WO2015098111A1

WO2015098111A1 - METHOD FOR ESTABLISHING iPS CELLS AND METHOD FOR LONG-TERM MAINTENANCE OF STEM CELLS

Info

Publication number: WO2015098111A1
Application number: PCT/JP2014/006454
Authority: WO
Inventors: 哲治岡本; 佐知子山崎; 顕嶋本; 栄俊田原
Original assignee: 国立大学法人広島大学
Priority date: 2013-12-26
Filing date: 2014-12-25
Publication date: 2015-07-02

Abstract

　The problem is to produce iPS cells under serum-free culture conditions without using feeder cells, and to provide a culture method capable of maintaining the undifferentiated state and pluripotent differentiation ability of iPS cells and other such stem cells over an extended period of time under serum-free culture conditions without using feeder cells. The present invention provides: a method for producing induced pluripotent stem cells (iPS cells), characterized in that iPS cells are induced by culturing reprogrammed somatic cells in serum-free medium without using feeder cells, preferably using fibronectin as an adhesion factor; and a method for maintaining the undifferentiated state and pluripotent differentiation ability of stem cells, characterized in that a protein belonging to the tumor growth factor-β (TGF-β) family is added and stem cells are subcultured in serum-free medium without using feeder cells.

Description

iPS cell establishment method and stem cell long-term maintenance method

The present invention relates to a method for establishing induced pluripotent stem cells (iPS cells) and a method for long-term maintenance of the undifferentiated state and differentiation pluripotency of stem cells. Specifically, the present invention relates to a method for inducing iPS cells under serum-free culture conditions without using feeder cells, and passage of stem cells by adding tumor growth factor-β (TGF-β) to the medium. The present invention relates to a method for long-term maintenance of the undifferentiated state and differentiation pluripotency of stem cells, characterized by culturing.

In recent years, embryonic stem cells (embryonic stem cell: hereinafter referred to as ES cells) and induced pluripotent stem cells (induced pluripotent stem cell: hereinafter referred to as iPS cells) have been used in the fields of developmental biology, regenerative medicine research, drug discovery and disease research. Attention has been paid. Stem cells are self-replicating and multipotent cells and have the ability to differentiate into various cell lineages. Since Yamanaka et al. Of Kyoto University reported the establishment of mouse iPS cells in 2006, it has attracted a great deal of attention as an alternative to ES cells that remain ethical problems using fertilized eggs. iPS cells are also referred to as induced pluripotent stem cells. Reprogramming genes (Oct3 / 4, Sox2, Klf4, c-Myc) are introduced into terminally differentiated cells such as skin using viruses, plasmid vectors, etc. By programming, it becomes a universal cell that can be differentiated into all cell types except for extraembryonic tissues.

These stem cells generally use an alternative serum-supplemented medium containing serum or animal-derived components, and increase feeder cells such as inactivated mouse embryonic fibroblasts (MEF) on the dish and support them. It has been cultured as. By the day before the passage of iPS cells, MEF cells were treated with mitomycin C or γ-irradiated in advance to prepare feeder cells with suppressed proliferation (inactivated), and then seeded on gelatin-coated dishes. After sufficient adhesion and extension on the dish, iPS cells are seeded thereon and co-cultured. In addition, it is necessary to separate feeder cells and stem cells such as ES cells and iPS cells as much as possible during passage. In this way, not only culturing stem cells but also adjustment of feeder cells is necessary, which requires very complicated operations. In addition, somatic differentiated cells (somatic cells) before being induced by iPS cells are also cultured in a medium supplemented with fetal bovine serum, human serum or human autoserum. Furthermore, under such culture conditions, there is a possibility of unknown components in the serum or alternative serum containing animal-derived components and the possibility of contamination with pathogens, so there are large lot differences, and culture under constant conditions is always difficult. Also, the feeder cells to be used have a great variety, kind of treatment, and lot difference, and the state of the stem cells is considerably influenced. Therefore, it was impossible to culture the stem cells under stable conditions.

Stem cells cultured under conditions with many indeterminate elements contain many unknown factors in the medium, making standardization of basic research such as tissue / organ development and regeneration difficult, and maintaining undifferentiation. It is difficult to compare the functions of necessary growth factors and differentiation-inducing factors. In addition, due to the action of various components contained in the medium, it is difficult to induce differentiation into specific cells. Furthermore, considering clinical application to regenerative medicine, there are problems with safety such as contamination of unknown factors and risk factors such as pathogens. It becomes. Therefore, in order to eliminate these elements, it is necessary to standardize the culture method under the culture conditions whose composition is clear. In particular, development of a method for producing iPS cells using a serum-free medium with a clear composition has been desired.

Furthermore, it has also been desired to passage and maintain stem cells such as iPS cells while maintaining an undifferentiated state and differentiation pluripotency over a long period of time. In this respect, a medium composition for maintaining primate embryonic stem cells in an undifferentiated state has been studied (see Patent Document 1). Furthermore, in order to maintain the undifferentiated nature of iPS cells, a tumor (transforming) growth factor (TGF) is also added to the medium (see Non-Patent Document 1). However, the serum-free medium used in Patent Document 1 does not contain TGF, and that used in Non-Patent Document 1 is a serum-containing medium, which is different from the present invention.

Special table 2009-542247 gazette

Stem cells cultured under conditions with many indefinite elements such as serum-containing medium contain many unknown factors in the medium, making standardization of basic research such as tissue and organ development and regeneration difficult and undifferentiated It is difficult to compare and examine the functions of growth factors and differentiation-inducing factors that are necessary for maintenance of cancer. In addition, it is difficult to induce differentiation into specific cells due to the action of various components contained in the medium. In addition, when considering clinical application to regenerative medicine, it is necessary to eliminate risk factors such as unknown factors and pathogens from the viewpoint of safety. There is. Therefore, in order to solve these problems, it is necessary to establish and standardize a culture method using a medium whose composition is clear and does not contain risk factors.

Therefore, a specific problem to be solved by the present invention is to cultivate somatic cells before induction into iPS cells under a condition that does not include an uncertain element and a risk factor, and iPS under a condition that does not include an uncertain element and a risk factor. It was to produce cells and to maintain stem cells such as iPS cells in culture. Specifically, specific problems to be solved by the present invention include culturing somatic cells under serum-free culture conditions, producing iPS cells under serum-free culture conditions without using feeder cells, and feeders. It was to provide a culture method that can maintain the undifferentiation and differentiation pluripotency of stem cells such as iPS cells for a long period of time without using cells.

The inventors of the present invention have made extensive studies to solve the above-mentioned problems, a method for serum-free culture of somatic cells before induction into iPS cells, and iPS cells by culturing without using feeder cells in a serum-free medium. Established the establishment method. In particular, it has been found preferable to use fibronectin as an adhesion factor. According to the method of the present invention, the induction efficiency of human iPS cells was 10 times or more that of the conventional method (culture conditions with serum). In addition, the present inventors have identified TGF-β as a cell growth factor capable of maintaining stem cell undifferentiation and pluripotency even after long-term passage in the above culture system. Based on these matters, the present invention has been completed.

Accordingly, the present invention provides the following:
(1) Production of iPS cells characterized by inducing induced pluripotent stem cells (iPS cells) by culturing reprogrammed somatic cells without using feeder cells in a serum-free medium. Method.
(2) The method according to (1), wherein fibronectin is used as an adhesion factor.
(3) The method according to (1) or (2), wherein the reprogramming treatment of somatic cells is performed in a serum-free medium.
(4) The method according to any one of (1) to (3), wherein the somatic cells before reprogramming are cultured from a primary culture in a serum-free medium.
(5) The method according to any one of (1) to (4), wherein the iPS cell is a human iPS cell.
(6) A serum-free culture substrate for inducing iPS cells containing fibronectin as an adhesion factor (however, the culture substrate does not contain feeder cells).
(7) A kit for producing a serum-free culture substrate for inducing iPS cells, comprising a serum-free medium component and fibronectin as components (however, the culture substrate does not contain feeder cells).
(8) An undifferentiated state of a stem cell, characterized in that a stem cell is subcultured by adding a protein belonging to the tumor growth factor-β (TGF-β) family to a serum-free medium without using a feeder cell And methods of maintaining pluripotency.
(9) The method according to (8), wherein the subculture is performed using fibronectin as an adhesion factor.
(10) The method according to (8) or (9), wherein the stem cell is an iPS cell.
(11) The iPS cells obtained by the method according to any one of (1) to (5) are subcultured without using feeder cells in a serum-free medium to which a protein belonging to the TGF-β family is added. A method for maintaining the undifferentiated state and pluripotency of iPS cells,
(12) Serum-free culture substrate for subculture for maintaining the undifferentiated state and differentiation pluripotency of stem cells, including proteins belonging to the TGF-β family (however, the culture substrate does not include feeder cells) .
(13) The culture substrate according to (12), further comprising fibronectin as an adhesion factor.
(14) The culture substrate according to (12) or (13), wherein the stem cells are iPS cells.
(15) A kit for producing a serum-free culture substrate for subculture for maintaining the undifferentiated state and differentiation pluripotency of stem cells, comprising a serum-free medium component and a TGF-β family protein (however, The culture substrate does not contain feeder cells).
(16) The kit according to (15), further comprising fibronectin as an adhesion factor.
(17) The kit according to (15) or (16), wherein the stem cell is an iPS cell.

According to the present invention, somatic cells are cultured using a serum-free medium with a clear component, and in the culture environment using a serum-free medium with a clear component, without using feeder cells, A method for producing iPS cells by gene transfer and induction, etc., and a culture substrate material and a culture solution capable of maintaining and culturing stem cells such as ES cells and iPS cells while maintaining an undifferentiated state and differentiation pluripotency A method for culturing stem cells using the above is provided. According to the method for producing iPS cells of the present invention, it was found that iPS cells can be induced with an efficiency 10 times or more that of the conventional method using a serum-containing medium.

Therefore, iPS cells can be induced with high efficiency in the present invention. And the undifferentiated state and differentiation pluripotency of a stem cell can be maintained over a long period of time. In addition, there are almost no unknown factors in the culture medium, and it is easy to standardize basic research such as tissue and organ development and regeneration, and the functions of growth factors and differentiation-inducing factors necessary for maintaining undifferentiation are compared. It is also easy to do. In the present invention, since the types of components contained in the medium are relatively few and known, differentiation induction into specific cells is easy, and highly safe cells can be obtained considering clinical application to regenerative medicine. can get.

FIG. 1 is a microscopic image showing the morphology of TIG-3 cell-derived hiPS cells induced on various extracellular matrices using serum-free medium hESF9. FIG. 1A shows the results of examining the influence of adhesion factors when inducing human iPS cells using a serum-free medium. FIG. 1B shows the form of human iPS cells (hiPS cells) 2 days after seeding on each extracellular matrix using serum-free medium hESF9. FIG. 1C is an ALP-stained image (36 days after infection) of hiPS cells on each extracellular matrix. The scale bar in FIGS. 1A-C is 200 μm. FIG. 2 is a microscopic image showing cell morphology on various extracellular matrices after induction. The upper row is a cell morphology image of TIG-3 cell-derived human iPS cells induced on various extracellular matrices before colony recovery. The lower row is a cell morphology image after seeding on each extracellular matrix using serum-free medium hESF9. The scale bar is 200 μm. FIG. 3 is a micrograph showing the examination results of serum-free culture conditions when virus infection is established and a graph showing virus infection efficiency. The results of transfection into PLAT-A using EGFP and infection into TIG-3 cells are shown. The top row shows the cell morphology two days after introduction of the pMXs- (EGFP) gene under the conditions of serum culture conditions (left) or hESF9 serum-free culture conditions (right) with 10% FBS added to DMEM medium. The lower row shows the virus infection efficiency to TIG-3 cells. The scale bar is 200 μm. FIG. 4 is a scheme and microscopic image showing the induction of hiPS cells using dental pulp-derived cells under serum-free culture conditions without using feeder cells. FIG. 4A is a scheme showing an outline up to hiPS cell induction. Day-7-7: Primary culture of dental pulp cells on type I collagen-coated dish using serum-free medium RD6F. Day 0 to 4: Reprogrammed gene transfer by retrovirus infection using serum-free medium hESF9 (Oct3 / 4, Sox2, KLF-4, c-Myc). Day 5: Induction of human iPS cells on a fibronectin-coated dish using a serum-free medium hESF9 medium. Day 6-30: Medium exchange every other day. FIG. 4B is a phase contrast microscopic image of dental pulp cells in serum-free medium RD6F before introduction of reprogramming gene. FIG. 4C is a phase-contrast microscope image of pulp cells that have been passaged 4 times on a type I collagen-coated dish using serum-free medium RD6F. FIG. 4D is an ALP-stained image 43 days after infection. The scale bar is 200 μm. FIG. 5 shows the results of analyzing the characteristics of human iPS cells derived from dental pulp-derived cells under culture conditions using serum-free medium hESF9 without using feeder cells. FIG. 5A is a phase contrast image of human iPS cells maintained in serum-free medium hESF9 or hESF9T medium (serum-free medium obtained by adding TGF-β1 to hESF9). DP-A-iPS-CL1: Phase contrast image of cells maintained for 2 generations (passage 2) or 21 generations (passage 21) on fibronectin-coated dishes using serum-free medium hESF9. The right side shows a phase contrast image of cells maintained for 5 generations (passage 5) on feeder cells using a serum-supplemented medium for human ES cells. DP-F-iPS-CL4, -CL6, -CL16: Cells maintained in the 58s (passage 58), 59s (passage 59) or 21s (passage 21) on the fibronectin-coated dish using the serum-free medium hESF9T Phase contrast image. The right side shows a phase contrast image of a cell (CL31) maintained for 19 generations (passage 19) on a feeder cell using a serum-supplemented medium for human ES cells. The scale bar is 200 μm. FIG. 5B shows flow cytometry using cells that have been passaged and maintained on a fibronectin-coated dish for a long time using serum-free medium hESF9 or hESF9T, and antibodies against Oct3 / 4 and SSEA-4 that are undifferentiated markers. The result of having analyzed by is shown. FIG. 5C shows the results of comprehensive gene expression analysis. Cluster analysis using a microarray was performed. DP cell (dental pulp cell), DP-iPS cells (dental pulp-derived human iPS cells) maintained on a fibronectin-coated dish using serum-free medium hESF9 or hESF9T, and feeder medium using serum medium for human ES cells The maintained human iPS cells (Tic) were analyzed. Lanes 1-7 in the figure are as follows. 1. DP cell (DP-A): passage 2 = before infection 2. DP cell (DP-F): passage 4 = before infection 3. DP-A-iPS-CL1: passage 14 = serum-free condition (hESF9 / on FN) 4. DP-F-iPS-CL12: passage 36 = KSR-based condition (KSR / on MEF) 5. DP-F-iPS-CL6: passage 37 = serum-free condition (hESF9T / on FN) 6 DP-F-iPS-CL8: Passage 35 = Serum-free condition (hESF9T / on FN) 7. Tic (hiPSC: JCRB1331): Passage 58 (= KSR-based condition (KSR / on MEF) FIG. 6 shows the results of comprehensive gene expression analysis (scatter-plot analysis). FIG. 7 shows the results of examining the effect of TGF-β1 under serum-free culture conditions. FIG. 7A shows the cell morphology when various concentrations of TGF-β1 (0, 0.1, 1, 2, 5, 10 ng / ml) are added. FIG. 7B shows the results of gene expression analysis of human iPS cells using Droplet® Digital-PCR when TGF-β1 was added. Each expression intensity was corrected by GAPDH. FIG. 7C shows the results of undifferentiated marker gene expression analysis of human iPS cells induced and maintained under serum-free culture conditions. # 1: DP cell (DP-A): passage 2 = before infection # 2: DP cell (DP-F): passage 4 = before infection # 3: DP-A-iPS-CL1: passage 14 = serum-free condition (hESF9 / on FN) # 4: DP-F-iPS-CL4: passage 37 = serum-free condition (hESF9T / on FN) # 5: DP-F-iPS-CL6: passage 35 = serum-free condition (hESF9T / on FN) # 6: DP-F-iPS-CL8: passage 35 = serum-free condition (hESF9T / on FN) # 7: DP-A-iPS-CL1: passage 8 = KSR-based condition (KSR / on (MEF) # 8: DP-F-iPS-CL12: passage 36 = KSR-based condition (KSR / on MEF) # 9: Tic (hiPSC: JCRB1331): passage 103 = KSR-based condition (KSR / on MEF) Figure 7D shows the result of expression analysis of undifferentiated marker protein in human iPS cells induced under serum-free culture conditions and subcultured and maintained in hESF9T medium. The scale bar is 100 μm. FIG. 8 shows the results of pluripotency analysis of human iPS cells induced and maintained under serum-free culture conditions. FIG. 8A shows the results of studies on the expression of various differentiation markers. FIG. 8B shows the results of examination of teratoma formation ability. The scale bar is 100 μm. FIG. 9 shows the results of cell proliferation ability and karyotype analysis. FIG. 9A shows the results of examining the cell doubling time of human iPS cells induced under serum-free culture conditions and passaged and maintained for 21 passages with hESF9T. FIG. 9B shows the result of karyotype simplified analysis of human iPS cells induced under serum-free culture conditions and maintained for 20 passages in serum-free medium hESF9T. FIG. 10 shows the results of studies on universality in passage and maintenance of human iPS cells using serum-free culture conditions. The scale bar is 200 μm. The upper part of FIG. 10 shows a phase contrast microscopic image of human iPS cells maintained on fibronectin using hESF9T medium. The lower panel shows a phase contrast microscopic image of human iPS cells maintained on fibronectin using DF8T medium. It is shown that both serum-free media maintain human ES cell-like morphology.

In the first aspect, the present invention is characterized in that induced pluripotent stem cells (iPS cells) are induced by culturing somatic cells subjected to reprogramming treatment in a serum-free medium without using feeder cells. A method for producing iPS cells is provided. As described below, an iPS cell refers to a cell that has been subjected to reprogramming by introducing a plurality of genes into a somatic cell to have differentiation pluripotency and self-replication ability. In the present invention, iPS cells may be derived from somatic cells of any animal, and iPS cells derived from somatic cells such as humans, monkeys, mice, rats, dogs, cats, horses, pigs and the like are exemplified. However, the present invention is not limited to these.

Reprogramming is dedifferentiation of differentiated somatic cells to change them into undifferentiated cells, which is also called initialization. Differentiated cells become iPS cells by the reprogramming process. In general, four types of genes (Oct3 / 4, Sox2, Klf4, c-Myc (or L-Myc)), or some of these genes (for example, Nanog, Lin28, etc.) plus differentiated cells The reprogramming process is performed by introducing into the above. A vector is generally used for gene introduction, and for example, a retrovirus vector is used. The reprogramming process is not limited to the above method, and can be performed using a method known to those skilled in the art. The medium on which the reprogramming process is performed is not particularly limited, but in the present invention, the reprogramming process is preferably performed in a serum-free medium.

The feature of the present invention is to induce iPS cells by culturing somatic cells subjected to reprogramming treatment in a serum-free medium. By using a serum-free medium, concerns that uncertain elements, unknown factors, or risk factors exist in the medium can be eliminated, and various analyzes can be easily performed with good reproducibility. In addition, since a harmful factor derived from a serum-containing medium can be eliminated by using a serum-free medium, the safety of the obtained iPS cells is ensured.

In addition, it is necessary to separate iPS cells and feeder cells at the time of passage, the operation becomes complicated, various factors derived from the feeder cells affect the culture, and the variation of the feeder cells themselves is large. Problems such as poor reproducibility of cell induction are also solved by the present invention that does not use feeder cells.

Serum-free medium is a medium that does not contain serum. Specifically, a serum-free medium is a basic culture solution containing amino acids, inorganic salts, vitamins, trace elements, sugars, etc., with known hormones and protein factors such as insulin and iron-binding protein. A medium containing only factors and capable of maintaining functions such as cell growth and differentiation and hormone secretion ability in the same manner as in vivo. Examples of the basal medium for animal cell culture that does not contain serum include Dulbecco's modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640 medium, F12 medium, MCDB medium, In addition, mixed media thereof and the like are known.

Examples of preferable serum-free medium used in the iPS cell induction method of the present invention include hESF9 medium, hESF9T medium, RD8F medium, DME / F12-8F medium, RDF8F medium, and modified media thereof. The serum-free medium used in the present invention is not limited to these media, and those skilled in the art can appropriately select and use known serum-free media, or can modify and use known serum-free media as appropriate.

Preferably, fibronectin is used as an adhesion factor in the induction of iPS cells in a serum-free medium without feeder cells. By using fibronectin as an adhesion factor, the induction efficiency of iPS cells can be dramatically increased, and iPS cell induction efficiency compared to the case of inducing iPS cells by a conventional method (in a serum-containing medium, using feeder cells). Rises about 10 times.

Fibronectin is a protein known to those skilled in the art. Various biological sources of fibronectin are known and can be used in the present invention. The fibronectin used in the present invention may be isolated from cells or may be produced by a genetic engineering method such as a recombinant method. The fibronectin used in the present invention may be full length or a fragment thereof.

The somatic cells that have undergone the reprogramming treatment are seeded in a culture vessel coated with fibronectin, and cultured to induce iPS cells. When reprogramming is performed in a medium other than a serum-free medium, somatic cells are seeded in a culture vessel coated with fibronectin together with the medium, and then cultured while exchanging the medium with the serum-free medium. Can be guided. When reprogramming is performed in a serum-free medium, somatic cells are seeded in a culture vessel coated with fibronectin together with the serum-free medium, and then cultured while changing the medium to the same or different serum-free medium. Cells can be induced. Methods of seeding and medium exchange, and culture conditions for induction of iPS cells are known to those skilled in the art. For example, the culture temperature can be 30 to 40 ° C., preferably about 37 ° C., and for example, the culture can be performed in an incubator filled with air containing 5 to 10% carbon dioxide.

A method for coating fibronectin on a culture vessel is known to those skilled in the art. For example, the surface of the culture container is treated with poly-L-ornithine or poly-L-lysine, washed, and then the surface of the culture container is treated with a buffer containing fibronectin to coat fibronectin on the culture container. can do. A person skilled in the art can easily determine the amount of fibronectin to be used. In the above method of the present invention, an adhesion factor other than fibronectin may be used in combination with fibronectin.

Methods for coating culture vessels with fibronectin are known. An example of a preferable coating method is as follows. The culture vessel is coated with fibronectin at a concentration of ² μg / cm ² and allowed to stand at 37 ° C. for 3 hours or longer and overnight. If not used immediately, seal with parafilm to prevent the coating solution from drying, store at 4 ° C., and use within 1 week. During use, the fibronetin solution is aspirated and washed once with PBS before use.

In the method for producing iPS cells of the present invention, subculture of somatic cells to be reprogrammed may be performed from primary culture in a serum-free medium, and somatic cell reprogramming may be performed in a serum-free medium. . Such embodiments are described in the Examples herein by conducting subculture of somatic cells from primary culture. Primary culture and subculture of somatic cells can be performed by those skilled in the art by appropriately selecting the medium composition and culture conditions according to the type of somatic cells.

In the second aspect, the present invention provides a serum-free culture substrate for inducing iPS cells, which contains fibronectin as an adhesion factor. The culture substrate of this aspect of the invention includes a serum-free medium and fibronectin. Therefore, one specific example of the culture substrate of this aspect of the present invention is a serum-free medium containing fibronectin. Further embodiments of the culture substrate of this aspect of the invention may include a culture vessel coated with fibronectin and a serum-free medium contained therein. However, the culture substrate does not contain feeder cells. The shape of the culture container includes a dish, a bottle, a tube, a flask, a bag and the like, and is not particularly limited.

In the third aspect, the present invention provides a kit for producing a serum-free culture substrate for inducing iPS cells, comprising a serum-free medium component and fibronectin as essential components. However, the culture substrate does not contain feeder cells.

Examples of the serum-free medium include those exemplified above and those obtained by modifying them. The components are also known or can be appropriately selected by those skilled in the art. The form of the serum-free medium component contained in the kit is not particularly limited. For example, it may be a liquid that can be transferred to a culture vessel as it is, may be in the form of a concentrate, or may be a solid such as a powder. The serum-free medium components included in the kit may be included in the kit, for example, divided into vitamins, minerals, amino acids, saccharides and the like.

The form of fibronectin contained in the kit of the present invention is not particularly limited. For example, the fibronectin may be a solid such as a powder or a liquid such as an aqueous fibronectin solution containing an appropriate buffer.

If desired, a culture vessel may be included in the kit of the present invention. The culture vessel may be made of any material such as glass or plastic. There are no particular limitations on the shape of the plate, bottle, tube, flask, bag, or the like. The culture container contained in the kit of the present invention may be coated with fibronectin in advance.

Usually, an instruction manual is attached to the kit.

In the fourth aspect of the present invention, the stem cell is subcultured by adding a tumor (transforming) growth factor-β (TGF-β) superfamily protein to a serum-free medium without using feeder cells. A method for maintaining the undifferentiated state and differentiation pluripotency of stem cells is provided. In the present invention, by adding a protein belonging to the TGF-β superfamily, particularly a protein belonging to the TGF-β family, to a serum-free medium, the cells can be passaged over a long period of time while maintaining the undifferentiated state and differentiation pluripotency of stem cells. It becomes possible to culture.

Proteins belonging to the TGF-β superfamily include TGF-β family, activin family, and bone morphogenetic protein (BMP) family proteins. Preferred in the present invention are proteins belonging to the TGF-β family. Proteins belonging to the TGF-β family are known to those skilled in the art, and TGF-β forms a family with activin and BMP and is currently composed of 33 types of family molecules in humans, including TGF-β1, Activin- A, BMP-2 and the like are exemplified. A particularly preferred protein belonging to TGF-β used in the above method of the present invention is TGF-β1.

In the method of this aspect of the present invention, the concentration of the TGF-β family protein in the serum-free medium is usually 1 ng / ml to 10 ng / ml, preferably 1 ng / ml to 5 ng / ml, but may be changed as appropriate. Can do. The TGF-β family protein used may be one type or two or more types.

TGF-β family proteins are known to those skilled in the art. Various biological TGF-β family proteins are known and can be used in the present invention. The TGF-β family protein used in the present invention may be isolated from cells or may be produced by a genetic engineering method such as a recombinant method. Further, the TGF-β family protein used in the present invention may be a full-length protein or a fragment thereof.

Preferred serum-free media used in the method for maintaining the undifferentiated state and differentiation pluripotency of the stem cells of the present invention include media such as hESF9 media, hESF9T media, RD8F media, DME / F12-8F media, RDF8F media, ESF7 media, etc. As well as their modified media.

In the above method of the present invention, it is preferable to use fibronectin as an adhesion factor. The method for coating fibronectin on the culture container and other explanations regarding fibronectin are as described above.

The stem cell to which the above-described method of the present invention can be applied is not particularly limited, and includes all stem cells including those exemplified below.

Stem cells are cells that have the ability to differentiate into cells of multiple lineages (differentiation pluripotency) and the ability to maintain differentiation pluripotency even after cell division (self-renewal ability). Stem cells include embryonic stem cells (ES cells) produced from fertilized eggs, somatic stem cells present in in vivo tissues, and induced pluripotent stem cells (iPS cells) created by introducing a specific gene. ES cells and iPS cells have the property of being able to differentiate into all types of cells (totipotency). Examples of somatic stem cells include hematopoietic stem cells, neural stem cells, hepatic stem cells, skin stem cells, and reproductive stem cells. In the present invention, the stem cells may be derived from any animal, and examples thereof include, but are not limited to, stem cells derived from humans, monkeys, mice, rats, dogs, cats, horses, pigs, and the like. The methods for producing and obtaining these stem cells are known to those skilled in the art, and some are stored in research institutions and others are commercially available. ES cells can be established by taking an inner cell mass from a blastocyst of a fertilized egg of a subject animal and culturing it on feeder cells such as fibroblasts. The method for producing iPS cells is as described above.

Subculture refers to the growth and maintenance of transferred cultured cells by transferring them to a new culture vessel. Methods for subculturing stem cells are known to those skilled in the art. In general, before the cells become confluent, a part of the cells can be detached from the culture vessel using a digestive enzyme such as trypsin, and subculture can be performed by culturing using a new culture vessel. . Selection of medium components and other culture conditions, timing of medium replacement, stripping conditions, culture time per passage, etc., techniques and techniques for subculture are appropriately determined by those skilled in the art according to the cells to be subcultured, You can choose.

The above method of the present invention is characterized in that stem cells are subcultured by adding TGF-β family protein in a serum-free medium without using feeder cells. Preparation and selection of a serum-free medium is easy for those skilled in the art. According to the method of the present invention, by adding a TGF-β family protein to the medium, even when many passages are repeated over a long period of time, the undifferentiated state and the differentiation level of the stem cells are stably maintained. Performance can be maintained.

Confirmation of the undifferentiated state of stem cells can be performed by examining the expression of undifferentiated markers such as Oct3 / 4, Nanog, Sox2, and SSEA-4 in addition to morphological observation. The method for confirming the undifferentiated state of stem cells is not limited to the above method, and can be performed by methods known to those skilled in the art.

Confirmation of the differentiation pluripotency of stem cells can be performed by forming embryonic rods from passaged and maintained stem cells, inducing differentiation, and examining the expression of various differentiation markers. The method for confirming the pluripotency of stem cells is not limited to the above method, and can be performed by methods known to those skilled in the art.

In one specific example of this aspect of the present invention, iPS cells obtained by the method for producing iPS cells of the present invention are passaged without using feeder cells in a serum-free medium supplemented with a protein belonging to the TGF-β family. By culturing, the undifferentiated state and pluripotency of iPS cells can be maintained. In the iPS cell production method of the present invention, when iPS cells are induced using a serum-free medium containing a TGF-β family protein, the induction efficiency may decrease. It is preferred not to use family proteins.

In the fifth aspect, the present invention provides a serum-free culture substrate for subculture for maintaining the undifferentiated state and differentiation pluripotency of stem cells, which comprises a TGF-β family protein. The culture substrate of this aspect of the invention includes a serum-free medium containing a TGF-β family protein. Thus, one specific example of this aspect of the invention is a serum-free medium containing a TGF-β family protein. A serum-free medium containing a TGF-β family protein may be provided as a liquid that can be used as it is by adding it to a culture vessel, or may be provided as a solid such as a concentrate or powder that can be prepared at the time of use. . Further specific examples of the culture substrate of this aspect of the invention may include a culture vessel and a serum-free medium containing the TGF-β family protein contained therein. The kind of TGF-β family protein and the concentration in the serum-free medium are as described above. The shape of the culture container includes a dish, a bottle, a tube, a flask, a bag and the like, and is not particularly limited. Preferably, in the culture substrate, the culture vessel is coated with fibronectin. However, the culture substrate does not contain feeder cells.

In a sixth aspect, the present invention provides a serum-free culture substrate for subculture for maintaining the undifferentiated state and differentiation pluripotency of stem cells, comprising a serum-free medium component and a TGF-β family protein. Providing a kit for The concentration of the TGF-β family protein contained in the culture medium in the culture substrate is as described above. However, the culture substrate does not contain feeder cells.

In the culture substrate prepared with the kit of the present invention, it is preferable that fibronectin is included as an adhesion factor. The form of fibronectin contained in the kit of the present invention is not particularly limited. For example, the fibronectin may be a solid such as a powder or a liquid such as an aqueous fibronectin solution containing an appropriate buffer.

Usually, an instruction manual is attached to the kit.

) Except for specially explained terms, the terms used in this specification are understood to have the meaning normally understood by those skilled in the art through publications, textbooks, dictionaries, and the like.

Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the Examples are not intended to limit the present invention.

Serum-free medium hESF9 (Furue MK, Na J, Okamoto T, et al. (2008) Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium. Proc Natl Acad Sci USA -13414), each process until induction of iPS cells was examined under serum-free culture conditions. Furthermore, using human dental pulp-derived cells, we attempted to establish and maintain hiPS by performing the entire process from primary culture to viral infection and iPS cell establishment in a complete serum-free culture system.

1. Cell culture method 1-1 Cell culture solution 1) Serum-free medium The serum-free medium used contains hESF-GRO medium (Nipro) (L-ascorbic acid-2-phosphate (100 μg / ml)) developed for human ES cells. ) (Table 1), human recombinant insulin (10 μg / ml) (CSTI 0105, Japan), human transferrin (5 μg / ml) (Sigma T-1147), 2-ethanolamine (10 μM) (Sigma E -0135), 2-mercaptoethanol (10 μM) (Sigma M-7522), sodium selenate (20 nM) (Sigma S-9133), and human recombinant albumin (0.5 mg / ml) (Cell Prime Albumin: Millipore 9301 HESF6 medium supplemented with oleic acid (4.7 μg / ml) (Sigma O-1383) contained in) was added to heparan sulfate sodium salt (100 ng / ml) (Sigma H-7640) and Human recombinant fibroblast growth factor -2 (10ng / ml) (FGF -2: R & D, 3718FB) using hESF9 medium supplemented with (Table 2), bovine plasma-derived fibronectin (2 [mu] g / ^cm 2) (Sigma F -1141) was cultured on the dish coated. In addition, the serum-free medium RD6F used for dental pulp cell culture was prepared by mixing RPMI1640 medium (Sigma) and DMEM medium (Sigma) at a ratio of 1: 1, bixillin (90 mg / ml) (Meiji, Japan), kanamycin (90 mg / day). ml) (GIBCO), sodium pyruvate (110 mg / ml) (Sigma), 15 mM HEPES (Dojindo), sodium bicarbonate (2 g / L) (Sigma) as a basal medium, and six additional factors (6F) That is, human recombinant insulin (10 μg / ml) (CSTI 0105, Japan), human transferrin (5 μg / ml) (Sigma T-1147), 2-ethanolamine (10 μM) (Sigma E-0135), 2 Mercaptoethanol (10 μM) (Sigma M-7522), sodium selenate (20 nM) (Sigma S-9133), and human recombinant albumin (500 μg / ml) (Cell Prime Albumin: Millipore 9301) Inclusion oleic acid (4.7μg / ml) (Sigma O-1383) is a medium supplemented with, it was cultured in pulp cells on type I collagen coated dish using the culture medium.

2) Serum supplemented medium for human ES cells 20% Knock Out Serum Replacement in DMEM-F12 (GIBCO 12660-012) medium: KSR (Invitrogen 12828-028), 0.1 mM 2-mercaptoethanol (GIBCO 21985-023), MEM C57 / BL6 inactivated by treatment with mitomycin C (10 ug / ml) using a medium supplemented with non-essential amino acids (GIBCO 11140-050) and recombinant human FGF-2 (4 ng / ml) (R & D 3718FB) The cells were seeded on mouse fetal fibroblasts (Millipore: EmbryoMax® PMEF-H) and cultured.

1-2 Cultured cell line and established human iPS cell line and culture method 1) Human fetal lung-derived normal fibroblasts (TIG-3 cells)
TIG-3 cells, which are Japanese fetal lung-derived fibroblasts established by the Tokyo Metropolitan Institute of Health and Longevity Medical Center, have 4 genes (Oct3 / 4, Sox2, Klf4, c) using retrovirus. -Myc) was introduced and human iPS cells were induced. TIG-3 cells were prepared by adding 10% fetal bovine serum (Hyclone® Thermo Scientific, US) and 1% penicillin-streptomycin (GIBCO) to DMEM medium (Sigma) at a ratio of 1: 4 every 2-3 days. The cells were seeded on a 10 cm dish (Falcon) at a split ratio of 5% CO ₂ /95% in a gas phase and cultured in a 37 ° C. incubator.

2) PLAT-A virus-packaging cell line Virus-packaging cell line Platinum-A Retroviral Packaging that is capable of producing retrovirus structural proteins (gag, pol, env) stably for a long period of time. Retrovirus production was performed using Cell, amphotropic (PLAT-A: Cell Biolabs Inc. CA, USA). Every two days using a medium supplemented with 10% FBS, 1 μg / ml puromycin (Sigma) and 10 μg / ml blasticidin (Funakoshi, Tokyo, Japan), 1% penicillin-streptomycin (GIBCO) in DMEM medium (Sigma) Cells were seeded on a 10 cm dish (Falcon) at a split ratio of 1: 4 and cultured in a CO ₂ incubator at 37 ° C. in a 5% CO ₂ /95% gas phase.
When transfection is performed, a medium in which 10% FBS is added to DMEM medium the day before is used, and 2 × 10 ⁶ cells are seeded in a collagen-coated 25 cm ² flask (BioCoat Collagen I Cellware, Falcon) After 16 to 24 hours, when the cells became 70% to 80% confluent, the cells were used for gene transfer (transfection), and the virus supernatant 48 to 72 hours after the start of transfection was collected and infected with target cells.

3) Established human iPS cell line (Tic) and its culturing method Tic (JCRB1331), established by the National Center for Child Health and Development and established by the National Institute of Biomedical Innovation, was used as a control iPS cell. Inoculate Tic with 0.1% gelatin in advance on inactivated mouse fetal fibroblasts (Millipore: PMEF-H) seeded as feeder cells and culture using serum-supplemented ES cell medium. went. The cell dispersion was subcultured using Despase II (1 mg / ml) (Roche 4942078, Basel, Switzerland).

2. Examination of serum-free culture conditions for human iPS induction from TIG-3 cells Using human fetal lung fibroblast TIG-3 cells, 4 genes of Oct3 / 4, Sox2, Klf4 and c-Myc were converted into retrovirus (PLAT). -A packaging cells) were used to induce human iPS cells.

1) Preparation of retrovirus 1-2 A retrovirus was prepared by introducing the reprogramming 4 gene into PLAT-A cells passaged and maintained according to the method of 2). The plasmids used were pMXs- (hOct3 / 4), pMXs- (hSox2), pMXs- (hKlf4), pMXs- (hc-Myc) (Cell Biolabs Inc. CA, USA) prepared in Yamanaka Laboratory, Kyoto University. Was used.
The day before transfection, cells were seeded at a density of 2 × 10 ^{6 in} 25 cm ² flasks (BioCoat Collagen I Cellware, Falcon) treated with type I collagen from PLAT-A cells, and 70% -80% confluent after 16-24 hours. Then, a mixed solution obtained by diluting 4 μg of a plasmid solution and a transfection reagent in DMEM medium was added, and this complex solution was added to a flask seeded with PLAT-A cells, and transfection was performed. Further, serum-added medium was added after 4 hours, and the medium was replaced with DMEM medium supplemented with 10% FBS on the next day. The virus supernatant 24 to 48 hours after the start of transfection was collected, and then infected with target cells. A total of four 25 cm ² flasks were prepared for transfection for each plasmid.

2) Examination of serum-free culture conditions during the reprogramming process (1) Virus infection and seeding on the extracellular matrix PIG (population cell doubling number) 24-40 TIG-3 cells cultured in 10% FBS-added DMEM medium Infected with retrovirus, 4 genes were introduced. Thereafter, the medium was replaced with a serum-free medium hESF9, seeded on various extracellular matrices, and serum-free culture conditions in the reprogramming process were examined.
The day before the virus infection (Day-1), TIG-3 cells cultured in DMEM medium supplemented with 10% FBS were seeded on a 6 cm dish with 3 × 10 ⁵ cells. The next day (Day 0), pMXs- (4F) mixed with an equal amount of each virus supernatant was added with polybrene to a final concentration of 8 μg / ml, filtered through a 0.45 μm filter, and then maintained in a serum-added medium. TIG-3 cells were infected. In order to attenuate the toxicity of polybrene 4 hours after the infection, the same amount of medium as the virus supernatant was added. The medium was changed 24 hours after infection (Day 1), and culture was performed for 4 days (until Day 4). The medium was changed every 2 days. Four days after infection (Day 4), TIG-3 cells were dispersed into single cells by 0.05% trypsin / EDTA treatment. Subsequently, 0.1% gelatin (Millipore ES-006B), type I collagen (0.3 mg / ml) (Nitta gelatin) or fibronectin (2 μg / cm ² ) (Sigma) was coated with various extracellular matrices in advance. It seed | inoculated so that it might become the cell number of 1x10 < ⁵ > / dish on a 10 cm dish. The day after re-seeding (Day 5), the medium was changed to the serum-free medium hESF9, and the induction of human iPS cells was attempted by seeding on various extracellular matrices without using feeder cells. The effect was examined (Figure 1). Medium exchange after re-seeding was performed every 2 days.
On the extracellular matrix of any of gelatin, type I collagen, and fibronectin, from around 13 days after infection, human ES cell-like colony formation with a large nucleus compared to the cytoplasm and small cell boundaries was observed. (FIG. 1). From 20 days after the infection, colonies were collected using a pipette under a microscope, seeded on a 4-well multi-dish previously coated with each extracellular matrix, and subcultured. In the dishes seeded on gelatin, cell adhesion was weak, and even if they adhered, colonies tended to differentiate significantly. Furthermore, in the dishes seeded on type I collagen, colonies adhered, but showed a tendency to differentiate significantly from the edges. On the other hand, in the dish seeded on fibronectin, it was possible to maintain and maintain the morphology of human iPS cells after cell attachment (FIG. 2). In addition, ALP staining, which is an undifferentiated index, was performed 36 days after infection, and the colonies on any dish showed a positive reaction. Cells seeded on fibronectin and collagen were most morphologically similar to human iPS cells.
As a control, TIG-3 cells 4 days after virus infection (Day 4) were cultured on a 10 cm dish in which feeder cells previously inactivated with mitomycin C (10 μg / ml) were seeded, and human ES from the next day (Day 5). The serum medium for cells was changed, and thereafter the medium was changed every two days. Although colonies appeared even under the main culture conditions, many colonies were incompletely initialized, and the colony appearance time was delayed as compared to serum-free culture conditions without using feeder cells (FIG. 1).

(2) Evaluation of infection efficiency (FACS analysis)
Simultaneously with the infection of the 4 genes, FACS analysis was performed using EGFP in order to evaluate the infection efficiency of the retroviral packaging cell PLAT-A used.

(I) Factor 1 infection efficiency According to the method of 2-1 1) and 2) described above, PLAT-A cells were transfected with pMXs- (EGFP) or pMXs-(-), and each virus supernatant was collected. Thereafter, each TIG-3 cell was infected, and 4 days later, the TIG-3 cell was collected in a FACS tube with cell strainer (BD Falcon) and analyzed with a flow cytometer (FACS Calibur ™, Beckton Dickinson).

(Ii) Four-factor infection efficiency According to the method of 2-1 1) and 2) described above, PLAT-A cells were treated with pMXs- (hOct3 / 4), pMXs- (hSox2), pMXs- (hKlf4), pMXs- ( hc-Myc) each transfected virus supernatant mixture (equal to pMXs- (4F)), or pMXs- (4F) and pMXs- (EGFP) virus supernatant in a ratio of 3: 1 Infect the TIG-3 cells with each of the virus mixtures mixed in 1. After 4 days, collect the TIG-3 cells in a FACS tube with cell strainer (BD Falcon) and analyze with a flow cytometer. 4 factor infection efficiency.
When the single factor infection efficiency was evaluated by FACS, the average was 43.4%. Next, when the four-factor infection efficiency was evaluated, the infection efficiency was slightly reduced to 26.6%, but average efficiency was obtained, so we tried to induce human iPS cells (hiPS cells) using this system. .

3) Examination of serum-free culture conditions when virus infection is established Retrovirus supernatant is collected in serum-free medium hESF9, and 24 hours after virus infection is established as serum-free medium hESF9. The effect was examined (Figure 3).
PLAT-A cells were transfected with pMXs- (EGFP) or pMXs-(−) under serum-added culture conditions with 10% FBS added to DMEM medium, and serum medium conditions with 10% FBS added to DMEM medium ( The virus supernatant 24 to 48 hours after transfection was collected under each condition of condition A) or hESF9 serum-free medium condition (hereinafter referred to as condition B), and infected with TIG-3 cells as target cells. I let you. 24 hours after virus infection is established under the above conditions A or B, and thereafter, both conditions are cultured under the condition of serum-added medium A. Three days later, TIG-3 cells are collected in a FACS tube with a cell strainer. The analysis was performed with a flow cytometer (FACS Calibur ™, Beckton Dickinson).
In the serum-added culture condition A, the infection efficiency was 62.6%, and in the serum-free culture condition B, the infection efficiency was 46.4%. Although serum-free culture condition B showed a slightly lower efficiency than condition A, it was generally considered that the titer (titer) necessary for hiPS induction was maintained.

3. Induction of human iPS cells from dental pulp-derived cells under serum-free culture conditions without feeder cells Based on the results of previous experiments using TIG-3 cells, retrovirus supernatant was used under serum-free culture conditions. Thus, the entire process from primary culture to viral iPS cell induction through human infection was performed in a complete serum-free culture system to examine whether human iPS cells could be induced (FIG. 4).

1) Primary culture of dental pulp-derived cells Based on a research plan approved by the Hiroshima University Human Genome / Gene Analysis Research Ethics Committee, after undergoing jaw and oral surgery at Hiroshima University Hospital, after extraction of a patient with consent The pulp-derived cells were separated from the dental pulp tissue and primary culture was performed using the serum-free medium by the expand culture method.
Dish coated with type I collagen (0.15 mg / ml) (Nita gelatin) using the RD6F medium described below after immersing the pulp separated from the extracted tooth of the mandibular impacted dentition in 70% ethanol. On top, the cells were cultured in a CO ₂ incubator at 37 ° C. in a 5% CO ₂ /95% gas phase. The medium is exchanged every 2 to 3 days, and after cell growth by treatment with 0.05% trypsin / EDTA at the stage of growth to sub-confluence, 0.1% trypsin inhibitor is added to neutralize trypsin action, and the cells on the dish are neutralized. And subcultured every 3-4 days.
RD6F shown below was used as a serum-free medium. Specifically, DMEM medium (Sigma) and RPMI 1640 medium (Sigma) were mixed at a ratio of 1: 1, and bivicillin (90 mg / ml) (Meiji, Japan), kanamycin (90 mg / ml) (GIBCO), sodium pyruvate (110 mg / ml). ) (Sigma), 15 mM HEPES (Dojindo), sodium bicarbonate (2 g / L) (Sigma) added as a basal medium, 6 kinds of additive factors insulin (10 μg / ml) (CSTI 0105), transferrin ( 5 μg / ml) (Sigma T-1147), 2-ethanolamine (10 μM) (Sigma E-0135), 2-mercaptoethanol (10 μM) (Sigma M-7522), sodium selenate (10 nM) (Sigma S-9133) ) And oleic acid (4.7 μg / ml) conjugated to fatty acid-free bovine serum albumin (Sigma O-3008) and RD6F. , And culture was performed.

2) Retrovirus infection and human iPS cell induction Dental pulp-derived cells that were primarily cultured in serum-free medium RD6F were virus-infected under serum-free conditions throughout the entire process, seeded on fibronectin, and induced.
Cells that had been subcultured for about 2 weeks from the start of the primary culture were seeded on a 60 mm dish at 3 × 10 ⁵ cells on the day before virus infection (Day-1). The next day (Day 0), polybrene (8 μg / ml) was added to the retroviral supernatant pMXs- (4F) collected in the serum-free medium hESF9, and then infected with dental pulp-derived cells. An equal amount of serum-free medium hESF9 was added 4 hours after infection, the medium was changed 24 hours later, and each cell was dispersed into single cells by 0.05% trypsin-EDTA treatment 5 days after infection (Day 5). On a dish coated with fibronectin (2 μg / cm ² ), seeding was performed so that the number of cells was 1.0 × 10 ⁵ cells / 10 cm. After re-seeding, the medium was changed every 2 days using a serum-free medium hESF9 medium (FIG. 4). An iPS-like colony appeared 15 days after the infection, and the colony 20 days after the infection was collected, seeded on a fibronectin-coated dish, and medium hESF9T supplemented with serum-free medium hESF9 or hESF9 with TGF-β1 (2 ng / ml). Was used for subculture. In the complete serum-free culture system, the number of colonies positive for ALP activity was large (FIG. 4D), and iPS cell induction efficiency was as high as 0.39%. Moreover, high induction efficiency was also shown compared with the literature using the same vector (Table 3). Furthermore, the conditions were also examined in which the medium was replaced with hESF9 without re-seeding 5 days after virus infection, but the change in cell morphology was poor and no colonies were formed. In addition, human iPS cell-like colonies induced under KSR-added culture conditions are seeded on feeder cells inactivated with mitomycin C, cultured using serum medium for human ES cells, and before differentiation. Colonies were collected and subcultured under a microscope.

3) Maintenance of undifferentiated human iPS cells derived from dental pulp cells (1) Effect of TGF-β1 on maintenance of proliferative ability and undifferentiation of human iPS cells HiPS cells established from dental pulp-derived cells were used using a micropipette. After mechanical reduction, the seeds were plated on fibronectin-coated dishes using a serum-free medium hESF9 or hESF9T medium supplemented with TGF-β1 (FIG. 5A). Although the hiPS cells maintained in the serum-free medium hESF9 adhere to the dish, it became difficult to sufficiently maintain undifferentiation along with long-term subculture, and migration of cells gradually differentiated from the margin was observed. On the other hand, human iPS cells maintained in serum-free medium hESF9T containing TGF-β1 could be subcultured while maintaining undifferentiation (FIG. 7A). In addition, various concentrations (0, 0.1, 1, 2, 5, 10 ng / ml) of TGF-β1 were added to hESF9 medium, and total RNA was recovered after 4 days of culture. After cDNA synthesis, QX100 ^™ Droplet Digital Gene expression analysis was performed using ^TM PCR system (Bio-RAD). Droplet Digital ^TM PCR uses ddPCR ^TM supermix (Bio Rad) according to the attached protocol, creates droplets with QX100 ^TM Droplet Generator (Bio Rad), amplifies it with PCR reaction, then QX100 ^TM Droplet Reader (Bio Rad) ) Was used for analysis. The number of cells that remained undifferentiated increased depending on the TGF-β1 concentration, and the expression of Oct3 / 4 and Nanog undifferentiated marker genes was enhanced (FIG. 7B). The TGF-β1 concentration was 2 to 10 ng / ml, and the most undifferentiated marker was highly expressed. However, in the absence of TGF-β1, the expression of the undifferentiated marker gene was remarkably reduced, and the mesoderm differentiation marker was positive. Expression of differentiation markers such as minogen activator inhibitor-1 (PAI-1) and GATA-binding protein 4 (GATA4), which is an endoderm differentiation marker such as cardiac muscle, was observed. In addition, when human iPS cells passaged and maintained for 30 generations using serum-free medium hESF9 or hESF9T were analyzed by flow cytometry, the SSEA4 positive cell rate was 91.7% in hESF9T medium, and in hESF9 medium It was significantly higher than the percentage (12%). The Oct3 / 4 positive cell rate was 67.5% in the hESF9T medium, but decreased to 0.6% in the hESF9 medium (FIG. 5B). By using serum-free medium hESF9, it is possible to establish iPS cells and to pass through and maintain undifferentiated human iPS cells in a short period of time. However, by adding TGF-β1, undifferentiation can be achieved. It was shown that the retained cells can be passaged and maintained for a long time.

(2) Characterization of human iPS cells passaged in medium hESF9T supplemented with TGF-β1 RNA was extracted from human iPS cells passaged and maintained on fibronectin using serum-free medium hESF9 or hESF9T, and Agilent Sure Print Gene-wide gene expression profiling analysis using Genespring 12.0 (Agilent Technologies, Santa Clara, CA) with G3 Human GE 8x60K v2 Microarray Compared with the human iPS cells used and maintained on the feeder cells, the gene expression pattern was almost the same (FIG. 5C, FIG. 6). On the other hand, in these pathway analyses, cells subcultured and maintained in hESF9 medium were compared with cells cultured in hESF9T-added medium, compared with TGF-β signaling pathway (WP560), WNT signaling and pluripotent pathway (WP399), WNT. It showed significantly different expression patterns in the Singnaring pathway (WP428) and apoptosis modulation and signaling pathway (WP1772).
From the above, human iPS cells passaged and maintained on fibronectin using serum-free medium hESF9T show the same gene expression as human iPS cell culture maintained on feeder cells using conventional serum-added medium. It was confirmed that the undifferentiation was retained.

4) Characteristic analysis of dental pulp-derived human iPS cells (1) Examination of undifferentiation (i) Analysis of undifferentiated marker protein expression using fluorescent immunostaining After induction on fibronectin-coated dishes using serum-free medium hESF9, TGF -For cells (DP-F-iPS-CL16 passage19) maintained in passage with β1-added medium, various undifferentiated marker antibodies (Nanog, Oct3 / 4, SSEA-4, Tra-1-60 and Tra-1-81) ) Was used for fluorescent immunostaining to analyze expression of undifferentiated markers. Each antibody was visualized with Alexa Fluor (R) 488 and combined with nuclear staining with DAPI. It was confirmed that all of Oct3 / 4, Nanog, SSEA-4, TRA-1-60 and TRA-1-81, which are undifferentiated markers of human ES cells and iPS cells, were expressed (FIG. 7D).

(Ii) Examination of Undifferentiated Marker Gene Expression Using RT-PCR Method RNA was extracted from cells subcultured under the same culture conditions, and expression of various undifferentiated marker genes was analyzed by RT-PCR. For RNA extraction, illustra RNA spin Mini Isolation kit (GE Healthcare UK Ltd, England) was used to extract total RNA of the cells cultured as described above according to the attached protocol. Nucleic acid quantification was performed using Nano Drop® (Nano Drop Technologies, Inc., USA). Total RNA extracted from cells (1 μg) using High Capacity RNA-to-cDNA Master Mix (Applied Biosystems, CA, USA) and thermal cycler (PTC-0220 DNA Engine Dyad: MJ Japan, Tokyo) A reverse transcription reaction was performed by incubation at 25 ° C. for 5 minutes, 42 ° C. for 30 minutes, and 85 ° C. for 5 minutes to synthesize cDNA. RT-PCR is performed using KOD FX Neo (Toyobo, Osaka, Japan) under the conditions of denaturation reaction 98 ° C., 10 seconds, annealing 62 ° C., 30 seconds, extension reaction 68 ° C., 30 seconds. 35 cycles were performed to obtain a PCR product. This PCR product was electrophoresed on a 1.5% agarose gel (Invitrogen) and then visualized with SYBR Safe DNA gel stain (Invitrogen).
The expression of undifferentiated marker genes of human ES cells, Sox2, Nanog, Oct3 / 4, Esg1, Rex-1 (Reduced-expression 1; Zfp42) was examined. As a control, expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was examined (FIG. 7C). Human iPS cells induced and maintained under serum-free culture conditions expressed undifferentiated markers, Sox2, Nanog, Oct3 / 4, Esg1, and Rex-1. On the other hand, these undifferentiated markers were not expressed in the pulp-derived cells before gene introduction (FIG. 7C).

(Iii) ALP staining After fixing human iPS cells with ALP fixative (4.5 mM citrate, 2.25 mM trisodium citrate, 3 mM sodium chloride, 65% methanol, 3% formaldehyde), alkaline phosphatase substrate kit (hist Fine First Red II Substrate Kit (Nichirei, Tokyo, Japan) was allowed to react for 15 minutes at room temperature in the dark, washed several times, and then observed with a phase contrast microscope (Nikon ECLIPSE TE300).

(2) Examination of pluripotency (i) Induction of differentiation using embryoid bodies Human iPS cells passaged and maintained in hESF9 medium or hESF9T medium under serum-free culture conditions (DP-A- maintained in hESF9 medium) A pseudo embryo called an embryoid body was formed from iPS cells or DP-F-iPS cells maintained in hESF9T medium) to induce differentiation. 10% FBS is added to the DMEM medium as a differentiation induction medium, seeded on a 96-well cell low adsorption plate (SUMILON Prime Surface (R) U plate, Sumitomo Bakelite), and suspended for 4 days to form embryoid bodies. Thereafter, the cells were seeded on a gelatin-coated plate, and differentiation induction was further performed for 10 days. Fluorescent immunostaining was performed on the cells 14 days after induction using various differentiation marker antibodies. Each antibody was visualized with Alexa Fluor (R) 594 and combined with nuclear staining with DAPI. As a result, neural stem cell line marker nestin, nerve cell marker βIII-tubulin, mesoderm marker α-smooth muscle actin (SMA),
Expression was observed for α-fetoprotein (AFP), an endoderm marker. On the other hand, the undifferentiated marker Oct3 / 4 was not expressed (FIG. 8A).

(Ii) Evaluation of teratoma (teratoma) formation ability using immunodeficient mice Human iPS cells subcultured and maintained in serum-free medium, hESF9 medium or hESF9T medium are treated with immunodeficient mice (CB17 / Icr-Prkdcscid / CrlCrlj). ) Was implanted subcutaneously in the back and examined for the ability to form teratomas. As a control, cells obtained by inducing human iPS cells on feeder cells with a standard medium for KSR-added ES cell culture were used. Under each condition, 1 × 10 ⁶ cells were suspended in 50 μl PBS, mixed with the same amount of type I collagen, and 50 μl was implanted subcutaneously in the back of immunodeficient mice. Since tumors formed in both conditions about 10 weeks after transplantation, the excised tumors were fixed overnight at 4 ° C. with 4% paraformaldehyde, degreased and embedded in paraffin according to the usual method, and 6 μm serial sections were prepared. It was divided into two groups, and hematoxylin and eosin staining (HE staining) was performed on one group according to a common method. One side was stained with Alcian blue / PAS. After deparaffinization, it was treated with 3% acetic acid for 3 minutes and then with a pH 2.5 Alcian blue solution (Wako Pure Chemicals, Osaka, Japan) for 30 minutes. After dyeing, wash with water, treat with 0.5% periodic acid solution (Wako Pure Chemical Industries) for 5 minutes, cold Schiff solution (Wako Pure Chemical Industries) for 30 minutes, and treat with sulfurous acid (Wako Pure Chemical Industries) for 3 minutes. Was repeated three times and then washed with water. Nuclear staining was performed with Mayer's hematoxylin solution, dehydrated and sealed, and then observed with a microscope. The sections showed ectoderm tissues such as epidermis and nerves, mesoderm tissues such as cartilage, muscle and connective tissue, and tissues that differentiated into endoderm such as the digestive tract and liver, and the excised tumor was shown to be teratoma. (FIG. 8B). A tissue differentiated into three germ layers was also observed in the cells subcultured on the feeder cells in the KSR-added ES cell culture standard medium.

(3) STR (Short Tandem Repeat) analysis Genomic DNA was extracted from dental pulp-derived cells collected from patients and human iPS cells prepared by introducing reprogramming genes under serum-free culture conditions. Powerplex 16 system (Promega Corporation , Madison, Wis.) And STR analysis using ABI PRISM® 3100 Genetic analyzer (Applied Biosystems) and Gene Mapper v3.5, allele patterns of 16 loci matched.

(4) Cell proliferation ability and karyotype analysis The population doubling time of human iPS cells induced in serum-free culture conditions and passaged and maintained for 21 passages in hESF9T was 16.6 ± 0.8 hours (FIG. 9A).
Karyotype analysis of human iPS cells maintained for 20 generations in serum-free medium hESF9T was performed. A colcemid solution (Karyo Max®, GIBCO) is added to the cells in culture to a final concentration of 0.06 μg / ml. After culturing for 6 hours, the cells are recovered, and 10 μM Rock inhibitor (Y- 27632: Wako Pure Chemicals) was added, and then the cells were dispersed. Subsequently, the hypotonic solution was treated with a 0.075 M potassium chloride solution at 37 ° C. for 10 minutes, fixed with a Carnoy fixative, centrifuged, and fixed again with a Carnoy fixative. It was dropped on a slide glass, air-dried, stained with 4% Giemsa staining solution (Mudo Chemical, Tokyo, Japan), and then examined with an optical microscope for karyotype analysis. A simple karyotype analysis of human iPS cells maintained for 20 passages in serum-free medium hESF9T showed the number of normal chromosomes. 2n = 46, XX (FIG. 9B).

4). Examination of universality in passage and maintenance of human iPS cells using the serum-free culture conditions of the present invention Based on the above research results, using human iPS cells maintained using serum-free medium hESF9T, -Instead of GRO, Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham medium (mixed at a ratio of 1: 1) was used as the basal medium, and FGF-2, Heparin, TGF-β1 were added to the above six types of additive factors. The DF8FT medium supplemented with DF8FT was examined for maintenance of undifferentiation. When human iPS cells were seeded on fibronectin using serum-free medium DF8FT, passage and maintenance were possible without changing the morphology, while maintaining undifferentiation (FIG. 10).

5. Summary Generally, stem cells such as ES cells and iPS cells are cultured under conditions containing inactivated feeder cells and animal-derived components such as serum. In such a culture system, various foreign antigens may be mixed, and it is difficult to apply to regenerative medicine. It is also very difficult to clarify the growth / differentiation control mechanism and its control factors of these stem cells. . According to the present invention, it has become possible to induce human iPS cells from normal human dental pulp-derived cells without using feeder cells using a serum-free medium. Human iPS colonies appeared approximately 2 weeks after infection and were undifferentiated and multipotent. In addition, it was revealed that human iPS cells can be induced for the first time by performing the entire process from primary culture to viral infection and human iPS cell establishment in a complete serum-free culture system. In addition, the method of subculture while maintaining undifferentiation and pluripotency of the present invention is applicable not only to human iPS cells but also to stem cells of all animals.

When using conventional human iPS cell culture systems, various known and unknown factors coexist due to the use of feeder cells and animal-derived components, making it difficult to standardize culture methods, maintaining undifferentiation, and maintaining specific tissues and organs. Differentiation control is also difficult. Furthermore, there is a risk of infection due to contamination with animal-derived proteins. In contrast, since the serum-free culture system of the present invention comprises only known factors, it is easy to identify and examine various factors that control the maintenance of proliferation and differentiation of stem cells, and to develop development, tissue, and organ regeneration. It becomes possible to elucidate the mechanism, apply it to drug discovery screening, and realize safe and reliable regenerative medicine.

The present invention can be used in the development of pharmaceuticals and medical materials, biochemical research fields, livestock industry, and the like.

Claims

A method for producing iPS cells, comprising inducing induced pluripotent stem cells (iPS cells) by culturing somatic cells subjected to reprogramming treatment in a serum-free medium without using feeder cells.
The method for producing iPS cells according to claim 1, wherein fibronectin is used as an adhesion factor.
The method according to claim 1 or 2, wherein the reprogramming treatment of somatic cells is performed in a serum-free medium.
The method according to any one of claims 1 to 3, wherein the somatic cells before reprogramming are cultured from a primary culture in a serum-free medium.
The method according to any one of claims 1 to 4, wherein the iPS cell is a human iPS cell.
Serum-free culture substrate for inducing iPS cells containing fibronectin as an adhesion factor (however, the culture substrate does not contain feeder cells).
A kit for producing a serum-free culture substrate for inducing iPS cells, comprising a serum-free medium component and fibronectin as constituent components (however, the culture substrate does not contain feeder cells).
A stem cell is subcultured without adding feeder cells and subcultured with a protein belonging to the tumor growth factor-β (TGF-β) family in a serum-free medium. How to maintain performance.
The method according to claim 8, wherein the subculture is performed using fibronectin as an adhesion factor.
The method according to claim 8 or 9, wherein the stem cell is an iPS cell.
6. The iPS cells obtained by the method according to any one of claims 1 to 5 are subcultured without using feeder cells in a serum-free medium to which a protein belonging to the TGF-β family is added. And a method for maintaining the undifferentiated state and pluripotency of iPS cells.
A serum-free culture substrate for subculture for maintaining the undifferentiated state and differentiation pluripotency of stem cells, including proteins belonging to the TGF-β family (however, the culture substrate does not include feeder cells).
The culture substrate according to claim 12, further comprising fibronectin as an adhesion factor.
The culture substrate according to claim 12 or 13, wherein the stem cells are iPS cells.
A kit for producing a serum-free culture substrate for subculture for maintaining the undifferentiated state and differentiation pluripotency of stem cells, comprising a serum-free medium component and a TGF-β family protein (however, the culture substrate) Does not contain feeder cells).
The kit according to claim 15, further comprising fibronectin as an adhesion factor.
The kit according to claim 15 or 16, wherein the stem cell is an iPS cell.