Although a chemical method can be applied, the method by adsorption is preferable because of the ease of operation. For example, the molecule can be easily adsorbed by bringing the molecule solution into contact with the surface of the culture substrate solid phase such as a plate and removing the solvent after a predetermined time. More specifically, for example, a protein molecule solution using distilled water, PBS or the like as a solvent is filtered and sterilized, and then contacted with a culture substrate such as a plate. For example, it is allowed to stand for several hours to one week, preferably overnight. By simply doing, a cell culture substrate on which the adhesive molecule is fixed or coated can be obtained. Preferably, it is used after being washed several times with distilled water, PBS, or the like and replaced with an equilibrium salt solution such as PBS.

  In addition, when an antigenic molecule is artificially added and fused to the protein to be immobilized in advance, it is possible to utilize the binding with a specific antibody against the protein, and to efficiently modify the protein on the substrate surface. Can do. In this case, the specific antibody must be fixed or coated on the surface of the culture substrate in advance by a physical method such as adsorption or a chemical method such as covalent bonding. For example, antibodies that specifically recognize IGFBP or (GVGVP) n can be used as the antibody that is previously modified on the culture substrate. In the case of a recombinant protein in which various proteins or tag sequence peptides are fused to the target protein molecule, an antibody specific for the fused molecule can be used by previously modifying the culture substrate.

  In the practice of the present invention, there is at least one protein or fusion protein belonging to the IGFBP family immobilized or coated on the solid phase surface of the cell culture substrate, but two or more proteins or fusion proteins belonging to the IGFBP family are combined. May be used. In that case, each protein solution may be mixed and the mixed solution may be modified based on the above-described method.

  The concentration of the protein belonging to the above IGFBP family or the fusion protein solution needs to be appropriately examined depending on the adsorption amount and / or affinity of the protein, and further the physical properties of the protein. The concentration range is about ˜1000 μg / mL, preferably about 0.1 to 200 μg / mL, more preferably 1 to 50 μg / mL, and most preferably 5 to 20 μg / mL.

  As described later, the cell culture substrate of the present invention is a culture that maintains the undifferentiation of various pluripotent stem cells, a culture that differentiates pluripotent stem cells, and a pluripotent stem cell that has been differentiated. It can use suitably for the culture method which selects and concentrates a desired cell from a cell group.

[Cell culture method]
The cell culture method of the present invention is characterized in that pluripotent stem cells are proliferated using the cell culture substrate and a liquid medium while maintaining undifferentiation and differentiation pluripotency.

  In practicing the present invention, the practitioner, unless otherwise indicated, for genetic engineering methods such as molecular biology and recombinant DNA technology and general cell biology methods and prior art, Reference books can be referenced. These include, for example, Molecular Cloning: A Laboratory Manual 3rd Edition (Sambrook & Russell, Cold Spring Harbor Laboratory Press, 2001); Current Protocols in Molecular Bio. in Enzymology Series (Academic Press); PCR Protocols: Methods in Molecular Biology (Bartlett & Styling, Humana Press, 2003); Animal Cell Culture: A Practure cal Approach Third Edition (Masters ed., Oxford University Press, 2000); Antiboides: A Laboratory Manual (. Harlow et al & Lane eds., Cold Spring Harbor Laboratory Press, 1987) refer to such. In addition, reagents and kits for cell culture and cell biology experiments referred to in this specification are available from commercial vendors such as Sigma, Aldrich, Invitrogen / GIBCO, Clontech, and Stratagene.

  In addition, regarding a general method of cell culture using pluripotent stem cells and development / cell biology experiments, the practitioner can refer to standard reference books in the field. These include Guide to Techniques in Mouse Development (Wasserman et al., Academic Press, 1993); Embryonic Stem Cell Differentiation in Vitro (M.V. Embryo: Includes A laboratory manual (Edited by Hogan et al., Cold Spring Harbor Laboratory Press, 1994); Reagents and kits for cell culture, development and cell biology experiments referred to in this specification are available from commercial vendors such as Invitrogen / GIBCO and Sigma.

  A standard protocol has already been established for the generation, passage, and storage of mouse and human pluripotent stem cells. In addition to the reference books listed in the previous section, the practitioner can use multiple references (Matsui). et al., Cell 70: 841, 1992; Thomson et al., US Patent No. 5,843,780; Thomson et al., Science 282: 114, 1998; Shamlott et al., Proc. Natl. USA 95: 13726, 1998; Shamblott et al., US Pat. No. 6,090,622; Reubinoff et al., Nat. Biotech. 18: 399, 2000; International Publication No. 00/27995), etc. By doing these It may be used pluripotent stem cells. As for other animal species, for example, monkeys (Thomson et al., US Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA, 92, 7844, 1996) and rats (Ianaccone et al. Biol. 163: 288, 1994; Loring et al., International Publication No. 99/27076), chicken (Pain et al., Development 122: 2339, 1996; US Pat. No. 5,340,740; US Pat. No. 5,656,479), swine (Wheeler et al., Reprod. Fertil. Dev. 6: 563, 1994; Shim et al., Biol. Reprod. 57: 1089, 1997) etc. Establishment method of S-like cells are known, according to the method of the described, can be manufactured ES cells used in the present invention.

  An ES cell is an undifferentiated stem cell population obtained by transferring a cell mass called an inner cell mass inside an blastocyst stage embryo to an in vitro culture and repeating dissociation and passage of the cell mass. Is a pluripotent stem cell isolated as Various mouse-derived ES cells such as E14, D3, CCE, R1, J1, and EB3 are known. Some are from American Type Culture Collection, Cell & Molecular Technologies, Thromb-X, etc. It is also possible to purchase. Currently, over 50 types of human-derived ES cells have been established all over the world, and are listed in the list of the National Institutes of Health (NIH) (http://stemcells.nih.gov/registry/index.asp). More than 20 strains are registered. Some of them can be purchased from ES Cell International or Wisconsin Alumni Research Foundation.

  Although ES cells are generally established by culturing early embryos, ES cells can also be produced from early embryos obtained by nuclear transfer of somatic cell nuclei (Munsie et al., Curr. Biol. 10). : 989, 2000; Wakayama et al., Science 292: 740, 2001; Hwang et al., Science 303: 1669, 2004). In addition, a cell structure of a blastocyst stage embryo by transplanting the cell nucleus of a desired animal into a cell vesicle (referred to as cytoplasts or ooplastoids) obtained by dividing an egg cell of a heterogeneous animal or an enucleated egg cell into a plurality of cells And methods for producing ES cells based on the above have also been devised (International Publication Nos. 99/45100; 01/46401; 01/96532; US Patent Publication No. 02/90722; 02). / 194637). Also, parthenogenetic embryos are developed to a stage equivalent to the blastocyst stage and ES cells are produced therefrom (US Patent Publication No. 02/168863; Vrana K et al., Proc. Natl. Acad. Sci). USA 100: 11911-6) and a method for producing ES cells having genetic information of somatic cell nuclei by fusing ES cells and somatic cells (International Publication No. 00/49137; Tada). et al., Curr. Biol. 11: 1553, 2001). ES cells used in the present invention include ES cells produced by such a method or those obtained by modifying genes on the chromosome of ES cells by genetic engineering techniques.

An EG cell is a fetal germ cell called a primordial germ cell, and, as in the case of an ES cell, LIF and bFGF / FGF-2 on a feeder such as a MEF cell, STO cell, or Sl / Sl ⁴ -m220 cell, Or prepared by stimulation with a drug such as forskolin (Matsui et al., Cell 70: 841, 1992; Koshimazu et al., Development 122: 1235, 1996), and properties very similar to those of ES cells (Thomson & Odorico, Trends Biotechnol. 18:53, 2000). As in the case of ES cells, EG cells (Tada et al., EMBO J. 16: 6510, 1997; Andrew et al.) Prepared by fusing EG cells and somatic cells and genes on the chromosomes of EG cells are genetically engineered. Those modified by a technique can also be used in the method of the present invention.

  An iPS cell (induced pluripotent stem cell) is a pluripotent cell obtained by reprogramming somatic cells. It can be produced by the method described in Patent Documents 3, 4 and 5. In addition to the methods described in the above-mentioned patent documents, methods for producing iPS cells by many modified methods are known. International Publication No. WO2007 / 069666 discloses somatic cell nuclear reprogramming factors including Oct family gene, Klf family gene, and Myc family gene gene product, as well as Oct family gene, Klf family gene, Sox family gene and Myc family. A somatic cell nuclear reprogramming factor containing a gene product of a gene is described, and further, the induced pluripotent stem cell is produced by somatic cell nuclear reprogramming, comprising the step of contacting the nuclear reprogramming factor with the somatic cell. A method is described. In addition to this, there is known a method of using another substance or gene without using one or more of the above-mentioned reprogramming factors, using other factors, or replacing or in addition to the reprogramming factors. However, any method can be used as long as it falls within the definition of iPS cells.

  IPS cells used in the present invention can be produced by reprogramming somatic cells. The type of somatic cell used here is not particularly limited, and any somatic cell can be used. The somatic cell includes all cells other than the internal germ cells of the cells constituting the living body, and may be a differentiated somatic cell or an undifferentiated stem cell. The origin of the somatic cell may be any of mammals, birds, fishes, reptiles and amphibians, but is not particularly limited, but is preferably a mammal (for example, a rodent such as a mouse, or a primate such as a human). A mouse or a human is preferable. In addition, when human somatic cells are used, any fetal, neonatal or adult somatic cells may be used.

  In addition, pluripotent stem cells are not limited to ES cells, EG cells, and iPS cells, but are derived from mammalian embryos, fetuses, umbilical cord blood, or adult tissues such as adult organs and bone marrow, blood, etc. ES / EG All pluripotent stem cells having traits similar to cells are included. For example, ES-like cells obtained by culturing germ cells under special culture conditions exhibit very similar characteristics to ES / EG cells (Kanatsu-Shinohara et al., Cell 119: 1001, 2004). Can be used as pluripotent stem cells. Other examples include pluripotent adult progenitor / stem cells (MAPCs) isolated from bone marrow cells and capable of differentiating into cells of all three germ layers. In addition, hair sheath cells, keratinocytes (International Publication No. 02/51980), intestinal epithelial cells (International Publication No. 02/57430), inner ear cells (Li et al., Nature Med. 9: 1293, 2003), etc. Pluripotent stem cells obtained by culturing the cells under special culturing conditions, or mononuclear cells in blood (or stem cells contained in the cell nuclei) are expressed as M-CSF (Macrophage-Colony Stimulating Factor; macrophage colony Stimulation factor) + PMA (phorbol 12-myristate 13-acetate) treatment (Zhao et al., Proc. Natl. Acad. Sci. USA 100: 2426, 2003), or CR3 / 43 antibody treatment (Abuljadayel, Curr) Med.Res.Opinion 19: 355,2003) with regard pluripotent stem cells or the like which is manufactured by, all included as long as stem cells having traits similar to ES / EG cells. In this case, traits similar to ES / EG cells include the presence of surface (antigen) markers specific to the cells, expression of the cell-specific genes, and the ability to form teratomas and chimeric mice. / Defined with cell biology specific to EG cells.

  In the practice of the present invention, pluripotent stem cells are seeded on the cell culture substrate of the present invention. The pluripotent stem cell culture method and conditions can be used as they are, except that the culture substrate is used. The normal culture method and culture conditions of pluripotent stem cells are described in the above-mentioned reference books, namely Guide to Techniques in Mouse Development (Edited by Wasserman et al., Academic Press, 1993); Embryonic Stem Cell Div. Wiles, Meth. Enzymol. 225: 900, 1993); s, 2002) and references (Matsui et al., Cell 70: 841, 1992; Thomson et al., US Pat. No. 5,843,780; Thomson et al., Science 282: 114, 1998; Shamblott et al. USA 95: 13726, 1998; Shamblott et al., US Patent No. 6,090,622; Rebinoff et al., Nat.Biotech.18: 399, 2000; / 27995)) can be referred to, but is not particularly limited thereto.

  Any liquid medium for culturing pluripotent stem cells can be used as long as it can be applied to a conventional method for subculturing pluripotent stem cells. Specific examples thereof include Dulbecco's Modified Eagle's Medium (DMEM), Glasgow Minimum Essential Medium (GMEM), RPMI 1640 medium, etc. Usually, about 2 mM glutamine and / or about 100 μM of 2-mercaptoethanol is used. Add and use. KnockOut DMEM (Invitrogen), ES cell-qualified DMEM (Cell & Molecular Technologies), TX-WES (Thromb-X), etc. which are marketed as ES cell culture media can also be used. Although it is preferable to add about 5 to 25% of FBS to these media, serum-free culture is also possible, and for example, 15 to 20% KnockOut Serum Replacement (Invitrogen) can be substituted. In addition, a culture supernatant of MEF cells or a medium supplemented with bFGF / FGF-2, SCF or the like may be used, and detailed methods thereof are known (Xu et al., Nature Biotech. 19: 971, 2001). International Publication No. 01/51616; International Publication No. 03/020920; Amit et al., Biol. Reprod., 70: 837, 2004).

  A substance / factor having an action of maintaining the undifferentiated state of the pluripotent stem cells can be added to the liquid medium for culturing the pluripotent stem cells. Specific substances / factors are not particularly limited, but in the case of mouse ES / EG cells, LIF is preferred. LIF has been reported in published papers (Smith & Hooper, Dev. Biol. 121: 1, 1987; Smith et al., Nature 336: 688, 1988; Rathjen et al., Genes Dev. 4: 2308, 1990), NCBI's. It is a known protein factor in the public DNA database with access numbers X13967 (human LIF), X06181 (mouse LIF), NM_022196 (rat LIF), etc. The recombinant protein is, for example, the trade name of ESGRO (Chemicon) Can be purchased at. In addition, by adding a GSK-3 inhibitor to the culture solution, it is possible to efficiently maintain the undifferentiation of mouse and human ES cells without particularly adding other growth factors or physiologically active factors. (Sato et al., Nature Med. 10:55, 2004). In this case, any substance having an action capable of inhibiting the activity of GSK-3 can be used. For example, a Wnt family molecule (Manookian & Woodgett, Adv. Cancer Res. 84: 203, 2002; J. Cell Sci. 116: 1175, 2003) and the like.

  In the practice of the present invention, pluripotent stem cells that have been passaged and maintained based on conventional methods are seeded on a culture substrate prepared by the above method and cultured based on the above culture conditions and methods. The cells can be subcultured in a dispersed state while maintaining the undifferentiated state inherent to the cells. Pluripotent stem cells cultured in such a state are not physically suppressed during cell division and / or cell growth suppression mechanisms due to cell-cell contact do not act and / or cell survival. As the sex increases and the number of dead cells decreases, there is a marked increase and proliferation of cells. As one example, when mouse ES cells are cultured by the method of the present invention, they are at least 1.25 times or more, preferably 1.5 times or more, more preferably 2 times as compared with the case of culturing by conventional methods. It is possible to exhibit the above proliferation ability. Further, when passage is performed for about 4 generations under the above conditions, it is possible to recover at least 3 times, preferably 10 times or more, cells as compared with the conventional method. The proliferative ability can be expressed by an index such as the rate of increase in cell number per unit time or doubling rate, and the measurement / calculation method is a known method used in experiments using general cells. Any of them can be used.

  As described above, an undifferentiated state in a pluripotent stem cell means that the pluripotent stem cell is capable of almost permanent or long-term cell growth, exhibits a normal nuclear (chromosomal) type, and has an appropriate condition. Below, it means a state with the ability to differentiate into cells of all three germ layers. Moreover, it is preferable that at least one of the other properties of the pluripotent stem cell, for example, properties such as maintenance of telomerase activity, teratoma formation ability, or chimera formation ability is desirable. A standard protocol has already been established as a method for examining these properties and characteristics. For example, the above-mentioned reference book, that is, Guide to Technologies in Mouse Development (Edited by Wasserman et al., Academic Press, 1993); Embryonic Stem Cell. Differentiation in Vitro (MV Wiles, Meth. Enzymol. 225: 900, 1993); n, ed., by referring to the Humana Press, 2002), etc., can be carried out easily, but are not limited to particular methods described in these.

  An undifferentiated pluripotent stem cell can also be defined as a cell in which the presence of at least one, preferably a plurality of marker molecules can be confirmed by at least one, preferably a plurality of methods described below. Expression of various markers specific to undifferentiated pluripotent stem cells is detected by conventional biochemical or immunochemical techniques. The method is not particularly limited, but preferably an immunochemical technique such as immunohistochemical staining or immunoelectrophoresis is used. In these methods, marker-specific polyclonal or monoclonal antibodies that bind to undifferentiated pluripotent stem cells can be used. Antibodies that target individual specific markers are commercially available and can be readily used. As specific markers for undifferentiated pluripotent stem cells, for example, ALP activity and expression of gene products such as Oct-3 / 4 or Rex-1 / Zfp42 can be used. Various antigen molecules can also be used. In mouse ES cells, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, GCTM-2, etc. are undifferentiated in human ES cells. Listed as a marker. The expression of these undifferentiated markers decreases and disappears as ES cells differentiate.

  Alternatively, the expression of the undifferentiated pluripotent stem cell marker may be any marker protein such as reverse transcriptase-mediated polymerase chain reaction (RT-PCR) or hybridization analysis, although the method is not particularly limited. This can be confirmed by a conventional molecular biological method for amplifying, detecting and analyzing mRNA. Nucleic acid sequences of genes encoding marker proteins specific to undifferentiated pluripotent stem cells (for example, Oct-3 / 4, Rex-1 / Zfp42, Nanog, etc.) are known, NCBI public databases, etc. The marker-specific sequences that are available in and required for use as primers or probes can be readily determined.

[Method for producing cardiomyocytes by inducing differentiation]
The method for producing cardiomyocytes of the present invention is characterized in that pluripotent stem cells are differentiated using the cell culture substrate of the present invention and a liquid medium.

  The culture method and culture conditions for inducing differentiation of pluripotent stem cells are the same as those used for normal differentiation induction culture and culture conditions of pluripotent stem cells, except that the cell culture substrate of the present invention is used. be able to. Moreover, about a liquid culture medium, the thing similar to what was mentioned above can be used.

  Differentiation-inducing factors (also called Growth Factors) are compounds such as peptides, hormones, cytokines, proteins, glycoproteins, etc. that are added to the medium to induce differentiation of pluripotent stem cells. Different types of differentiation-inducing factors are used depending on the type of differentiation and the stage of differentiation. In the present invention, a known differentiation-inducing factor can be added to a liquid medium according to a target cell according to a known method or protocol. In the differentiation induction method of the present invention, the IGFBP family protein promotes the differentiation of pluripotent stem cells into cardiomyocytes, and therefore is expected to cause differentiation into cardiomyocytes without any addition of a differentiation inducer from the outside. it can.

  The pluripotent stem cells prepared by the present invention and / or differentiated cells prepared from the cells are useful for pharmacological evaluation and activity evaluation of various physiologically active substances (for example, drugs) and new gene products whose functions are unknown. . For example, it can be used for screening of substances and drugs related to the functional regulation of pluripotent stem cells and various differentiated cells, and / or substances and drugs that are toxic or toxic to pluripotent stem cells and various differentiated cells. it can. In particular, at present, few screening methods using human cells have been established, and various differentiated cells derived from the pluripotent stem cells prepared according to the present invention are useful cell sources for carrying out the screening method. Become. In addition, if the cardiomyocytes differentiated by the method of the present invention can be separated as a cardiomyocyte sheet, it is useful as a regenerative medical method for the heart without using heart transplantation.

[experimental method]
<Preparation of (GVGVP) n plasmid>
The plasmid is based on the description of non-patent literature (McPherson DT et al., Biotechnol. Prog. 1992; 8: 347-52. McPherson DT et al., Protein Expr. Purif. 1996; 7: 51-7.). Made. Oligonucleotides encoding Xho I, PflMI, Not I, and BamHI sites and a PflMI- (GVGVP) ₁₁ -PflMI sequence were purchased from Operon Biotechnology. These oligonucleotides were subcloned into pMD20-T (manufactured by Takara Bio Inc.) using the Mighty TA-cloning kit for PrimeSTAR (Takara Bio Inc.). This vector was digested with PflMI to obtain a PflMI fragment of (GVGVP) ₁₁ and a PFlMI fragment of pMD20-T having XhoI, PflMI, NotI and BamHI sites. The PFlMI fragment of pMD20-T was dephosphorylated to prevent self-ligation. The enzyme-treated fragment and the vector were ligated using T4 ligase. Similarly, pMD20 vectors containing (GVGVP) ₁₂ , (GVGVP) ₂₃ , (GVGVP) ₄₅ , and (GVGVP) ₆₇ were obtained. Since both the fragment and the vector had PflMI sites on both sides, and the vector was dephosphorylated, a concatamer of the fragment could be obtained. The resulting plasmid was treated with Xho I and Bam HI. The Xho I / Bam HI-treated fragment of (GVGVP) n and the Xho I / Bam HI-treated fragment of pET14b were ligated, and His tag- (GVGVP) ₁₂ , (GVGVP) ₂₃ , (GVGVP) ₄₅ , (GVGVP) ₆₇ A recombinant plasmid expressing

<Production of (GVGVP) n-IGFBP4>
A cDNA fragment encoding the Thy domain of IGFBP4 was amplified by PCR from a cDNA library of P19CL6 cells. PCR of a DNA fragment encoding the Thy domain (amino acid positions: 167-245) was performed using primers having Not I, Xho I, and Bam HI sites. The primers used were 5′-ATAAGAAATGCGCCGCCTAAACTATCAGGGTTCCTGCCCAGAGCGGCTG-3 ′ and 5′-CGCTCGAGCGGCAGGGTTTCCTGCCAGAGCGA-3 ′ as IGFBP sense primers. As an antisense primer, 5′-CGGGATCCCCTCACTCTTGGAAGCTGTCAGCCCAG-3 ′ was used.
The cDNA fragment was subcloned into the pMD20-T vector using the Mighty TA-cloning kit for PrimeSTAR (manufactured by Takara Bio Inc.). The resulting plasmid was treated with Not I or Xho I and Bam HI. The Not I / Bam HI treated fragment containing the Thy domain sequence was inserted into the Not I / Bam HI fragment of pMD20 containing (GVGVP) n. A plasmid containing (GVGVP) n-IGFBP4 was treated with Xho I and Bam HI to obtain a (GVGVP) n-IGFBP4 fragment. By ligating the Xho I / Bam HI-treated fragment of (GVGVP) n-IGFBP4 or the Xho I / Bam HI-treated fragment of IGFBP4 (Thy) with the Xho I / Bam HI-treated fragment of the pET14b vector, 6- A recombinant plasmid expressing His-tagged (GVGVP) ₁₂ -IGFBP4, (GVGVP) ₂₃ -IGFBP4, (GVGVP) ₄₅ -IGFBP4, (GVGVP) ₆₇ -IGFBP4, or IGFBP4 (Thy) was constructed.

<Expression and purification of recombinant protein>
The recombinant protein is E. coli. It was expressed using E. coli KRX cells (Promega). In order to prevent aggregation and insolubilization in E. coli, protein expression was performed at 20 ° C., which is lower than the phase transition temperature. The expressed protein was purified using a COSMOGEL His Accept purification system (manufactured by Nacalai Tesque). The protein was dissolved in phosphate buffer (PBS).

<Determination of Tg (t) of purified (GVGVP) n protein and temperature-dependent aggregation profile>
Since the (GVGVP) n protein is a protein that exhibits the behavior that its hydrophobicity changes in a temperature-dependent manner, the aggregation behavior at temperatures exceeding Tg (t) was examined. Tg (t) depends on the number of GVGVP repeats. The Tg of (GVGVP) ₁₂ , (GVGVP) ₂₃ , (GVGVP) ₄₅ , (GVGVP) ₆₇ was determined by a turbidity assay. The aggregation temperature profile used to determine the Tg (t) value was measured using a spectrophotometer manufactured by Shimadzu Corporation. The wavelength was fixed at 350 nm. The turbidity of each protein (1 mg / mL) was measured between 15 ° C. and 60 ° C. while increasing or decreasing the temperature at 1 ° C./min. The Tg (t) value was defined as the temperature at which 50% turbidity was measured.

<Immobilization of (GVGVP) n-IGFBP4 and IGFBP4 (Thy) on a polystyrene dish and evaluation of adsorption stability of recombinant protein using ELISA>
(GVGVP) n-IGFBP4 and IGFBP4 (Thy) solutions (0-50 μg / mL) were incubated on polystyrene dishes at temperatures higher than Tg (t) at 37 ° C. and 4 ° C. for 3 hours, respectively. In order to evaluate the optimal concentration and adsorption stability of each protein, an ELISA assay using an anti-IGFBP4 antibody (Millipore) was performed. After incubation, the dishes were blocked with PBS containing 1% bovine serum albumin for 1 hour and incubated with rabbit polyclonal anti-IGFBP4 antibody (1: 20000 dilution) for 2 hours at room temperature. Detection was performed using ULTRA-SENSITIVE TMB LIQUID HORSERADISH SUBSTRATE SYSTEM (manufactured by Millipore). Absorption at 450 nm was measured with a microplate reader (GE Healthcare).

<Cell culture>
P19CL6 cells were cultured on Dulbecco's Modified Eagle's Medium (DMEM) containing 10% bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL streptomycin at 37 ° C., 95% air / 5% CO2. ₂ under culture. In order to maintain ES cells undifferentiated, ES cells (1 × 10 ⁶ cells / 35 mm dish) were cultured on the feeder layer. The feeder layer was formed by mouse embryo fibroblasts using COMPLETE ES CELL MEDIA W / 15% bovine serum and LIF as a medium.

<Differentiation of ES cells into cardiomyocytes>
The cardiomyocyte differentiation of ES cells in the early and late stages on (GVGVP) n-IGFBP4 immobilized dishes was examined. Initially, ES cells (1 × 10 ⁵ cells / 35 mm dish) were cultured on (GVGVP) n-IGFBP4 immobilized dishes or 0.1% gelatin-coated dishes. Later, after formation of embryoid bodies (EB, embryoid body) (hang drop specimen inspection method, 5 days), embryoid bodies were placed on (GVGVP) n-IGFBP4 immobilized dishes or 0.1% gelatin coated dishes And cultured. Iscove's Modified Dulbecco's Medium (IMDM, Gibco) containing 20% FBS, 1 mM sodium pyruvate, 0.1 mM MEM non-essential amino acid (Gibco) and 0.05 mM 2-mercaptoethanol (Sigma) Was used as a medium for cardiomyocyte differentiation of ES cells.

<Immunoprecipitation and Western blot>
After P19CL6 cells were cultured on (GVGVP) n-IGFBP4 for 3 hours, the cells were lysed (25 mM Tris-HCl 2.5 mM EDTA, 137 mM NaCl, 2.7 mM KCl, 1% sodium deoxycholate, 0.1 % SDS, 1% Triton X-100 and protease inhibitor cocktail (Nacalai Tesque)). The crushed liquid was centrifuged at 20000 × g for 30 minutes at 4 ° C. The supernatant was taken out, incubated with PureProteome protein G-conjugated magnetic beads (Millipore) and bound to anti-LRP5 / 6 antibody (Acris antibody GmbH). After incubation, the proteins bound to the beads were separated by 5-20% SDS-PAGE gradient gel (manufactured by DRC CO. Ltd). The separated protein was transferred to a PVDF membrane. The membrane was incubated with a rabbit polyclonal anti-IGFBP4 antibody (1: 10000 dilution), and then incubated with an HRP-conjugated anti-rabbit IgG antibody (manufactured by Jackson ImmunoResearch Laboratories). Detection was performed using Immobilon Western detection reagents (Millipore) on the membrane.

<RT-PCR>
Total RNA extraction was performed using Tripure Isolation Reagent (Roche Diagnostics). CDNA synthesis was carried out using Transscriptor High Fidelity cDNA Synthesis kit (Roche). RT-PCR was performed using the following protein detection primers. Mouse β-actin, mouse GATA 4, mouse α-myosin heavy chain (αMHC), mouse E-cadherin, mouse N-cadherin, mouse Oct-3, mouse Sox1, mouse Sox17, mouse BrachyuryT, mouse Wnt3a, mouse IGFBP4, mouse Frizzled8. PCR was performed using Ex-Taq polymerase (manufactured by Takara Bio Inc.).

<Immune staining and confocal microscope>
Cells were cultured on 24 × 24 mm cover glasses. Cells were fixed with 4% paraformaldehyde / PBS for 15 minutes, washed with 50 mM glycine-PBS, permeabilized with 0.1% Triton X-100 / PBS for 5 minutes, and blocked with 1% BSA / PBS for 1 hour. Did. After fixation and permeabilization, the cells were incubated with anti-human αMHC monoclonal antibody (MF20, R & D systems, 1: 100 dilution) or anti-IGFBP4 antibody (1: 100 dilution) for 1 hour at room temperature. did. The cells were washed with PBS and incubated for 1 hour at room temperature using FITC-conjugated second antibody or Cy3-conjugated second antibody (manufactured by Jackson ImmunoResearch Laboratories). Cell nuclei were stained with 0.1 μg / mL DAPI. All cells were observed using a confocal microscope (A1 / A1R system, manufactured by Nikon). The fluorescence image was processed using NIS-Elements Imaging Software (manufactured by Nikon Corporation). The ratio of the MF20 positive area was determined from the average of the ratio of the MF20 stained area / DAPI stained area in 20 random locations of each sample.

<Statistical analysis>
The cell experiment was performed 3 times, and the results are shown as mean ± standard deviation.

[Experimental result]
<Determination of Tg (t) and temperature-dependent aggregation profile of (GVGVP) n-IGFBP4>
_{IGFBP4 (Thy), (GVGVP)} 12 -IGFBP4, (GVGVP) 23 -IGFBP4, (GVGVP) 45 -IGFBP4, (GVGVP) 67 -IGFBP4, (GVGVP) 45, the production of _{(GVGVP) 67,} SDS-PAGE and Confirmed by Western blot (FIG. 1A).
To determine the Tg (t) of (GVGVP) n-IGFBP4, a turbidity assay was performed to determine the temperature dependence of aggregation of (GVGVP) ₁₂ , (GVGVP) ₂₃ , (GVGVP) ₄₅ , (GVGVP) ₆₇ . The embodiment was investigated. Temperature-dependent aggregation of (GVGVP) ₄₅ and (GVGVP) ₆₇ was observed at 50 ° C. or higher, but (GVGVP) ₁₂ and (GVGVP) ₂₃ were not (FIG. 1B). It is thought that 23 repetitions of GVGVP are too short to cause aggregation. _(GVGVP) 45, the temperature profile _{(GVGVP) 67, (GVGVP)} 45, was calculated to be 32 ° C., 42 ° C. respectively Tg of _{(GVGVP) 67.}

<Adsorption stability of IGFBP4 (Thy), (GVGVP) _12- IGFBP4, (GVGVP) _23- IGFBP4, (GVGVP) _45- IGFBP4, (GVGVP) _67- IGFBP4 to polystyrene dishes>
In order to examine the adsorption stability of IGFBP4 (Thy), (GVGVP) ₁₂ -IGFBP4, (GVGVP) ₂₃ -IGFBP4, (GVGVP) ₄₅ -IGFBP4, (GVGVP) ₆₇ -IGFBP4 with each protein, they were coated with each protein. The dishes were incubated at 4 ° C. or 37 ° C. for 1 day or 1 week. The amount of adsorbed protein was measured by ELISA. (GVGVP) ₆₇ -IGFBP4 adsorbed most. After incubation 1 _{day, (GVGVP) 12 -, (} GVGVP) 23 -, (GVGVP) 45 -IGFBP4 was equivalent to IGFBP4 (Thy) (Fig. 2 A). All these proteins were saturated in adsorption above 10 μg / mL at both 4 ° C. and 37 ° C. (FIGS. 1A and B). (GVGVP) ₆₇ -IGFBP4, (GVGVP) ₄₅ -IGFBP4 has a lower adsorption amount at 4 ° C. than Tg, and at 37 ° C., the adsorption amount of (GVGVP) ₆₇ -IGFBP4 is (GVGVP) ₄₅ -Less than IGFBP4 (Figure 2A). It was found that IGFBP4 (Thy) alone did not cause stable adsorption over a week. On the other hand, (GVGVP) ₆₇ -IGFBP4 was stably adsorbed on the dish for 1 week at 37 ° C. (FIGS. 2B and C). From the above results, it was found that IGFBP4 (Thy) can be stably immobilized on a polystyrene dish via (GVGVP) ₆₇ .

<Interaction of IGFBP4 (Thy), (GVGVP) ₁₂ -IGFBP4, (GVGVP) ₂₃ -IGFBP4, (GVGVP) ₄₅ -IGFBP4, or (GVGVP) ₆₇ -IGFBP4 and LRP5 / 6>
To examine whether IGFBP4 (Thy), (GVGVP) ₁₂ -IGFBP4, (GVGVP) ₂₃ -IGFBP4, (GVGVP) ₄₅ -IGFBP4, (GVGVP) ₆₇ -IGFBP4 binds to the Wnt receptor LRP5 / 6. Immunoprecipitation using anti-LRP5 / 6 antibody was performed. Immunoprecipitation was cultured on dishes coated with 10 μg / mL IGFBP4 (Thy), (GVGVP) ₁₂ -IGFBP4, (GVGVP) ₂₃ -IGFBP4, (GVGVP) ₄₅ -IGFBP4, or (GVGVP) ₆₇ -IGFBP4 P19CL6 cells were used. P19CL6 cells express Lnt5 / 6 and Frizzled 8 which are Wnt receptors, and it has been reported that these receptors can bind to IGFBP4 (Reference 4). Whether P19CL6 cells adsorbed to (GVGVP) n-IGFBP4-coated dishes through the interaction of IGFBP4 and Wnt receptors was examined. P19CL6 cells adsorbed to dishes coated with these proteins, but not IGFBP4 (Thy) domain (GVGVP) ₆₇ alone coated dishes (no data). In P19Cl6 cells cultured on these dishes, (GVGVP) _12,23,45,67- IGFBP4 and IGFBP4 (Thy) precipitated together with anti-LRP5 / 6 antibody-bound beads were detected by Western blot using anti-IGFBP4 antibody. (FIG. 3). These results revealed that P19CL6 cells are thought to be adsorbed to (GVGVP) n-IGFBP4-coated dishes through the interaction of IGFBP4 (Thy) and the Wnt receptor.

<Differentiation into cardiomyocytes by culturing mouse ES cells on (GVGVP) _67- IGFBP4-coated dish>
To examine the differentiation of mouse ES cells into cardiomyocytes using (GVGVP) ₆₇ -IGFBP4-coated dishes, mouse ES cells were treated with 0.1% gelatin-coated dishes (control), 10 μg / mL (GVGVP) ₆₇ − Cultures were performed for 24 days using IGFBP4-coated dishes, 1 μg / mL IGFBP4 (Thy) -coated dishes, or gelatin-coated dishes supplemented with 1 μg / mL (GVGVP) ₆₇ -IGFBP4 as a soluble factor. The medium was changed every other day. Mouse ES cells were similarly adsorbed and proliferated under all conditions (FIG. 4A). Cardiac gene expression was examined by RT-PCR of GATA4 and αMHC. In mouse ES cells cultured on (GVGVP) ₆₇ -IGFBP4-coated dishes, the expression levels of GATA4 and αMHC after 24 days of culture were high (FIG. 4B). Moreover, it became positive by immunostaining with an anti-αMHC antibody (MF20) (FIG. 4C). The ratio of the MF20 positive region to the DAPI positive region was 5%, 7%, and 7% in the order of control, IGFBP4 (Thy) added, (GVGVP) ₆₇ -IGFBP4 added, and (GVGVP) ₆₇ -IGFBP4 immobilized, respectively. 18% (FIG. 4C). From these results, it was revealed that immobilization of (GVGVP) ₆₇ -IGFBP4 promotes the differentiation of mouse ES cells into cardiomyocytes.

<Differentiation of mouse ES cells cultured on (GVGVP) ₆₇ -IGFBP4-coated dish into cardiomyocytes at a late stage after embryoid body formation>
IGFBP4 has been reported to promote the differentiation of mouse ES cells into cardiomyocytes in the late stage after embryoid body formation (Reference 4). It was examined whether differentiation of mouse ES cells that formed embryoid bodies into cardiomyocytes was promoted by (GVGVP) ₆₇ -IGFBP4 immobilized dishes. The gene expression profile of ES cells in the process of embryoid body formation in the hanging drop specimen inspection method was examined by RT-PCR. Embryoid bodies were formed in the hanging drop medium on the third day. Embryoid bodies expressed N-cadherin, Sox17, BrachyuryT, GATA4, Wnt3a, and IGFBP4 (FIG. 5). From these results, it was revealed that ES cells differentiate into endoderm mesoderm cells through the formation of embryoid bodies in hanging drop medium. Moreover, in the culture medium on the 5th day, IGFBP4 was strongly expressed, while the expression of Wnt3a was weakened (FIG. 5B). This suggests that on the fifth day, cardiac development has begun. Therefore, 5 days after the hanging drop culture method, the embryoid bodies were cultured on 10 μg / mL (GVGVP) ₆₇ -IGFBP4 immobilized dish or 0.1% gelatin-coated dish (control). 1 μg / mL IGFBP4 (Thy), 10 μg / mL (GVGVP) ₆₇ -IGFBP4 was added as a solubility factor to the gelatin-coated dish for 20 days (FIG. 6A). The medium was changed every other day. The gene expression of GATA4 and αMHC was confirmed using RT-PCR. High expression of both GATA4 and αMHC was observed in embryoid bodies cultured on (GVGVP) ₆₇ -IGFBP4 for 20 days (FIG. 6C). After 20 days, autonomous pulsation of myocardial differentiated ES cells was observed. The pulsatile region of the embryoid body cultured on (GVGVP) _67- IGFBP4 is shown in FIG. 6B. In the immunostaining using MF20, the ratio of the MF20 positive region to the DAPI positive region was approximately in the order of control, IGFBP4 (Thy) addition, (GVGVP) ₆₇ -IGFBP4 addition, and (GVGVP) ₆₇ -IGFBP4 immobilized dish, respectively. 30%, 40%, 40% and 65%. FIG. 7 shows the observation result of the MF20 positive area | region of the ES cell cultured on the (GVGVP) _67- IGFBP4 fixed dish by the confocal microscope.

Claims (11)

  A polypeptide comprising all or part of a protein belonging to the insulin-like growth factor binding protein (IGFBP) family, or a fusion protein comprising all or part of the protein belonging to the insulin-like growth factor binding protein family, A cell culture substrate characterized by being fixed or coated on.   The cell culture substrate according to claim 1, wherein the protein belonging to the insulin-like growth factor binding protein (IGFBP) family is IGFBP4 or a protein having homology with IGFBP4.   A polypeptide comprising a region of all or part of the protein belonging to the insulin-like growth factor binding protein (IGFBP) family, or a fusion protein comprising all or part of the protein belonging to the insulin-like growth factor binding protein family, The cell culture substrate according to claim 1 or 2, comprising an amino acid sequence homologous to the Thy domain of IGFBP4 or the Thy domain of IGFBP4.   The cell culture substrate according to any one of claims 1 to 3, wherein the fusion protein comprising all or part of a protein belonging to the insulin-like growth factor binding protein family comprises all or part of an elastin-like polypeptide.   The fusion protein comprising all or part of the protein belonging to the insulin-like growth factor binding protein family comprises a repetitive sequence represented by (GVGVP) n (n is a number of 2 or more). The cell culture substrate according to any one of the above.   The cell culture substrate according to claim 5, wherein the repetitive sequence is represented by (GVGVP) n (n is a number of 65 or more).   The fusion protein is a protein containing (GVGVP) n (n is a number of 65 or more) on the N-terminal side and all or part of IGFBP4 on the C-terminal side. The cell culture substrate according to claim 1.   The fusion protein is a protein containing (GVGVP) n (n is a number of 65 or more) on the N-terminal side and an amino acid sequence homologous to the Thy domain of IGFBP4 or the Thy domain of IGFBP4 on the C-terminal side. The cell culture substrate according to claim 7.   A cell characterized by proliferating pluripotent stem cells using the cell culture substrate according to any one of claims 1 to 8 and a liquid medium while maintaining undifferentiation and differentiation pluripotency. Culture method.   A method for producing cardiomyocytes, comprising using the cell culture substrate according to any one of claims 1 to 8 and a liquid medium to differentiate pluripotent stem cells into cardiomyocytes.   The method for producing cardiomyocytes according to claim 10, wherein pluripotent stem cells are differentiated into cardiomyocytes by inhibiting the Wnt / β-catenin signaling pathway.