JP2013126405A - Cell culture substrate, and method for culturing cell and method for inducing differentiation of multipotent stem cell using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method a cell culture substrate capable of culturing multipotent stem cells without using feeder cells, differentiating the multipotent stem cells to specific cells at high purity, and also being used for selecting cells after differentiation, and to provide a method for culturing cells using the same, and a method for inducing differentiation of the multipotent stem cells.SOLUTION: The cell culture substrate includes fixing or coating a protein belonging to cadherin family or a fusion protein containing all or a part of region of the protein belonging to cadherin family, and a polymer having a glycosylated chain on a surface thereof.

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 without using feeder cells. The present invention relates to a cell culture substrate capable of differentiating sex stem cells into specific cells with high purity, a cell culture method using the same, and a method for inducing differentiation of pluripotent stem cells.

  Living organisms have the ability to quickly complement and repair cells and tissues lacking in order to maintain their own lives, and this ability is called “regenerative ability”. As an example of “regenerative ability” in higher animals, wound healing such as skin and blood vessels is generally well known, but even in large parenchymal organs such as the liver and kidney, the tissue homeostasis is promptly against tissue injury. It is known that cell proliferation and tissue remodeling occur in order to restore the disease. In recent years, attempts have been made to heal and improve various diseases and wounds by utilizing the “regenerative ability” inherent to this individual organism, which is called “regenerative medicine” and is a new medical technology. Attention has been paid.

  “Stem cells” play a central role in carrying out “regenerative medicine”. In general, a “stem cell” can be defined as an undifferentiated cell having the ability to differentiate into a specific or multiple functional cells and the ability to self-replicate to repeatedly produce the same cells as itself. Various tissues / cells have their own stem cells. For example, a series of blood cells such as erythrocytes, lymphocytes, megakaryocytes, and the like are progenitor cells derived from the stem cells called hematopoietic stem cells. skeletal muscle cells are produced from stem / progenitor cells called satellite cells and myoblasts. In addition, neural stem cells that exist in neural tissues such as the brain and spinal cord and produce nerve cells and glial cells, epidermal stem cells that produce epidermal cells and hair root cells, oval cells (hepatic stem cells) that produce hepatocytes and bile duct cells, Cardiac stem cells that form cardiomyocytes have been identified so far.

  Some regenerative medicine methods using stem cells or progenitor cells derived from such cells have already been put into practical use for the treatment of diseases caused by deficiency or dysfunction of blood cells such as leukemia and aplastic anemia. A method of injecting and transplanting hematopoietic stem cells or hematopoietic progenitor cells is well known. However, in the case of stem cells present in the parenchymal organs such as the brain, heart and liver, it is technically difficult to collect them from living tissues and / or in vitro culture, and these stem cells generally have low proliferation ability. On the other hand, it is possible to recover the stem cells from cadaver tissue, but using the cells obtained in this manner for medical treatment is ethically problematic. Therefore, in order to carry out regenerative treatment targeting neurological diseases, heart diseases, etc., it is necessary to develop a technique for producing a desired cell type using cells other than stem cells present in such target tissues. It is.

  As an attempt based on such a viewpoint, first, a method using “pluripotent stem cells” can be mentioned. A “pluripotent stem cell” refers to a normal nuclear (chromosomal) type that can proliferate almost permanently or for a long period of time while maintaining an undifferentiated state in vitro (hereinafter referred to as in vitro). And are defined as cells capable of differentiating into cells of all lineages of the three germ layers (ectoderm, mesoderm, and endoderm) under appropriate conditions. At present, pluripotent stem cells include embryonic stem cells (Embronic Stem cells: ES cells) isolated from early embryos and similar cells, and embryonic germ cells (Embryonics) isolated from embryonic primordial germ cells. Germ cells (EG cells) are the best known and are used in various studies.

  ES cells are undifferentiated by transferring cell clusters called inner cell mass inside the blastocyst stage embryo to in vitro culture, and repeating dissociation and passage of the cell mass. It can be isolated as a stem cell population. In addition, the cells are suitable for use on feeder cells prepared using primary culture fibroblasts derived from mouse fetal tissue (Murine Embryonic Fibroblasts; hereinafter referred to as MEF cells) or stromal cells such as STO cells. By maintaining the cell density and repeating the subculture while frequently changing the culture medium, it is possible to establish a cell line while retaining the properties of the undifferentiated stem cells. Another characteristic of ES cells is that they have telomerase. ES cells have almost unlimited cell division ability in vitro due to the presence of the enzyme exhibiting the activity of maintaining the telomere length of the chromosome. .

  The ES cell line produced in this way repeats proliferation and passage almost indefinitely while maintaining a normal karyotype, and has the ability to differentiate into various types of cells, that is, pluripotency. . For example, when ES cells are transplanted subcutaneously, intraperitoneally, in the testis, etc. of an animal individual, a tumor called a teratoma is formed. The tumor is a nerve cell, bone / chondrocyte, intestinal cell, muscle cell. Various types of cells / tissues are mixed together. Further, in the case of mice, all of the body can be obtained by injecting and transplanting ES cells into blastocyst stage embryos or collecting embryos aggregated with 8-cell stage embryos into the uterus of pseudopregnant mice. Alternatively, it is possible to produce offspring individuals, so-called chimeric mice, that contain differentiated cells derived from ES cells in a certain proportion in some organs / tissues. This technique is frequently used as a basic technique for producing a mouse individual in which a desired gene is artificially disrupted or modified, that is, a so-called knockout mouse.

  Furthermore, it is known that ES cells can be induced to differentiate into various cell types even in vitro culture. Although the detailed method differs depending on the cell type, differentiation is induced by agglutination of ES cells by suspension culture to form a cell mass in a pseudo-embryonic state, so-called embryoid body (hereinafter abbreviated as EB). The method is commonly used. In this way, cells with fetal embryonic endoderm, ectoderm, and mesoderm properties, blood cells, vascular endothelial cells, chondrocytes, skeletal muscle cells, smooth muscle cells, cardiomyocytes, glial cells, nerves Differentiated cells such as cells, epithelial cells, melanocytes, keratinocytes, and adipocytes can be prepared. Differentiated cells produced in this way under in vitro culture have structurally and functionally almost the same traits as cells present in organs and tissues. By transplantation experiments using experimental animals, It has been shown that ES cell-derived cells settle in organs and tissues and function normally.

  As described above, as a review on the characteristics and culture methods of ES cells and their in vivo / in vitro differentiation ability, a plurality of reference books such as Guide to Techniques in Mouse Development (Wasserman et al., Academic Press, 1993); Cell Differentiation in Vitro (MV Wiles, Meth. Enzymol. 225: 900, 1993); Manipulating the Mouse Embryo: A laborary manual 19 (Hogen et al., Cold non-patent literature). ); Embryo Reference can be made to nic Stem Cells (Turksen edited by Humana Press, 2002) (see Non-Patent Document 2).

  In addition, EG cells are obtained by using embryonic germ cells called primordial germ cells on leukemia inhibitory factor (Leukemia Inhibitory Factor) on feeder cells such as MEF cells and STO cells as in the case of ES cells. (Hereinafter abbreviated as LIF) and basic fibroblast growth factor (hereinafter referred to as bFGF / FGF-2) or forskolin (Matsui et al.,). Cell 70: 841, 1992; Koshimazu et al., Development 122: 1235, 1996). EG cells have properties very similar to ES cells and have been confirmed to have pluripotency (Thomson & Odorico, Trends Biotechnol. 18:53, 2000). Therefore, hereinafter, “ES cell” may include “EG cell”.

  In 1995, Thomson et al. Established ES cells from primates (rhesus monkeys) for the first time, and practical application of regenerative medicine using pluripotent stem cells has become a reality (US Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92: 7844, 1995). Subsequently, by a similar method, they succeeded in isolating and establishing ES cell lines from human early embryos (Science 282: 114, 1998). Since then, similar reports have been made by research groups in Australia and Singapore (Rebinoff et al., Nat. Biotech. 18: 399, 2000; International Publication No. 00/27995). The list of NIH) (http://stemcells.nih.gov/registry/index.asp) contains 20 or more human ES cell lines. Meanwhile, Garhart et al. Has successfully established a human EG cell line from human primordial germ cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998; US Pat. No. 6,090,622). ).

  When these pluripotent stem cells are used for the production of research materials or regenerative medicine products, it is indispensable to subculture them in a manner that retains the undifferentiated nature and high proliferation ability of the cells. In general, ES / EG cells can maintain the undifferentiation and proliferative ability of the cells by using MEF cells or similar cells (such as STO cells) as feeders. The presence of fetal bovine serum (hereinafter referred to as FBS) to be added to the culture medium is also important, and an FBS suitable for ES / EG cell culture is selected, and the FBS is usually added in an amount of 10 to 20%. It is important. Furthermore, in the case of ES / EG cells derived from mouse embryos, LIF has been identified as a factor that maintains the undifferentiated state (Smith & Hooper, Dev. Biol. 121: 1, 1987; Smith et al., Nature). 336: 688, 1988; Rathjen et al., Genes Dev. 4: 2308, 1990), and by adding LIF to the culture medium, the undifferentiated state can be more effectively maintained (as a reference book) , Manipulating the Mouse Embryo: A laboratory manual (Hogan et al., Cold Spring Harbor Laboratory Press, 1994 (Non-patent Document 1), Embronic Stem Ce. ls (Turksen ed., Humana Press, 2002) see (Non-Patent Document 2)).

  However, these classical ES / EG cell culture methods are suitable methods, particularly when human ES (or EG) cells are used for regenerative medicine or other practical purposes. Absent. The reason for this is that, firstly, human ES cells do not have reactivity with LIF, and in the absence of feeder cells, the cells die or become undifferentiated and differentiate into heterologous cells ( Thomson et al., Science 282: 114, 1998). In addition, there is a problem in the use of feeder cells themselves, and such a co-culture system increases the production cost and makes it difficult to expand the culture scale. Moreover, when ES cells are actually used, the ES cells are removed from the feeder cells. It becomes necessary to separate and purify. In the future, when pluripotent stem cells such as human ES cells are used as a cell source for regenerative medicine, particularly cell transplantation treatment, cells and products derived from non-human animals such as MEF cells and FBS The use is not preferred because ES cells may be infected with a virus derived from a heterologous animal or contaminated with an antigen molecule that can be recognized as a heterologous antigen (Martin et al., Nature Med. 11: 228, 2005). .

  Therefore, in order to improve the ES / EG cell culture method to a more refined method suitable for future practical use, development of an FBS alternative (International Publication No. WO98 / 30679) and human instead of MEF cell Attempts to use cells as feeders (Richards et al., Nature Biotech. 20: 933, 2002; Cheng et al., Stem Cells 21: 131, 2003; Hovatta et al., Human Reprod. 18: 1404, 2003; Amit et al., Biol.Reprod. 68: 2150, 2003).

  In addition, the development of culture methods that do not use feeders is also remarkable. Carpenter and coworkers seeded ES cells in a culture plate coated with matrigel or laminin, and added the culture supernatant of MEF cells to the culture medium to undifferentiate human ES cells. It has been reported that long-term culturing can be performed while maintaining pluripotency (Xu et al., Nature Biotech. 19: 971, 2001 (Non-patent Document 3); International Publication No. 01/51616 (Patent Document 1) reference). Furthermore, the group succeeded in constructing a more effective ES cell culture system by developing a serum-free medium supplemented with bFGF / FGF-2 and stem cell growth factor (hereinafter referred to as SCF) ( International Publication No. 03/020920 (Patent Document 2)). A feeder-free ES cell culture system using a similar serum-free medium has also been reported by a research group in Israel (see Amit et al., Biol. Reprod. 70: 837, 2004 (Non-patent Document 4)). ).

  Recently, a method for maintaining undifferentiation of human ES cells by adding bFGF / FGF-2 and Noggin, which is a bone morphogenetic protein antagonist, has also been reported (Xu). et al., Nature Methods 2: 185, 2005). On the other hand, by adding a Glycogen Synthase Kinase (GSK) -3 inhibitor to the culture solution, it is possible to detect mouse and human ES cells without using feeder cells, especially without adding growth factors. It has also been shown that undifferentiation can be efficiently maintained (see Sato et al., Nature Med. 10:55, 2004 (Non-Patent Document 5)).

  As described above, new methods are being devised for culturing pluripotent stem cells that do not use feeder cells. For practical use and industrial use of the cells, the proliferation effect of pluripotent stem cells and It is necessary to further enhance the convenience of the culture method.

  The above-mentioned LIF is known as a factor that maintains the undifferentiated state of mouse ES / EG cells and enhances the proliferation ability thereof, and IL-6 family molecules similar to LIF are also included in the category (Yoshida et al. al., Mech. Dev. 45: 163, 1994; Koshimizu et al., Development 122: 1235, 1996), but few other examples have been reported. Recently, it has been reported that a serum-free medium supplemented with bFGF / FGF-2 or SCF significantly enhances the proliferation ability of human ES cells (see International Publication No. 03/020920 (Patent Document 2)). .

  In the past, the characteristics of ES cells, that is, the proliferative potential of the cells is vigorous compared to other cells, so there are few attempts to obtain knowledge about the proliferative ability. There is a need to increase proliferation.

  One of the problems related to the culture of pluripotent stem cells at present is that the cells generally form a solid colony and are difficult to handle during subculture. Undifferentiated ES / EG cells usually exhibit a state in which the cells are strongly adhered to each other, and form cell clumps, that is, colonies such that the boundaries of each cell become unclear. Therefore, when subjected to experiments such as subculture of ES / EG cells or differentiation induction, it is necessary to disperse colonies in as short a time as possible by treating with a proteolytic enzyme solution such as trypsin. However, in that case, in order to disperse the colonies of ES / EG cells into single cells, it is necessary to apply a relatively high concentration of proteolytic enzyme treatment and / or vigorous mechanical agitation. It significantly reduces the viability and engraftment of ES / EG cells.

  Furthermore, ES / EG cells will spontaneously differentiate if they continue to be cultured in a dense state, so they are dispersed until they become single cells at the time of passage, and colonies grow to an excessive size. It is necessary to perform the passage before. For example, in the case of mouse ES cells, it is usually necessary to pass every 2 to 3 days. However, if passage is not performed by an appropriate method, cells that have deviated from an undifferentiated state appear in the population. Thus, the cells become unsuitable for use. This cannot be solved simply by adding a sufficient amount of a factor that maintains the undifferentiation of ES / EG cells, such as the above-mentioned LIF and GSK-3 inhibitors, and excessive colony growth has a differentiation trait. Induce the appearance of damaged cells. Therefore, the method of growing in a state where ES / EG cells do not form colonies, that is, in a state where the cells are dispersed one by one, is considered to be extremely useful when the ES / EG cells are used for industrial use. . However, there has been no such attempt and / or success.

In recent years, so-called induced pluripotent stem cells (iPS cells, induced pluripotent stem cells) that can be prepared from skin and organ cells without destroying the embryo have been prepared (patents). Document 3, Patent Document 4, Patent Document 5).
iPS cells have been established in mice and humans. Because it can be obtained without the ethical problem of embryo destruction, and human iPS cells can be differentiated into cells of each tissue after being prepared using patient-derived cells to be treated In particular, in the field of regenerative medicine, it is expected as a transplant material without rejection. iPS cells have the same properties as ES cells and have the same problems as ES cells described above.

  Previously, the inventors used a culture plate coated with E-cadherin (Nagaoka et al., Biotechnol), which is a type of embryonal carcinoma cell and is known to grow normally in colonies. Lett. 16, Taipei, Taiwan; 1st Japan Society for Regenerative Medicine, 2002.18-18., Kyoto, Japan). That is, on a culture plate (hereinafter referred to as E-cad plate) in which E-cadherin known as a cell adhesion molecule of F9 cells is solid-phased on an untreated polystyrene culture plate, the F9 cells exhibit a dispersed cell form. did.

  F9 cells express traits similar to those of ES cells, such as expressing alkaline phosphatase (hereinafter referred to as ALP), SSEA-1, Oct-3 / 4, etc., which are known as specific markers of ES / EG cells. (Lehtonen et al., Int. J. Dev. Biol. 33: 105, 1989, Alonso et al., 35: 389, 1991). However, in the case of F9 cells, feeder cells, LIF, and the like are not required to maintain the undifferentiated nature of the cells, and there are differences in their undifferentiated maintenance mechanisms. In addition, ES cells have the ability to differentiate into three germ layers, whereas F9 cells are limited to endoderm cells and do not have chimera-forming ability. That is, F9 cells may be used as a model system for ES / EG cells in certain experiments, but there are many differences from ES / EG cells with respect to culture methods and culture conditions.

  Therefore, the E-cad plate can be applied to an ES cell culture method that does not require feeder cells, or the ES cells cultured by such a method retain undifferentiation and pluripotency. Whether it could be subcultured or further promote the proliferation ability of the ES cells could not be predicted based on scientific evidence. In fact, the proliferative ability of F9 cells cultured on an E-cad plate is almost the same as that of F9 cells cultured on a conventional cell culture plate, and the fact that analogy can promote the proliferative ability of ES cells is obtained. There wasn't.

  E-cadherin is known to be expressed in undifferentiated mouse ES cells, and in ES cells in which the expression of E-cadherin gene has been deleted by genetic engineering, its cell-cell adhesion is significantly inhibited. (Larue et al., Development 122: 3185, 1996). However, there has been no attempt to use E-cadherin as an adhesion substrate in the ES / EG cell culture method.

  In addition to the above efficient proliferation method, when pluripotent stem cells such as ES cells are used for the production of research materials or regenerative medical products, the desired foreign gene can be efficiently transferred to the cells. There is also a need for construction of methods for introduction and expression in cells. In particular, in order to apply ES cells to regenerative medicine such as treatment of various diseases, it is considered as one means to change cell characteristics such as proliferation ability and differentiation ability of the cells, or drug sensitivity. For realization, a method of introducing an appropriate foreign gene into a cell and expressing it is preferable. In addition, in the case of mouse ES cells, it is widely known that so-called transgenic mice and knockout mice can be prepared by artificially modifying the gene. In this case, an efficient gene transfer method is extremely useful. is there.

  In general, when a foreign gene is introduced into a cell, a method using calcium phosphate, DEAE-dextran, or a cationic lipid preparation is frequently used. However, when these methods are applied to ES cells, it is known that the efficiency thereof is low compared to other cell lines (Lakshmipathy et al., Stem Cells 22: 531, 2004 (Non-patent Document 8)). reference). In addition, methods using various viral vectors have been reported as methods for introducing foreign genes. For example, retrovirus vectors (Cherry et al., Mol. Cell Biol., 20: 7419, 2000), adenovirus vectors (Smith-Arica et al., Cloning Stem Cells 5:51, 2003), lentivirus vector (Hamaguchi et al. J. Virol. 74: 10778, 2000; Asano et al., Mol. Ther. 6: 162, 2002; International Publication No. WO02 / 101057), Sendai virus vector (Sasaki et al., Gene). Ther., 12: 203, 2005; Japanese Patent Publication No. 2004-344001) and the like are known. However, the construction and production of viral vectors require relatively complicated and long-term work, and depending on the virus used, there are concerns about biological safety problems, and this is not a simple and versatile method.

Therefore, when introducing a foreign gene into ES cells, a method called electroporation (electroporation) is frequently used. This method is highly versatile because it is a method in which pores are transiently opened in a cell membrane by applying an electric pulse to the cell and a foreign gene is introduced into the cell. Recently, as a method for improving the method, a method called Nucleofection has been established which introduces a foreign gene into the cell nucleus and significantly improves its expression efficiency (Lorenz et al., Biotech. Lett. 26: 1589, 2004; Lakshmipathy et al., Stem Cells 22: 531, 2004 (non-patent document 8)). However, these methods require a special electric pulse generator, and it is difficult to set optimum conditions. In addition, the cells need to be once treated with a protease such as trypsin and dispersed in a single cell, and the cytotoxicity is relatively high.
Therefore, in the gene transfer method for pluripotent stem cells including ES cells, a gene transfer agent that can be purchased at low cost or can be easily prepared is used, and the cells are efficiently transferred in a state where the cells are cultured on the incubator. A method that can introduce a gene is considered to be very useful.

International Publication No. 2001/51616 International Publication No. 2001/51616 International Publication No. 2007/069666 International Publication No. 2009/057831 International Publication No. 2009/075119

Manipulating the Mouse Embryo: A laboratory manual (Hogan et al., Cold Spring Harbor Laboratory Press, 1994 Embryonic Stem Cells (Turksen, HumanaPress, 2002) Xu et al., Nature Biotech. 19: 971,2001 Amit et al., Biol.Reprod. 70: 837,2004 Sato et al., Nature Med. 10: 55,2004 Nagaoka et al., Biotechnol. Lett. 24: 1857,2002 Nagaoka et al., Protein Eng. 16: 243,2003 Lakshmipathy et al., Stem Cells 22: 531,2004

In order to use pluripotent cells such as ES cells and iPS cells as described above as a transplant material without rejection, it is necessary to differentiate into specific cells. In order to differentiate a pluripotent cell into an arbitrary cell, not only the efficiency of differentiation induction but also uniformity of the cell after differentiation induction is required. That is, after differentiation induction, it is not preferable that cells other than the target cells, such as differentiated cells and undifferentiated cells, are mixed in to avoid the risk of canceration after transplantation.
For example, in the conventional differentiation induction methods including the formation of embryoid bodies and the use of feeder cells, cell colonies are formed, in which cells of all three germ layers are induced. As a result, there is a problem that only the target cells cannot be obtained with high purity.

  Therefore, an object of the present invention is to cultivate pluripotent stem cells without using feeder cells, and to differentiate pluripotent stem cells into specific cells with high purity. The object is to provide a cell culture substrate that can also be used for selection, a cell culture method using the same, and a method for inducing differentiation of pluripotent stem cells.

  As a result of intensive studies, the present inventors have found that the above-mentioned problems can be solved by using a cell culture substrate in which a specific protein and polymer are fixed or coated on the surface, and the present invention has been completed. .

  That is, the present invention relates to the following [1] to [11] relating to a cell culture substrate, a cell culture method using the same, and a differentiation induction method for pluripotent stem cells.

[1] A cell culture medium characterized by fixing or coating a surface of a protein belonging to the cadherin family or a fusion protein including all or part of a protein belonging to the cadherin family, and a polymer having a sugar side chain Wood.
[2] The cell culture substrate according to [1], wherein the sugar side chain is lactose or galactose.
[3] The cell culture substrate according to [1] or [2], wherein the polymer having a sugar side chain is poly (pN-vinylbenzyl-D-lactone amide).
[4] The protein belonging to the cadherin family is E-cadherin or a protein having homology with E-cadherin, and one or more of EC1 domain, EC2 domain, EC3 domain, EC4 domain and EC5 domain The cell culture substrate according to any one of [1] to [3], which is a protein containing and having a binding affinity with the same affinity as E-cadherin.
[5] The cell culture according to any one of [1] to [3], wherein the fusion protein containing all or part of the protein belonging to the cadherin family is a fusion protein of E-cadherin and an Fc region of an immunoglobulin. Base material.
[6] The protein belonging to the cadherin family or a fusion protein including all or a part of the protein belonging to the cadherin family and a polymer having a sugar side chain form a sea-island structure [1] to [5] Any cell culture substrate.
[7] Proliferating pluripotent stem cells using the cell culture substrate according to any one of [1] to [6] and a liquid medium while maintaining undifferentiation and differentiation pluripotency Cell culture method.
[8] Pluripotent stem cell differentiation characterized in that pluripotent stem cells are differentiated using the cell culture substrate according to any one of [1] to [6] and a liquid medium containing a differentiation-inducing factor. Guidance method.
[9] The method for inducing differentiation of pluripotent stem cells according to [8], wherein pluripotent stem cells are differentiated into hepatocytes.
[10] Using the cell culture substrate according to any one of [1] to [6] and a liquid medium containing a differentiation-inducing factor, pluripotent stem cells are differentiated into hepatocytes, and the liver is transformed from the cell culture substrate. A cell culture method comprising separating cells, seeding and culturing on a cell culture substrate fixed or coated with a polymer containing galactose or a lactose side chain on the surface.
[11] The cell culture method according to [10], wherein the polymer containing galactose or a lactose side chain is poly (p-N-vinylbenzyl-D-lactone amide).

  According to the present invention, pluripotent stem cells can be cultured without using feeder cells, and pluripotent stem cells can be differentiated into specific cells with high purity. It is possible to provide a cell culture substrate that can be used, a cell culture method using the same, and a method for inducing differentiation of pluripotent stem cells.

It is a schematic diagram showing the preparation method of the co-immobilization base layer of PVLA and E-cad-Fc, and the differentiation induction method to the hepatocyte of the ES cell on a co-immobilization base layer. (A) Represents the molecular formula of PVLA. Galactose residues bind to ASGPR on hepatocyte cell membranes. (B) It is a schematic diagram showing the co-immobilization method of PVLA and E-cad-Fc to the polystyrene surface. (C) Schematic diagram showing the step of inducing differentiation of mouse ES cells into hepatocytes. The differentiation state can be divided into four stages. Endoderm mesoderm, definitive endoderm, hepatic progenitor cells, hepatocytes. The number of days after the start of differentiation induction, the medium used, and the growth factors are as described in the upper table. The enlarged portions are schematic views showing the state of cell adhesion to the co-immobilized base layer of E-cad-Fc and PVLA and the PVLA single coat base layer, respectively. It is a graph showing the change of the adsorption amount of PVLA and E-cad-Fc on the polystyrene surface, (A) is PVLA, (B) is an adsorption isotherm prepared from the Langmuir adsorption equation of E-cad-Fc. is there. (C) is a graph showing co-adsorption of E-cad-Fc and PVLA. 100 μg / mL PVLA (slowest rising graph) is a graph showing the adsorption of 100 μg / mL PVLA alone, and 10 μg / mL E-cad-Fc (second rising graph) is 10 μg / mL E -A graph showing adsorption of cad-Fc alone. 7.5 μg / mL E-cad-Fc then 100 μg / mL PVLA (the fastest rising graph) adsorbs 7.5 μg / mL E-cad-Fc and then adsorbs 100 μg / mL PVLA. It is a graph when. Each was washed 3 times after adsorption, indicating that it was adsorbed stably after washing. (A) It is a photograph figure which shows culture | cultivation on various base layers of a primary culture hepatocyte. From the top, the photograph was cultured on gelatin base layer, collagen base layer, E-cad-Fc single base layer, PVLA single base layer, PVLA and E-cad-Fc co-immobilized base layer (1 day, 3 days, 5 days, 7 days A day later), a fluorescence micrograph of primary cultured hepatocytes. Each photograph in the column labeled ALB shows the results of immunostaining with anti-albumin antibody. Each of the photographs in the row labeled “Merged” is a merge of nuclear staining with DAPI and the above immunostaining. (B) It is a photograph figure showing the result of the Western blot using an anti-albumin antibody. Anti-β-actin antibody was used as a control. In each day (Day1, Day3, Day5, Day7), the lane labeled G is gelatin base layer, E is E-cad-Fc single base layer, P is PVLA single base layer, EP is E-cad-Fc and PVLA. Represents cells cultured on co-immobilized substrate. (C) Agarose electrophoresis photograph showing the results of RT-PCR in which the gene expression of albumin (ALB), HNF-4α, ASGPR, mTO, G6P, and E-cadherin was examined. The notation of each lane is the same as (B) above. (D) It is a graph which shows the result of the albumin ELISA assay performed in order to investigate the quantity of the albumin secreted in the culture medium. Each graph is normalized by the number of cells. (E) It is a graph which shows the result of PAS reaction performed in order to investigate glycogen accumulation amount. Each graph is normalized by the number of cells. (A) Photomicrograph of undifferentiated mouse ES cells (EB3) cultured for 2 days on gelatin base layer, E-cad-Fc single base layer, PVLA single base layer, co-immobilized base layer of E-cad-FC and PVLA. (B) It is a graph which shows the change of the cell number for every culture | cultivation time of an undifferentiated cell on a gelatin base layer, an E-cad-Fc independent base layer, and a co-immobilization base layer of E-cad-Fc and PVLA. (A) RT-PCR results of examining the gene expression levels of undifferentiated marker (Oct3 / 4), mesoderm marker (Bra), endoderm marker (Gata6, Sox17), and ectoderm marker (Pax6, TH) It is a photograph figure of agarose electrophoresis which shows. The lane labeled G represents a gelatin base layer, E represents an E-cad-Fc single base layer, P represents a PVLA single base layer, and EP represents cells cultured on a co-immobilized base layer of E-cad-Fc and PVLA. The lane labeled ES indicates an undifferentiated ES cell, Day 4 indicates that 4 days after the start of differentiation induction, and Day 6 indicates that 6 days later. PH is a lane of early hepatocytes and is a control. (B) It is a graph which shows what processed the result of RT-PCR of Sox17 gene using ImageQuant software, and digitized the relative expression level. The results of primary cultured hepatocytes cultured for 1 day on a gelatin base layer were used as controls. (C) Micrograph showing the results of immunostaining using anti-Sox17 antibody and anti-E-cadherin antibody. The cells were cultured on the gelatin base layer, the E-cad-Fc single base layer, and the co-immobilized base layer of E-cad-Fc and PVLA on the fifth day from the start of differentiation induction. (D) A graph showing the result of examining the proportion of cells expressing each protein by immunostaining using anti-Sox17 antibody and anti-Oct3 / 4 antibody using cells on the fifth day after initiation of differentiation induction FIG. For each antibody, the left bar represents cells cultured on the E-cad-Fc and PVLA co-immobilized base layer, and the right bar represents cells cultured on the gelatin base layer. (A) It is a photograph of the agarose electrophoresis which shows the result of RT-PCR performed about the hepatic progenitor cell marker gene (AFP, ALB, CK18, HNF-4 (alpha)). β-actin was used as a control. In each day (Day 4 to Day 18), the lane labeled G is a gelatin base layer, E is an E-cad-Fc single base layer, EP is a cell cultured on a co-immobilized base layer of E-cad-Fc and PVLA. Represent. (B) It is a graph which shows what processed the result of RT-PCR of the AFP gene using ImageQuant software, and digitized the relative expression level. Normalization was performed using the results of β-actin. Primary cultured hepatocytes (PH), undifferentiated ES cells (ES), and spontaneously differentiated cells (SD) were used as controls. (C) It is a graph which shows what processed the result of RT-PCR of ALB gene using ImageQuant software, and digitized the relative expression level. Normalization was performed using the results of β-actin. Primary cultured hepatocytes (PH), undifferentiated ES cells (ES), and spontaneously differentiated cells (SD) were used as controls. (D) It is a graph which shows what processed the result of RT-PCR of CK18 gene using ImageQuant software, and digitized the relative expression level. Normalization was performed using the results of β-actin. Primary cultured hepatocytes (PH), undifferentiated ES cells (ES), and spontaneously differentiated cells (SD) were used as controls. (E) It is a graph which shows what processed the result of RT-PCR of HNF-4 (alpha) gene using ImageQuant software, and digitized the relative expression level. Normalization was performed using the results of β-actin. Primary cultured hepatocytes (PH), undifferentiated ES cells (ES), and spontaneously differentiated cells (SD) were used as controls. Primary cultured hepatocytes (PH), undifferentiated ES cells (ES), and spontaneously differentiated cells (SD) were used as controls. (F) Fluorescence microscope photograph showing the results of immunostaining for cells on day 10 of differentiation induction using an anti-AFP antibody. (G) It is a photograph of the fluorescence microscope which shows the result of having carried out the immuno-staining using the anti- ALB antibody about the cell on the 16th day from the start of differentiation induction. (H) It is a graph which shows the result of having investigated the ratio of the cell which performed immunostaining using the anti- AFP antibody and the anti- ALB antibody, and expressed each protein. For each antibody, the left bar represents cells cultured on the E-cad-Fc and PVLA co-immobilized base layer, and the right bar represents cells cultured on the gelatin base layer. The immunostaining with anti-AFP antibody used cells on the 10th day from the start of differentiation induction, and the immunostaining with anti-ALB antibody used cells on the 16th day from the start of differentiation induction. (I) It is a photograph figure which shows the result of the Western blot performed using the anti-albumin antibody about the cell on the 16th day from the start of differentiation induction. β-actin was used as a control. The lane labeled G represents a gelatin base layer, E represents an E-cad-Fc single base layer, and EP represents cells cultured on a co-immobilized base layer of E-cad-Fc and PVLA. (A) Photograph of agarose electrophoresis showing the results of RT-PCR performed on the early hepatocyte differentiation marker (AFP, ALB, CK18, HNF-4α) gene and the final stage marker of hepatocyte differentiation (ASGPR, mTO) gene FIG. β-actin was used as a control. (B) It is a graph which shows what processed the result of RT-PCR of the ASGPR gene using ImageQuant software, and digitized the relative expression level. Normalization was performed using the results of β-actin. Primary cultured hepatocytes (PH), undifferentiated ES cells (ES), and spontaneously differentiated cells (SD) were used as controls. (C) It is a graph which shows what processed the result of RT-PCR of mTO gene using ImageQuant software, and digitized the relative expression level. Normalization was performed using the results of β-actin. Primary cultured hepatocytes (PH), undifferentiated ES cells (ES), and spontaneously differentiated cells (SD) were used as controls. (D) Micrograph showing the results of immunostaining using anti-ALB antibody and anti-ASGPR antibody. The cells were cultured on a gelatin base layer, an E-cad-Fc single base layer, and a co-immobilized base layer of E-cad-Fc and PVLA, respectively, on the 24th day from the start of differentiation induction. (E) It is a graph which shows the result of the albumin ELISA assay performed in order to investigate the quantity of the albumin secreted in the culture medium. Each graph is normalized by the number of cells. Cells cultured on the gelatin base layer, the E-cad-Fc single base layer, and the co-immobilized base layer of E-cad-Fc and PVLA on the 20th day (day 20) and 24th day (day 24) of differentiation induction were used. Primary cultured hepatocytes cultured for 3 days on a PVLA single substrate were used as controls. (F) It is a graph which shows the result of PAS reaction performed in order to investigate glycogen accumulation amount. Each graph is normalized by the number of cells. Cells cultured on the gelatin base layer, the E-cad-Fc single base layer, and the co-immobilized base layer of E-cad-Fc and PVLA on the 20th day (day 20) and 24th day (day 24) of differentiation induction were used. Primary cultured hepatocytes cultured for 3 days on a PVLA single substrate were used as controls. (G) It is a graph which shows the result of having measured the urea level in a culture medium using the Urea assay kit (made by Bioassay Systems). Each graph is normalized by the number of cells. Cells cultured on the gelatin base layer, the E-cad-Fc single base layer, and the co-immobilized base layer of E-cad-Fc and PVLA on the 20th day (day 20) and 24th day (day 24) of differentiation induction were used. Primary cultured hepatocytes cultured for 3 days on a PVLA single substrate were used as controls. (A) For cells cultured on gelatin base layer, E-cad-Fc single base layer, E-cad-Fc and PVLA co-immobilized base layer, differentiation induction on the 18th, 20th, 22nd, 24th day, It is a graph showing the result of separating the differentiated cells using a chelating agent (CDB), seeding them on a PVLA-coated culture dish, conducting a binding assay, and calculating the relative binding rate of the cells. The primary cultured hepatocytes cultured on the collagen base layer were separated and seeded on a PVLA-coated culture dish, and the result of the binding assay was used as a control, and the binding rate was 100% (C). As a control, cells obtained by culturing primary cultured hepatocytes on a PVLA single substrate (P) were used. (B) Mouse ES cells differentiated on a co-immobilized substratum of E-cad-Fc and PVLA on a gelatin substratum, separated after differentiation induction culture on a co-immobilized substratum of E-cad-Fc and PVLA, It is the microscope picture figure which shows the cell which seed | inoculated on the culture dish coated with PVLA, and was cultured for 3 days, and each form. (C) Agarose electrophoresis showing the results of RT-PCR performed on mesoderm markers (Bra), endoderm markers (Gata6, Sox17), hepatocyte marker (ASGPR, mTO, G6P), pancreatic marker (Pdx1) genes FIG. β-actin was used as a control. The lane labeled G represents a gelatin base layer, E represents an E-cad-Fc single base layer, P represents a PVLA single base layer, and EP represents cells cultured on a co-immobilized base layer of E-cad-Fc and PVLA. Before represents differentiation-incubated culture for 24 days on each substratum, After represents 22-day differentiation-inducing culture on co-immobilized substratum of E-cad-Fc and PVLA, separated, and re-applied to PVLA single substratum. Represents cells seeded and cultured for 3 days. Mouse ES cells (ES) maintained on the gelatin base layer and primary cultured hepatocytes (PH) cultured on the PVLA base layer for 1 day were used as controls. (D) It is a graph which shows the result of the albumin ELISA assay performed in order to investigate the quantity of the albumin secreted in the culture medium. Each graph is normalized by the number of cells. After differentiation induction culture on E-cad-Fc and PVLA co-immobilized substratum for 22 days, the cells were separated, re-seeded on PVLA single substratum, and cultured for three days (Re-seeding on PVLA) on PVLA single substratum Primary cultured hepatocytes cultured for 3 days were used. (E) It is a graph which shows the result of PAS reaction performed in order to investigate glycogen accumulation amount. After differentiation induction culture on E-cad-Fc and PVLA co-immobilized substratum for 22 days, the cells were separated, re-seeded on PVLA single substratum, and cultured for three days (Re-seeding on PVLA) on PVLA single substratum Primary cultured hepatocytes cultured for 3 days were used. The graph represents a relative value with the value of Re-seeding on PVLA being 100. (F) It is a graph which shows the result of having measured the urea level in a culture medium using the Urea assay kit (made by Bioassay Systems). Each graph is normalized by the number of cells. After differentiation induction culture on E-cad-Fc and PVLA co-immobilized substratum for 22 days, the cells were separated, re-seeded on PVLA single substratum, and cultured for three days (Re-seeding on PVLA) on PVLA single substratum Primary cultured hepatocytes cultured for 3 days were used.

[Definition]
As used herein, the term “pluripotent stem cell” refers to a normal nucleus (chromosome) that is capable of almost permanent or long-term cell growth while maintaining an undifferentiated state by in vitro culture. ) Refers to cells that have the ability to differentiate into cells of all three germ lines (ectodermal, mesoderm, and endoderm) under appropriate conditions. “Pluripotent stem cells” are ES cells, iPS cells and similar cells isolated from early embryos, including but not limited to EG cells isolated from embryonic primordial germ cells. In the present specification, the expression “ES cell” may also include “EG cell”.

  As used herein, the term “undifferentiated” refers to the expression of at least one or more undifferentiated markers, such as ALP activity or Oct-3 / 4 gene (product), in pluripotent stem cells, or It means the property of exhibiting an undifferentiated state that can be confirmed by the expression of various antigen molecules. 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 all three germ layers under appropriate conditions It means a state with the ability to differentiate into cells of the genealogy.

  As used herein, the term “pluripotency” refers to the ability to differentiate into various types of cells. The differentiated cell is not particularly limited as long as it is a kind of cell that can generally be induced to differentiate from a pluripotent stem cell. Specific examples include ectoderm cells or ectoderm-derived cells, mesoderm cells or mesoderm-derived cells, endoderm cells or endoderm-derived cells, and the like.

  As used herein, the term “liquid medium” includes any liquid medium that can be applied to conventional methods of subculturing pluripotent stem cells.

  As the culture substrate of the present invention, any of those conventionally used for cell culture, such as culture dishes (dish), microplates such as 96 holes and 48 holes, plates and flasks, can be used. These culture substrates can be made of either an inorganic material such as glass or an organic material such as polystyrene or polypropylene, but those made of a material having heat resistance and water resistance that can be sterilized are preferred. .

  As a method for immobilizing or coating a protein belonging to the cadherin family on the solid phase surface of a culture substrate, a physical method such as adsorption or a chemical method such as covalent bonding can be applied. The method by adsorption is preferred. Adsorption can be achieved by bringing a solution containing a protein belonging to the cadherin family into contact with the substrate surface for a certain period of time, preferably several hours to one day and more preferably 1 hour to 12 hours. Alternatively, an antigenic molecule can be artificially added to and fused with an adhesive molecule in advance, and the binding with a specific antibody against the antigenic molecule can be used. In this case, the specific antibody needs to be fixed or coated in advance on the solid phase surface of the culture substrate by a physical method such as adsorption or a chemical method such as covalent bonding.

  The culture substrate produced as described above can be used as it is in a normal culture method for pluripotent stem cells. That is, an appropriate number of pluripotent stem cells may be suspended in a commonly used liquid medium or cell culture solution and added to the culture substrate. Subsequent replacement and passage of the liquid medium can be performed as in the conventional method.

  As used herein, the term “homophilic binding” refers to the same type of adhesion molecules in cell adhesion, in cell-cell or cell-substrate bonding via adhesion molecules. Is made by binding or associating.

  As used herein, the term “feeder cell” is also referred to as a feeder cell. Another cell that plays a role such as replenishment. “Feeder cells” include, but are not limited to, stromal cells such as MEF cells and STO cells.

  As used herein, the term “dispersed state” means that when a cell grows in a state of adhering to the surface of a culture substrate, a distinct colony is not formed, and each cell is a cell other than the other. It means that the contact area is very small even if it is not in contact with or partly in contact.

  As used herein, the term “gene” refers to genetic material and refers to a nucleic acid comprising a transcription unit. The gene may be RNA or DNA, and may be a naturally-derived or artificially designed sequence. The gene may not encode a protein. For example, the gene may encode a functional RNA such as a ribozyme or siRNA (short / small interfering RNA).

  The polymer having a sugar side chain is preferably a polymer having a main chain obtained by polymerizing an ethylenically unsaturated bond and a sugar side chain. Examples of the sugar side chain include glucose, galactose, lactose and the like, but lactose or galactose is preferable. This is because β-galactose and β-galactose side chains in lactose are specifically recognized by asialoglycoprotein receptors and can contribute to cell binding. The polymer having a sugar side chain is preferably a styrene derivative polymer, and most preferably poly (p-N-vinylbenzyl-D-lactone amide) (hereinafter also referred to as “PVLA”).

  As a method for immobilizing or coating a polymer having a sugar side chain on the solid phase surface of a culture substrate, a physical method such as adsorption or a chemical method such as covalent bonding can be applied. Therefore, the method by adsorption is preferable. Adsorption can be achieved by bringing a solution containing a polymer having a sugar side chain into contact with the substrate surface for a certain period of time, preferably several hours to one day and more preferably 1 hour to 12 hours. Alternatively, an antigenic molecule can be artificially added to and fused with a polymer having a sugar side chain in advance, and a bond with a specific antibody against the antigenic molecule can be used. In this case, the specific antibody needs to be fixed or coated in advance on the solid phase surface of the culture substrate by a physical method such as adsorption or a chemical method such as covalent bonding.

  The above advantages and other advantages and features of the present invention are described in detail in the detailed description of the preferred embodiments below.

[Cell culture substrate]
The cell culture substrate of the present invention is obtained by immobilizing or coating a surface of a protein belonging to the cadherin family or a fusion protein including all or part of the protein belonging to the cadherin family, and a polymer having a sugar side chain. It is a feature.

Cadherin is an adhesion molecule involved in Ca ²⁺ -dependent cell-cell adhesion / bonding called adhesion bond or adherence junction, and is an E (epithelial) type, N (neural) type, P (placenta) Three types of molds are known as representative examples. These cadherin molecules are membrane-bound glycoprotein molecules consisting of 700 to 750 amino acid residues, and in the extracellular region thereof, a repetitive structure consisting of about 110 amino acid residues, a region called an extracellular cadherin (EC) domain There are five. For example, in the case of human E-cadherin (the amino acid sequence of which is shown in SEQ ID NO: 1), the domains of EC1, EC2, EC3, EC4, and EC5 are 157 to 262, 265 to 375, 378 to 486, and 487 to 595, respectively. Corresponds to 596-700 (the numerical value is the number of the residue in the amino acid sequence shown in SEQ ID NO: 1). In the case of mouse E-cadherin (the amino acid sequence of which is shown in SEQ ID NO: 2), the domains of EC1, EC2, EC3, EC4, and EC5 are 159 to 264, 267 to 377, 380 to 488, and 489 to 597, respectively. Corresponds to 598 to 702 (the numerical value is the number of the residue in the amino acid sequence shown in SEQ ID NO: 2). These EC domains have homology among different cadherin species, and in particular, the domains located on the N-terminal side (EC1, EC2) have high homology. As cadherin molecules having such a similar structure, at least 50 kinds of cadherin molecules are currently known, and these molecules are collectively referred to as a cadherin family. As described above, reviews on cadherins include Takeichi, Curr. Opin. Cell Biol. 7: 619, 1995; Marrs & Nelson, Int. Rev. Cytol. 165: 159, 1996; Yap et al. , Annu. Rev. Cell Dev. Biol. 13: 119, 1997; Yagi & Takeichi, Genes Dev. 14: 1169, 2000; Gumbiner, J. et al. Cell Biol. 148: 399, 2000, etc.

  E-cadherin (also known as cadherin-1) is widely expressed in parenchymal cells of visceral organs such as liver, kidney, and lung, and epithelial cells such as keratinocytes, and is known to be an important adhesion molecule responsible for cell-cell adhesion. (Reviewed: Mariel et al., Int. J. Dev. Biol. 37: 227, 1993; Mays et al., Cord Spring Harb. Symp. Quant. Biol. 60: 763, 1995; El-Bahrawy & Pignatelli, Microsc. Res. Tech. 43: 224, 1998; Nolet et al., Mol. Cell. Biol. Res. Commun. 2: 77, 1999). E-cadherin is also strongly expressed in undifferentiated mouse ES cells, and ES cells in which expression of the E-cadherin gene has been genetically deleted may significantly inhibit intercellular adhesion. (Larue et al., Development 122: 3185, 1996). Moreover, it can confirm that the E-cadherin gene is also expressed in the human ES cell line from the information registered in the public gene expression database of National Center for Biotechnology Information (NCBI).

  A method for producing a protein belonging to the cadherin 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 can be used as long as it exhibits the same effect. Alternatively, the peptide can be chemically synthesized and used.

  Regarding a protein belonging to the cadherin family, a standard protocol has already been established for a method for producing a recombinant protein and a method for obtaining a gene encoding the molecule. Reference books can be referred to, but this is not particularly limited. In the case of E-cadherin as an example, the E-cadherin gene has already been isolated and identified in animals such as humans (SEQ ID NO: 1), mice (SEQ ID NO: 2), rats, etc. (Access number: (human) NM_004360; (mouse) NM_009864; (rat) NM_031334). Therefore, those skilled in the art can obtain and use cDNA of the E-cadherin gene by designing a primer or probe specific to the E-cadherin gene and using a general molecular biological technique. It is. The cDNA of the E-cadherin gene can also be purchased from RIKEN Gene Bank (Tsukuba, Japan), American Type Culture Collection (ATCC), Invitrogen / ResGen, and the like. The gene encoding a protein belonging to the cadherin family to be used is preferably derived from the same species of animal as the species from which the pluripotent stem cells are derived. For example, when the present invention is performed using mouse ES cells, E -The use of cadherin cDNA is desirable. However, also use E-cadherin cDNA derived from heterologous animals, ie, humans, monkeys, cows, horses, pigs, sheep, or birds (eg, chickens, etc.), or amphibians (eg, Xenopus, etc.). Can do.

  An example of a suitable method for producing a recombinant protein of a protein belonging to the cadherin family is to introduce a gene encoding the molecule into mammalian cells such as COS cells, 293 cells, and CHO cells for expression. It is a feature. Preferably, the gene is linked to a nucleic acid sequence that enables transcription and expression of the gene in a wide range of mammalian cells, a so-called promoter sequence, in a form that allows transcription and expression under the control of the promoter. . Moreover, it is desirable that the gene to be transcribed / expressed is further linked to a poly A addition signal. Suitable promoters include promoters derived from viruses such as SV (Simian Virus) 40 virus, cytomegalovirus (CMV), Rous sarcoma virus (Rus sarcoma virus), β-actin promoter, and EF (Elongation). Factor) 1α promoter and the like.

  The gene used to produce the above recombinant protein does not need to include the full length region of the gene encoding the molecule, and even if it is a partial gene sequence, the protein or peptide molecule encoded by the partial sequence However, what is necessary is just to have the same or more adhesive activity as the original molecule concerned. For example, in the case of E-cadherin used in a preferred case of the present invention, a recombinant protein produced from a partial sequence containing 690 to 710 amino acid residues from the N-terminal side encoding the extracellular region, that is, EC1 to EC5. Proteins containing the domain can be used. Further, in general, in the cadherin molecule, the domain (EC1) located on the most N-terminal side defines the binding specificity of the molecule, that is, homophilicity (Nose et al., Cell 61: 147, 1990), it is also possible to produce and use a protein molecule containing at least EC1 and excluding one or several other domains. Furthermore, a protein having an adhesion activity with the above-described protein molecule that is 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more at the amino acid level is used. can do.

  The above recombinant protein can also be prepared as a fusion protein with another protein or peptide. For example, immunoglobulin Fc region, GST (Glutathione-S-Transferase) protein, MBP (Mannose-Binding Protein) protein, avidin protein, His (oligo-histidine) tag, HA (HemAgglutinin) tag, Myc tag, VSV- Recombinant protein can be easily and efficiently purified by preparing a fusion protein with a G (Vesicular Stromatis Virus Glycoprotein) tag and using a protein A / G column, a specific antibody column, or the like. In particular, Fc fusion proteins are suitable in the practice of the present invention because their ability to adsorb to a culture substrate made of polystyrene or the like is increased.

  Many genes encoding immunoglobulin Fc regions have already been isolated and identified in mammals including humans. Numerous nucleotide sequences have been reported. For example, sequence information of nucleotide sequences including Fc regions of human IgG1, IgG2, IgG3, and IgG4 can be used in public DNA databases such as NCBI, and can be accessed respectively. Numbers: AJ294730, AJ294731, AJ294732, and AJ294733 are registered. Therefore, those skilled in the art can obtain and use cDNA encoding the Fc region portion by designing a primer or probe specific to the Fc region and using a general molecular biological technique. is there. In this case, the gene encoding the Fc region to be used is not particularly limited to animal species and subtypes, but Fc such as human IgG1 and IgG2 or mouse IgG2a and IgG2b having strong binding to protein A / G. A gene encoding the region is preferred. In addition, a method for enhancing the binding property to protein A by introducing a mutation into the Fc region is also known (see Nagaoka et al., Protein Eng. 16: 243, 2003 (Non-patent Document 7)). Fc proteins with genetic modifications can also be used.

  In the case of E-cadherin that is suitably used for carrying out the present invention, as an example of a method for producing the recombinant protein, there can be mentioned a paper already reported by the inventors (Nagaoka et al., Biotechnol. Lett. 24: 1857, 2002 (Non-Patent Document 6); Protein Eng. 16: 243, 2003 (Non-Patent Document 7)).

  In addition, a fusion gene in which a cDNA encoding the extracellular region of mouse or human E-cadherin and a cDNA encoding the Fc region of human IgG and a cDNA of His tag sequence are introduced into mouse cells, Purified recombinant proteins (Recombinant Human / Mouse E-cadherin-Fc Chimera; R & D systems, Genzyme Techne) produced on the market are also commercially available, and may be used as mouse- or human-derived E-cadherin proteins Yes (E-cad-Fc protein).

  The polymer having a sugar side chain is preferably a polymer having a main chain obtained by polymerizing an ethylenically unsaturated bond and a sugar side chain. Examples of the sugar side chain include glucose, galactose, lactose and the like, but lactose or galactose is preferable. This is because β-galactose and β-galactose in lactose can be specifically recognized by the asialoglycoprotein receptor and contribute to cell binding. The polymer having a sugar side chain is preferably a styrene derivative polymer having a polystyrene main chain, and most preferably poly (pN-vinylbenzyl-D-lactone amide) (hereinafter also referred to as “PVLA”).

  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 base material 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 or the like and is not particularly treated for cell culture. 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 cadherin family on the above-mentioned culture substrate solid phase surface, physical methods such as adsorption, covalent bonds, etc. Although a chemical method can be applied, the method by adsorption is preferable because of the ease of operation. When the adhesive molecule is a proteinaceous or peptidic molecule or a polymer compound containing a sugar chain, the solution of the molecule is brought into contact with the surface of the culture substrate solid phase such as a plate, and the solvent is removed after a certain period of time. Thus, the molecule can be easily adsorbed. More specifically, for example, a solution of an adhesive molecule using distilled water or PBS as a solvent is filtered, sterilized, then contacted with a culture substrate such as a plate, and left alone for several hours to overnight. A cell culture substrate on which an adhesive molecule is fixed or coated is 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 to and fused with the adhesive molecule in advance, it is possible to utilize the binding with a specific antibody against the antigenic molecule, and the adhesive molecule is efficiently attached to the substrate surface. It is more preferable because it can be modified. 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, in the case of a recombinant protein in which an IgG Fc region protein is fused to an adhesive molecule, an antibody that specifically recognizes the IgG Fc region may be used as an antibody that is previously modified on the culture substrate. it can. In the case of a recombinant protein in which various proteins and tag sequence peptides are fused to an adhesive 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 cadherin family that is immobilized or coated on the solid phase surface of the cell culture substrate, but two or more proteins or fusion proteins belonging to the cadherin 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 cadherin family or the fusion protein solution needs to be appropriately determined depending on the adsorption amount and / or affinity of the protein, and further the physical properties of the protein, but E-cadherin cells In the case of a recombinant protein in which the Fc region is fused to the outer region, the concentration range is about 0.01 to 1000 μg / mL, preferably about 0.1 to 200 μg / mL, more preferably 1 to 50 μg / mL, And most preferably, it is 5-10 microgram / mL.

  As a method for immobilizing or coating a polymer having a sugar side chain on the solid phase surface of a culture substrate, as in the case of the protein belonging to the cadherin family, a physical method such as adsorption or a chemical method such as covalent bonding. Although a method can be applied, the method by adsorption is preferable from the viewpoint of ease of operation. Adsorption can be achieved by bringing a solution containing a polymer having a sugar side chain into contact with the substrate surface for a certain period of time, preferably several hours to one day and more preferably 1 hour to 12 hours. Alternatively, an antigenic molecule can be artificially added to and fused with a polymer having a sugar side chain in advance, and a bond with a specific antibody against the antigenic molecule can be used. In this case, the specific antibody needs to be fixed or coated in advance on the solid phase surface of the culture substrate by a physical method such as adsorption or a chemical method such as covalent bonding.

  As an aspect of the process of immobilizing or coating a protein belonging to the cadherin family, a fusion protein, or a polymer having a sugar side chain on the surface of the culture substrate, a schematic diagram in the case of using E-cad-Fc protein and PVLA As shown in FIG. In FIG. 1 (B), E-cad-Fc protein and PVLA are coated in this order. When coating is performed in this order, it is considered that PVLA is coated in the gaps between the coatings with the E-cad-Fc protein, and that an sea-island structure is formed in which the E-cad-Fc protein becomes the sea and PVLA becomes the island.

  As will be 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 by adding a differentiation-inducing factor, It can be suitably used in a culture method for selecting and concentrating desired cells from a cell group obtained by differentiating potent stem cells.

[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) 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 inner 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, and the like. 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).

  It is desirable to add a substance / factor having an action of maintaining the undifferentiated state of the pluripotent stem cells 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; Double & Woodgett, 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); Manipulating the Mouse Embryo: A laboratory manual, Hs et. 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.

[Differentiation induction method]
The method for inducing differentiation of pluripotent stem cells 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 containing a differentiation-inducing factor.

  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 inducers 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.

  For example, taking differentiation into hepatocytes as an example, hepatocytes are ES cells that are Mesendoderm (endodermal mesenchymal cells), Definitive Endoderm (endodermal cells), Hepatic Progenitor Cells (hepatic progenitor cells), Hepatocytes. It can be obtained by differentiating in the order of (hepatocytes). For differentiation into hepatocytes, Activin A, Nodal, bFGF (basic fibroblast growth factor, basic fibroblast growth factor), HGF (hepatocyte growth factor, hepatocyte growth factor), OSM (Oncostatin ex, X, DE). ), EGF (epidermal growth factor), and TGF-α (transforming growth factor-α) transforming growth factor-α), but other factors are used according to techniques described in known literatures. Also good. For cells other than hepatocytes, differentiation can be induced by using a differentiation-inducing factor necessary for differentiation into each cell.

[Other cell culture methods]
Another cell culture method of the present invention uses the above cell culture substrate and a liquid medium containing a differentiation-inducing factor to differentiate pluripotent stem cells into hepatocytes, and the hepatocytes are separated from the cell culture substrate. It is characterized by being separated, seeded on a cell culture substrate fixed or coated with a polymer containing galactose or a lactose side chain, and cultured. The polymer containing the galactose or lactose side chain is preferably poly (p-N-vinylbenzyl-D-lactone amide). About the culture method other than using a specific cell culture substrate, it can be based on a well-known cell culture method similarly to the above.

  When pluripotent stem cells are differentiated into hepatocytes using the cell culture substrate of the present invention, hepatocytes are obtained with high purity. After differentiation induction, hepatocytes are peeled off from the cell culture substrate, and seeded on the cell culture substrate fixed or coated with a polymer containing galactose or lactose side chains on the surface, and further cultured to further increase the purity of hepatocytes. be able to. This is because the ASGPR (Asialoglycoprotein Receptor) expressed on the cell surface of hepatocytes recognizes galactose side chains, so that only hepatocytes have a cell culture substrate fixed or coated with a polymer containing galactose side chains on the surface. This is for bonding.

Since cell adhesion by a protein belonging to the cadherin family and cell adhesion by a polymer containing a galactose side chain are both Ca ²⁺ dependent, a chelating agent can be used as a method of peeling hepatocytes from a cell culture substrate after differentiation induction. Is preferably used. Any known chelating agent can be used, and those that do not adversely affect cells are preferred.

Utilization as a method for gene transfer into pluripotent stem cells As another embodiment of the present invention, the method described in the present invention is used as a method for efficiently introducing a desired foreign gene into cells of pluripotent stem cells. be able to. The foreign gene to be introduced is not particularly limited. For example, it may be a natural protein such as a growth factor, receptor, enzyme, or transcription factor, or an artificial protein that has been modified and prepared using a genetic engineering technique. is there. The transgene may be a functional RNA such as a ribozyme or siRNA. Furthermore, as a foreign gene, a marker gene for evaluating gene introduction efficiency or expression stability, for example, a gene encoding GFP (Green Fluorescent Protein), β-galactosidase, luciferase, or the like can be used.

  In one preferred embodiment, the foreign gene to be introduced is linked to a nucleic acid sequence that enables transcription and expression of the gene, a so-called promoter sequence, in such a manner that transcription and expression are possible under the control of the promoter. Is done. In this case, the gene is preferably further linked to a poly A addition signal. Examples of promoters that enable transcription and expression of foreign genes in pluripotent stem cells include promoters derived from viruses such as SV40 virus, CMV, and Rous sarcoma virus, β-actin promoter, EF1α promoter, and the like. Depending on the purpose, a nucleic acid sequence that enables specific transcription and expression in a certain cell / tissue or a cell at a certain differentiation stage, a so-called cell / tissue-specific promoter sequence or a differentiation stage-specific promoter. Or Pol. Suitable for RNA expression. III promoters can also be used. The base sequences of these promoters can be used in public DNA databases such as NCBI, and gene vectors using desired gene sequences can be prepared by using general molecular biological techniques. . In addition, vectors that can use these promoters can be purchased from Invitrogen, Promega, Ambion, and the like.

  The method for introducing the gene (vector) is not particularly limited, and examples thereof include a transfection method using calcium phosphate or DEAE-dextran. In addition, a cell to which a gene is introduced can be taken into the cell, and a lipid preparation with low cytotoxicity, such as Lipofectamine (Invitrogen), Superfect (Qiagen), DOTMA (Roche), etc., A method of transfection after forming a liposome-nucleic acid complex with a target gene is also suitable. Furthermore, a method of incorporating a target gene into a virus vector such as a retrovirus or adenovirus and infecting cells with the recombinant virus can be used. In this case, a viral vector is a construct that allows a target gene to be incorporated and expressed in a nucleic acid sequence in which the entire length or part of viral DNA or RNA has been deleted or mutated.

Utilization of Pluripotent Stem Cells Proliferated by the Method of the Present Invention Pluripotent stem cells grown by the culture method (proliferation method) according to the present invention are continuously differentiated by using a cell recovery method by a known method. Pluripotent stem cells that maintain a stable state can be obtained efficiently and in large quantities. Moreover, the gene introduction method according to the present invention can efficiently obtain a large amount of pluripotent stem cells into which a desired gene has been introduced and expressed. The pluripotent stem cells thus obtained are hereinafter referred to as pluripotent stem cells prepared according to the present invention.

  Examples of the method for recovering pluripotent stem cells include known enzymatic digestion methods used in general subculture methods for pluripotent stem cells. Specific examples thereof include removing the medium from the culture vessel in which pluripotent stem cells are cultured, washing with PBS several times, preferably 2-3 times, and a solution containing an appropriate proteolytic enzyme (for example, trypsin). And a solution containing a proteolytic enzyme such as dispase) and incubation at 37 ° C. for a suitable time, preferably 1 to 20 minutes, more preferably 3 to 10 minutes, and then a suitable medium such as the above ES cell culture medium. A method of suspending in a solution to form a single cell can be mentioned. In addition, a method that does not use an enzyme can also be used. For example, the medium is removed from a culture vessel in which pluripotent stem cells are cultured, and after washing with PBS several times, preferably 2-3 times, ethylenediamine is used. A tetraacetic acid (EDTA) solution is added to a final concentration of 0.01 to 100 mM, preferably 0.1 to 50 mM, more preferably 1 to 10 mM, and an appropriate time at 37 ° C., preferably 1 to 60 minutes, preferably May be treated for about 10 to 30 minutes to detach the cells, and then suspended in a suitable solution such as an ES cell culture medium to form a single cell. Further, instead of EDTA, an ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA) solution can be used in the same manner.

  The present invention also provides differentiated cells prepared from the pluripotent stem cells prepared according to the present invention by appropriate differentiation induction treatment. The differentiated cell is not particularly limited as long as it is a kind of cell that can generally be induced to differentiate from a pluripotent stem cell. Specific examples include ectoderm cells or ectoderm-derived cells, mesoderm cells or mesoderm-derived cells, endoderm cells or endoderm-derived cells, and the like.

  The ectoderm-derived cells are cells constituting tissues and organs such as nerve tissue, pineal gland, adrenal medulla, plastid and epidermal tissue, but are not limited thereto. Mesodermal-derived cells are cells that constitute tissues and organs such as muscle tissue, connective tissue, bone tissue, cartilage tissue, heart tissue, blood vessel tissue, blood tissue, dermis tissue, urinary organs, and genital organs, but are not limited thereto. Not. Endoderm-derived cells are cells constituting tissues and organs such as digestive tract, respiratory tract, thymus, thyroid, parathyroid, bladder, middle ear, liver, and pancreas, but are not limited thereto.

  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.

  Furthermore, the present invention also provides a method for producing a chimeric embryo or chimeric animal using the pluripotent stem cells prepared by the method disclosed in the present invention, and the produced chimeric embryo or chimeric animal. A standard protocol has already been established for a method for producing a chimeric embryo or a chimeric animal. For example, Manipulating the Mouse Embryo: A laboratory manual (Edited by Hogan et al., Cold Spring Harbor Laboratory Press, 1994). However, this is not particularly limited.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited at all by these Examples.

  The experimental methods performed in the examples of the present invention are as follows.

<Synthesis of PVLA>
PVLA (FIG. 1 (A)) was obtained from Kobayashi A, Kobayashi K, Akake T. et al. J. et al. Biometer. Sci. Polym. Ed. 1992; 3: 499-508. p-vinylbenzylamine was synthesized by Gabriel synthesis, and oligosaccharide was oxidized to obtain glucoamide. N-p-vinylbenzyl-O-β-D-galactopylanosyl- (1 → 4) -D-glucoamide (VLA) is dissolved in water and 1% as an initiator at 60 ° C. for 2 hours under nitrogen atmosphere. Polymerization was carried out using ₂ S ₂ O ₈ (w / v). The precipitated polymer was extracted and freeze-dried to obtain a polymer powder.

<Synthesis of E-cad-Fc>
Expression and purification of the E-cad-Fc fusion protein is described in Nagaoka M, Akaike T. et al. , ProteinEng. 2003; 16: 243-245. In accordance with In this example, the extracellular domain cDNA obtained from the full-length mouse E-cadherin (RIKEN BRC DNA bank, code 1184) and the mutated IgG1-Fc domain cDNA (T252M / T254S) were ligated, and E-cad-Fc was ligated. The fusion protein was expressed.

<Quartz crystal microbalance measurement method (QCM)>
An AFFINIX Q4 device (manufactured by Initium Co. Ltd) was installed in the temperature control device. The electrode of the QCM cell was washed with pure water and chloroform, treated with sulfuric acid / hydrogen peroxide (volume ratio 3/1), and then treated with 2% SDS solution (Nacalai Tesque). The electrode was coated with a polystyrene thin film by a high-speed rotation method. The cell was set in the QCM apparatus and stabilized for 2 hours, and then a sample solution (500 μl) was added. The surface was stabilized for at least 30 minutes and rinsed 3 times with PBS to remove unstable adsorbates. In order to generate an adsorption curve, the concentration of the PVLA solution was changed to 2, 4, 6, 8, 10, 50, 100 and 200 μg / mL, and the concentration of the E-cad-Fc solution was changed to 1, 2, 4, 6 , 8, 10, and 15 μg / mL. After confirming the concentration required for monolayer formation, co-coating of PVLA and E-cad-Fc was tested. E-cad-Fc solution (7.5 μg / mL) was added to the QCM cell, stabilized and washed three times, then added with PVLA solution (100 μg / mL), and stabilized and washed in the same way. Went.
The mass change per unit area was calculated by the Sauerbrey equation. That is, in the 1 cm ² region, the change in frequency at 1 Hz is equivalent to a mass change of 0.606 ng / cm ² . Concentration-dependent adsorption was described using the Langmuir equation after linear regression using the Lineweaver-Burk equation for the double reciprocal of the data.

<Preparation of PVLA-coated culture dish>
After sterilization using a Millipore 0.22 μm filter, 1.5 mL of an aqueous PVLA solution (100 μg / mL) was added to an untreated polystyrene culture dish (35 mm, manufactured by Iwaki) and incubated at 37 ° C. for 2 hours. Thereafter, the culture dish was washed with phosphate buffer (PBS) and blocked with 1% BSA for 1 hour. Thereafter, the culture dish coated with PVLA was rinsed with PBS.
By the same method as described above, a gelatin-coated polystyrene culture dish was prepared using an aqueous gelatin solution (1 mg / mL). In addition, a collagen-coated culture dish was prepared using a collagen aqueous solution (type I, 0.1 mg / mL, pH 3.0).

<Preparation of culture dish co-immobilized with E-cad-Fc and PVLA>
In order to produce the co-immobilized base layer described in FIG. 1 (B), a purified E-cad-Fc solution (7.5 μg / mL) was added to an untreated polystyrene culture dish. After incubation at 37 ° C. for 2 hours, the culture dishes were washed with PBS, treated with PVLA solution (100 μg / mL), and incubated at 37 ° C. for 2 hours. The co-immobilized culture dish was blocked with 1% BSA, washed with PBS, and used.

<Isolation and culture of hepatocytes>
Seglen PO. , Exp. Cell Res. 1973; 82: 391-398, and Ise H, Sugihara N, Negishi N, Nikaido T, Akaike T .; Biochemical and biophysical research communications. 2001; 285: 172-182, primary culture hepatocytes were isolated from liver tissue of male ICR mice (6-8 weeks, SLC) by a two-step in situ collagenase perfusion method.
Mice were anesthetized with pentobarbital, the portal vein cannulated, and the liver was perfused with Ca ²⁺ free pre-perfusion solution (pH 7.4) containing EGTA, followed by trypsin inhibitor (0.05 mg / mL, Wako). And a collagenase solution (pH 7.4) containing collagenase (0.15 mg / mL, manufactured by Wako). The perfused tissue was separated with Hank solution and collected with a 100 μm nylon cell strainer (BD Falcon). Hepatocytes were purified by density gradient centrifugation (50 × g, 10 minutes, 4 ° C.) using a 10% Percoll solution (GE Healthcare Bioscience). Cell survival was confirmed using the trypan blue method. Viable primary cultured hepatocytes were suspended in Williams E medium (Invitrogen) containing 50 ng / mL penicillin and 50 μg / mL streptomycin (manufactured by Nacalai Tesque), and gelatin, collagen, E-cell at a density of 3 × 10 ⁴ cells / cm ^2. cad-Fc, PVLA, seeded on co-immobilized culture dishes. The medium was removed after 4 hours to remove unbound cells and dead cells.

<Maintenance of undifferentiated ES cells>
Feeder-free mouse ES cell line (EB3) was prepared using 10% FBS, 2 mM L-glutamine, 1% non-essential amino acid (NEAA, manufactured by Nacalai Tesque), 1 mM pyruvate, 0.1 mM β-mercaptoethanol, 1000 units / mL. Maintained in gelatin-coated culture dishes containing Grasgow minimal medium (GMEM, Sigma-Aldrich) supplemented with LIF (recombinant leukemia inhibitory factor, Chemicon), antibiotics (50 ng / mL penicillin and 50 μg / mL streptomycin). .
mES cells were separated by Accutase (manufactured by Millipore), subcultured every 3 days, changing the medium every day, and subcultured at least 3 times (9 days).

<Differentiation of mES cells into hepatocytes>
The differentiation process was performed in four steps as described in FIG. 1 (C). Differentiation into endoderm mesoderm, differentiation into definitive endoderm, differentiation into hepatic progenitor cells, differentiation into hepatocytes. Therefore, three types of differentiation induction media were used. GMEM containing 10% knockout serum substitute (KSR, manufactured by Invitrogen), 1% FBS, 2 mM L-glutamine, 1% NEAA, 1 mM pyruvate, 0.1 mM β-mercaptoethanol and antibiotics was used as the basic medium. .
First, a medium obtained by adding 10 ng / mL activin A (manufactured by R & D system) to the above basic medium was used as medium A and used for endoderm-type mesoderm differentiation. Co-immobilization with medium A added so that undifferentiated ES cells maintained on gelatin (after 0 days) were separated by Accutase and a concentration of 5000 cells / mL (10000 cells in the whole 35 mm culture dish) was obtained. The cells were seeded on a chemical culture dish and cultured for 3 days. Thereafter, medium B was used, which was obtained by adding activin A (10 ng / mL) and bFGF (50 ng / mL, manufactured by Promega) to the basic medium for differentiation into endoderm. The cells differentiated as described above (after 3 days) were further cultured in medium B for 2 days. As a control for spontaneous differentiation, the cells were cultured in a basic medium for 5 days in parallel with the above (5 days).
HGF (10 ng / mL, Sigma), Oncostatin M (10 ng / mL, OSM, Sigma) and dexamethasone (1 mM, DEX, Sigma) are added to the basic medium to form medium C, which differentiates into hepatocytes Used for.
On day 6, cells were separated using an enzyme-free cell separation buffer (CDB, manufactured by Gibco), and a density of 5000 cells / mL was added to an E-cad-Fc and PVLA co-immobilized culture dish containing medium C. Sowing.
After 14 days, the differentiated cells were separated using CDB, and seeded at a density of 5000 cells / mL in a co-immobilized culture dish containing E-cad-Fc and PVLA containing medium C.
For the culture dish coated with E-cad-Fc alone, mES cells were cultured in the same manner as described above. For gelatin-coated culture dishes, undifferentiated ES cells were maintained in gelatin-coated culture dishes for 2 days and then cultured in the same manner to form stable colonies.
On the 18th, 20th, 22nd, and 24th days after the start of differentiation induction, the differentiated cells were separated by CDB, and placed on a 35 mm PVLA-coated culture dish containing medium C at a density of 10,000 cells / mL (in the whole culture dish) 20000 cells). The percentage of cells bound to PVLA was used to evaluate the number of ASGPR positive cells and the optimal timing of reseeding.

<Binding assay>
After culturing the cells on a 96-well plate for 4 hours, cells that did not bind to the medium were removed. The bound cells were washed three times with PBS and fixed with Midform 20N (4% formaldehyde, pH 7.4, manufactured by Wako Pure Chemical Industries) for 15 minutes. Thereafter, it was stained with 0.1% crystal violet for 10 minutes. Finally, after rinsing, 2% SDS was added and incubated for 30 minutes, and the absorbance at a wavelength of 570 nm was measured with a microplate reader.

<Semi-quantitative RT-PCR>
RNA was extracted from the cell lysate using a TRIzol agent (Invitrogen), suspended in distilled water, and reverse transcribed into cDNA using dNTP, oligo-dT primer and Moloney murine leukemia virus reverse transcriptase (Invitrogen).
PCR is, dNTPs mix (manufactured by Takara), TopTaq polymerase (manufactured by Qiagen), was performed using a PCR buffer containing MgCl _2. PCR products were subjected to 2% agarose gel electrophoresis and scanned with a Molecular Dynamics Typhoon 8600 imager. Band intensities compared to housekeeping gene markers were calculated using ImageQuant software (ver. 5.2, Molecular Dynamics).

<Immunofluorescence assay>
The cells were fixed with Mildform 20N (8% formaldehyde, pH 7.4) for 20 minutes and washed with PBS. Fixed cells were treated with 0.2% Triton X-100 for 5 minutes and washed with PBS. Incubated for 30 minutes in the presence of Image-iT signal enhancer (Invitrogen) and blocked with Blocking One solution (Nacalai Tesque) for 1 hour. Cells were then incubated with primary antibody for 2 hours at room temperature, carefully washed with PBS, and then incubated with secondary antibody for 2 hours at room temperature. Thereafter, the cells were nuclear stained with DAPI. The stained cells were suspended in PBS and observed with an inverted fluorescence microscope (Olympus).
In order to calculate the number of cells expressing Oct3 / 4, Sox17, AFP, and ALB, 50-100 cells collected from 5 different places in 3 independent culture dishes were observed.

<Western blot>
Cellular proteins were extracted with lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 1% Triton X-100) containing 1% PMSF, 1% protease inhibitor cocktail. The cell lysate was centrifuged at 15000 × g and 4 ° C. for 15 minutes to obtain a protein sample solution. The protein treated with the sample buffer was subjected to SDS-PAGE (7.5%, 10% acrylamide gel) and transferred to a PVDF membrane (Immobilon-P, manufactured by Millipore). The PVDF membrane was incubated with mouse anti-β-actin antibody and goat anti-mouse albumin antibody (Abcam) for 2 hours at room temperature. Then, it was incubated with a HRP-conjugated secondary antibody (diluted 1/10000, Jackson ImmunoResearch Laboratories) for 2 hours at room temperature. HRP activity was detected by Immobilon Western detection agent (Millipore).

<Albumin ELISA assay>
Albumin secreted into the medium was detected using a mouse albumin ELISA kit (manufactured by Shibayaki). The medium was collected every 24 hours, centrifuged at 8000 × g for 5 minutes, and diluted to obtain a sample solution. Thereafter, the microplate treated with anti-albumin antibody was rinsed with 250 μl of washing buffer, and incubated with 50 μl of sample solution (diluted 10 times) or standard solution for 1 hour.
The microplate was rinsed and incubated with HRP-conjugated antibody solution for 1 hour at room temperature. After rinsing with a washing buffer, the substrate was treated with a chromogenic substrate solution (3,3 ′, 5,5′-tetramethylbenzidine, TMB) for 20 minutes and then with 50 μl of 1M sulfuric acid. Absorbance at a wavelength of 450 nm was measured with a microplate reader. A standard curve was created to calculate albumin concentration and normalized by cell number.

<Glycogen accumulation (PAS reaction)>
Primary cultured hepatocytes and differentiated cells were each fixed with Mildform 20N (8% formaldehyde, pH 7.4) for 10 minutes. The cells were washed twice with PBS, and the cells were oxidized with 1% periodic acid (manufactured by Wako Pure Chemical Industries) for 5 minutes and washed again. The cells were treated with a Schiff reaction solution (manufactured by Wako Pure Chemical Industries, Ltd.) for 10 minutes and washed with PBS. After treatment with 2% SDS solution for 30 minutes, the absorbance at a wavelength of 550 nm was measured using a microplate reader and normalized by the number of cells. Undifferentiated ES cells were used as a control.

<Urea assay>
The urea level in the medium was measured using the Urea assay kit (manufactured by Bioassay Systems). The medium was collected every day, centrifuged at 8000 × g for 5 minutes, and the supernatant was used as a sample. Water (blank), diluted standard solution (2 mg / dL), and sample solution were introduced into a 96-well plate. 200 μL of reaction reagent solution was added and incubated at room temperature for 50 minutes. The OD (optical density) at 492 nm was measured. Finally, the unit amount for every 10 ⁵ cells was calculated using a standard curve and normalized by the number of cells.

(result)
<Co-adsorption of PVLA and E-cad-Fc>
First, the binding ability of PVLA and E-cad-Fc to the polystyrene surface was examined using QCM. By adding the sample solution, the oscillation wavelength of the quartz crystal was reduced and stabilized after a certain point.
The change in frequency (frequency) decreased with increasing coating concentration until the saturation point was reached. Full surface saturation by extracellular matrix molecules can be seen as monolayer adsorption on a polystyrene surface. From the results of QCM, it was found that the monolayer concentration of PVLA was 100 μg / mL. Further, it was found that the monolayer concentration of E-cad-Fc was 10 μg / mL, which was the same as the value revealed by the ELISA assay. Thereafter, the mass change of the adsorbed part was calculated by the Sauerbrey equation and normalized by linear regression. The GFI value (Goodness of Fit, goodness-of-fit index) is described as a coefficient of determination (R ² value). The regression line of statistical values closely approximated the measured values. On the other hand, the maximum adsorption amount Δm _max and the constant Kα in the Langmuir adsorption equation were also obtained. According to the theory of adsorption isotherm, the adsorption energy of E-cad-Fc protein is larger than PVLA. That is, E-cad-Fc adsorbs faster than PVLA. The adsorption curves of PVLA and E-cad-Fc are shown by the Langmuir adsorption equation (FIGS. 2A and 2B).
In addition, co-adsorption of PVLA and E-cad-Fc for forming an immobilized base layer was also evaluated by QCM (FIG. 2 (C)). First, a 7.5 μg / mL E-cad-Fc solution lower than the monolayer concentration (10 μg / mL) was introduced into the QCM cell. After stabilization and rinsing, the PVLA solution (100 μg / ml) was added and the adsorption value increased. As a result, it was revealed that PVLA occupies the space remaining after immobilization of E-cad-Fc, and the inference that E-cad-Fc has higher adsorption capacity for polystyrene than PVLA was supported.

<Effects of PVLA and E-cad-Fc co-immobilized substrata on primary cultured hepatocyte function and adsorption>
E-cadherin performs an important function in spheroid formation of hepatocytes bound to PVLA. Cells in the agglomerate have a longer survival period and higher ability to express differentiation function than cells in a single cell layer culture. Considering these phenomena, the binding and function of primary cultured hepatocytes to the co-immobilized substratum of PVLA and E-cad-Fc were examined.

  The morphology and albumin expression of primary cultured hepatocytes on various matrices were examined by fluorescent staining (FIG. 3 (A)). Primary cultured hepatocytes formed a single cell layer on the surface of gelatin, collagen, E-cad-Fc alone, PVLA and E-cad-Fc co-immobilized base layer. On the other hand, on a dish coated with PVLA alone, a spheroid structure was formed. In all matrices, most of the cells showed high albumin expression levels. After 3 days in culture, hepatocytes on PVLA formed stable multilayered spherical structures. Cells on PVLA and E-cad-Fc co-immobilized substrata begin to aggregate into a single cell layer aggregate, and cells on gelatin, collagen, and E-cad-Fc are not particularly altered in morphology. I couldn't. The albumin expression of the cells in the aggregate was maintained at a high level. On the other hand, in the cells on gelatin, collagen and E-cad-Fc, the expression of albumin was remarkably lowered. On the existing culture system, cells were spread, whereas cells in aggregates on PVLA and E-cad-Fc co-immobilized substratum maintained high albumin expression levels over a long period of time. (FIGS. 3A and 3B). This suggests that PVLA plays an important role as a substratum in the formation of hepatocyte aggregates and maintenance of function.

  Using cells on the respective matrix, RT for albumin (ALB), hepatocyte nuclear factor 4α (HNF-4α), asialoglycoprotein receptor (ASGPR), mouse tryptophan oxygenase (mTO), glucose 6-phosphatase (G6P) -PCR was performed. As a result, cells on the co-immobilized base layer of PVLA and E-cad-Fc and the PVLA-only coating showed higher expression levels of hepatocyte-specific genes than cells on the surface immobilized with gelatin and E-cad-Fc alone. (Fig. 3 (C)).

  Furthermore, in order to evaluate the function of hepatocytes cultured on each matrix, the amount of albumin produced specifically in the hepatocytes was monitored. Primary cultured hepatocytes were cultured for 1 week in the presence of 10% FBS in gelatin, E-cad-Fc alone, PVLA alone, PVLA and E-cad-Fc co-immobilized base layer. As shown in FIG. 3D, in contrast to PVLA alone, a co-immobilized base layer of PVLA and E-cad-Fc, the amount of albumin produced by hepatocytes on a matrix of gelatin and E-cad-Fc alone is , Decreased rapidly, suggesting dedifferentiation.

  In freshly cultured hepatocytes, the amount of glycogen stored is almost the same level between PVLA and E-cad-Fc co-immobilized substrate and gelatin or E-cad-Fc single matrix. However, the co-immobilized base layer was maintained at a high level through long-term culture, whereas gelatin and E-cad-Fc single matrix clearly decreased through long-term culture. This suggested that PVLA plays an important role in maintaining the function of hepatocytes.

  In view of the knowledge that PVLA contributes to the maintenance of hepatocyte function and E-cadherin promotes the binding and differentiation of primary cultured hepatocytes and ES cells, highly active hepatocytes are collected with high purity in vitro. Therefore, it was decided to use a co-immobilized substrate matrix of PVLA and E-cad-Fc.

<Undifferentiated mouse ES cells on co-immobilized substrate>
E-cad-Fc, a fusion protein, was developed as a feeder cell-free extracellular matrix to maintain ES cells in a dispersed state and totipotency.
In this example, the behavior of mouse ES cells (mES cells) on the co-immobilized substratum matrix was examined in order to confirm the compatibility of the PVLA and E-cad-Fc co-immobilized substratum matrix system. It was.

  First, from the morphology of undifferentiated mES cells (EB3) after 2 days of culture, the co-immobilized substratum matrix can maintain a dispersed state at the single cell level as well as the E-cad-Fc matrix. Was found (FIG. 4A). On the other hand, undifferentiated mES cells did not bind to the PVLA single coating matrix. Thereafter, the proliferation of undifferentiated mES cells on various matrices was evaluated, and standardized by the number of bound cells on each matrix (FIG. 4B). As a result, the co-immobilized substrate matrix had higher cell proliferation ability than the gelatin matrix and was lower than the E-cad-Fc single matrix. This suggests that the presence of PVLA slightly affected the growth of some cells.

  By examining transcription using an undifferentiated marker (Oct3 / 4), it was confirmed that mES cells on each matrix were undifferentiated (FIG. 5 (A)). ES cells on the co-immobilized substratum matrix expressed similar levels of undifferentiated markers as on the E-cad-Fc single matrix.

<Differentiation of mES cells into hepatocytes>
In order to obtain hepatocytes derived from ES cells, mES cells (EB3) were seeded on a co-immobilized substratum matrix to form a uniform cell group. Cells dispersed on the co-immobilized substrate matrix were cultured in the presence of activin A and bFGF and differentiated into endoderm cells. Thereafter, the cells were further cultured in a low serum medium containing HGF, oncostatin M and dexamethasone. Differentiation induction using E-cadherin and gelatin matrix was always performed in parallel.

  RT-PCR was performed using cells collected over time, and the uniformity of the cells and the degree of differentiation into hepatocyte-like traits were examined. RT-PCR includes endoderm specific genes (Gata6 and Sox17), hepatic progenitor cell specific genes (AFP, albumin, cytokeratin 18 and HNF4α), hepatocyte specific genes (ASGPR, albumin, tryptophan oxygenase) And an undifferentiated mES cell marker (Oct3 / 4), a mesoderm marker (brachyury), and an ectoderm marker (Pax6, tyrosine hydroxylase).

  As shown in FIGS. 5A and 5B, the endoderm-specific gene expression increased at an early stage, and the expression level of Oct3 / 4 gradually decreased. As a result, when the E-cadherin single matrix and the co-immobilized base layer matrix were used, efficient and uniform differentiation was observed. On the other hand, the expression level of Sox17 was small in the cells on the gelatin coating matrix. Cells on the gelatin coating matrix also expressed ectoderm markers. Cells on the co-immobilized substratum matrix did not show ectoderm marker expression. This result suggested an important role of E-cadherin in differentiation into endoderm.

  In order to examine the proportion of cells differentiated into endoderm trait cells, immunostaining of Sox17, E-cadherin and Oct3 / 4 was performed. Cells on the co-immobilized substrate matrix remained dispersed and many expressed Sox17 and E-cadherin. Within 5 days after differentiation induction, the differentiated ES cells have enlarged nuclei, and 95.3 ± 3.7% of the cells on the co-immobilized substratum matrix expressed Sox17, while expressing Oct3 / 4. Only 41.4 ± 15.8% (FIGS. 5C and 5D). In contrast, cells on gelatin expressed 8.6 ± 5.9% expressed Sox17 and 85.3 ± 9.6% expressed Oct3 / 4. The stable expression of E-cadherin suggests that the E-cadherin matrix is suitable for endoderm differentiation. These results suggest that a co-immobilized substrate matrix that maintains the advantages of E-cad-Fc is a suitable substrate for endoderm differentiation.

Furthermore, the influence of the presence of PVLA on the differentiation into hepatic progenitor cells and hepatocytes was also examined.
In order to determine specific differentiation markers for early and intermediate hepatocyte differentiation, the expression of AFP, ALB, cytokeratin 18, HNF4α and E-cadherin was examined by RT-PCR (FIG. 6 (A)). All hepatocyte-specific genes except AFP gradually increased with the differentiation induction time course. The maximum expression level of AFP was recorded 10 days after differentiation induction (FIG. 6B). The expression levels of ALB, cytokeratin 18, and HNF4α were also examined semi-quantitatively every 4 days (FIGS. 6C, 6D, and 6E).

  In cells on the co-immobilized substratum matrix, albumin expression was observed 8 days after the start of induction, and cytokeratin 18 was observed after 4 days. Thereafter, the differentiated cells began to express HNF4α after 12 days. For all three markers, cells on the co-immobilized substrate had higher levels of transcription than those on gelatin.

In order to examine the proportion of cells differentiated into hepatocytes, immunostaining was performed for AFP and ALB (FIGS. 6F and 6G).
As shown in FIG. 6 (F), cells dispersed at a single cell level on the co-immobilized base layer at a high rate of 93.8 ± 6.0% strongly expressed AFP, while colonies on gelatin. A small percentage of cells expressed AFP (15.6% ± 7.1%). After 16 days of induction of differentiation, cells on the co-immobilized substratum were ALB positive at a high rate of 90.0% ± 9.6%. On the other hand, only 20.5% ± 5.3% of cells on gelatin expressed ALB (FIGS. 6 (G) and (H)). The co-immobilized base layer was the same result as that of the E-cad-Fc single matrix, and it was confirmed that the co-immobilized base layer has an excellent cell concentration performance as compared with gelatin.

  The expression of albumin in the cells on the co-immobilized substrate and on the E-cad-Fc single matrix was confirmed by Western blot (FIG. 6 (I)).

  As FIG. 7 (A) shows, the expression of mature hepatocyte-specific genetic markers including ASGPR and mTO increased in the latter stages of differentiation. As the differentiation progressed, the expression level of ASGPR peaked after 24 days (FIG. 7B). Interestingly, ASGPR expression began after 10 days for cells on the co-immobilized substratum, after 12 days for cells on the E-cad-Fc single matrix, and after 16 days for cells on the gelatin matrix. Was started. Cells on the co-immobilized substrate always had higher expression levels of ASGPR than the other two matrices. This suggests that PVLA may contribute to the promotion of ASGPR expression. In agreement with these results, the cells on the co-immobilized substrate also had higher mTO expression levels compared to the E-cad-Fc single matrix and gelatin. In particular, the difference was remarkable after 22 days (FIG. 7C).

  On the other hand, from the results of immunostaining performed 24 days after differentiation induction, the expression levels of ALB and ASGPR were clearly higher in the co-immobilized base layer and the E-cad-Fc single matrix than in gelatin (FIG. 7). (D)).

  Hepatocytes are mature cells with various metabolic functions. The functional level of mES cells at the final stage of differentiation (20th and 24th days after induction) was evaluated, and the influence of the co-immobilized substrate matrix on the differentiation induction efficiency was confirmed. Cellular albumin secretion (FIG. 7 (E)), glycogen accumulation (FIG. 7 (F)), and urea production level (FIG. 7 (G)) on the co-immobilized substrate layer were determined by gelatin or E-cad-Fc single matrix. Higher than.

<Concentration of mouse ES cell-derived hepatocytes based on ASGPR expression>
The present invention provides a matrix-dependent selective cell enrichment method that isolates ES cell-derived hepatocytes and removes undifferentiated cells and cells that may differentiate into other lineages. E-cad-Fc may support ASGPR selective binding by PVLA.

Since both the binding by E-cadherin and the binding of PVLA-ASGPR are Ca ²⁺ -dependent, differentiation cells were differentiated with a chelating agent (CDB) after 18 days, 20 days, 22 days and 24 days of induction of differentiation. Were separated and seeded on PVLA coated dishes. The number of bound cells was calculated (FIG. 8A), and the presence of ASGPR expressed on the cell membrane was evaluated. The ratio of ASGPR positive cells increased with time from the start of differentiation induction in each matrix. Cells on the co-immobilized substrate had a higher percentage of ASGPR positive cells than E-cad-Fc alone or gelatin-coated dishes. After 24 days, the binding rate of cells derived from the co-immobilized base layer was 59.7% ± 11.6%, and the binding rate of cells derived from the E-cad-Fc single matrix was 45.0 ± 2.5. %, And the binding rate of the gelatin-derived dish-derived cells was 23.2 ± 3.3%. The change in the percentage of attached cells was small between 22 and 24 days. Therefore, it can be said that the optimal re-seeding timing is 22 days after differentiation induction.

In the co-immobilized base layer, even a differentiated cell in the final stage maintained a single cell layer, but a tendency toward aggregation was observed (FIG. 8B).
However, when the cells were cultured for 3 days from the re-seeding on the PVLA-coated dish, some of the differentiated cells aggregated to form a multilayer aggregate. This was similar to tissue-like spheroids formed after 3 days of primary hepatocyte culture.

<Characteristics of ES cell-derived hepatocytes concentrated by ASGPR>
For cells 24 days after differentiation induction in each of gelatin, E-cad-Fc single matrix, and co-immobilized base layer, the amount of transcription of various markers was re-seeded on PVLA-coated dishes as described above. The cells were seeded and cultured for 3 days) (FIG. 8C). From the RT-PCR of brachyury (mesoderm-specific gene) and Pdx1 (pancreatic marker), it can be seen that cells that do not express markers other than hepatocytes were enriched by cell selection process by chelating agent and reseeding . In addition, since transcription of Gata6 and Sox17 (endoderm and hepatic progenitor cells) was not observed, it can be seen that cells at an early stage of differentiation were removed. From the fact that the amount of transcription of mature hepatocyte markers (ASGPR, mTO, G6P) was maintained or increased, it can be seen that selective binding of mature hepatocytes occurred.
From these results, it can be seen that selective enrichment of hepatocytes was achieved.
To investigate whether differentiated ES cells could function as primary cultured hepatocytes, albumin secretion, glycogen accumulation, and urea production were measured. Hepatocytes replated on PVLA-coated dishes maintained their metabolic functions.

Claims (11)

  A cell culture substrate characterized in that a surface thereof is fixed or coated with a protein belonging to the cadherin family or a fusion protein comprising all or part of a protein belonging to the cadherin family, and a polymer having a sugar side chain.   The cell culture substrate according to claim 1, wherein the main chain of the polymer is polystyrene and the sugar side chain is lactose or galactose.   The cell culture substrate according to claim 1 or 2, wherein the polymer having a sugar side chain is poly (p-N-vinylbenzyl-D-lactone amide).   The protein belonging to the cadherin family is E-cadherin or a protein having homology with E-cadherin, and includes one or more of EC1 domain, EC2 domain, EC3 domain, EC4 domain, and EC5 domain, and The cell culture substrate according to any one of claims 1 to 3, which is a protein having a binding affinity of the same kind as E-cadherin.   The cell culture substrate according to any one of claims 1 to 3, wherein the fusion protein comprising all or part of the protein belonging to the cadherin family is a fusion protein of E-cadherin and an Fc region of an immunoglobulin. .   The protein belonging to the cadherin family or a fusion protein including all or part of the protein belonging to the cadherin family and a polymer having a sugar side chain form a sea-island structure. Cell culture substrate.   A cell characterized in that pluripotent stem cells are proliferated while maintaining undifferentiation and differentiation pluripotency using the cell culture substrate according to any one of claims 1 to 6 and a liquid medium. Culture method.   A method for inducing differentiation of pluripotent stem cells, comprising differentiating pluripotent stem cells using the cell culture substrate according to any one of claims 1 to 6 and a liquid medium containing a differentiation-inducing factor. .   The method for inducing differentiation of pluripotent stem cells according to claim 8, wherein the pluripotent stem cells are differentiated into hepatocytes.   Using the cell culture substrate according to any one of claims 1 to 6 and a liquid medium containing a differentiation-inducing factor, pluripotent stem cells are differentiated into hepatocytes, and the hepatocytes are separated from the cell culture substrate. A cell culturing method comprising culturing by separating and culturing on a cell culture substrate fixed or coated with a polymer containing galactose or a lactose side chain on a surface.   The cell culture method according to claim 10, wherein the polymer containing galactose or a lactose side chain is poly (p-N-vinylbenzyl-D-lactone amide).