JP2016073323A - Culture method of human pluripotent stem cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method, which is more efficient than the conventional method, for culturing human pluripotent stem cells while maintaining undifferentiated condition; and to provide a kit therefor.SOLUTION: The method of the invention comprises (a) culturing human pluripotent stem cells in a first culture medium for human pluripotent stem cells containing activin; (b) replacing the first culture medium with a second culture medium for human pluripotent stem cells containing no activin to culture the human pluripotent stem cells; and (c) passaging the human pluripotent stem cells in the first culture medium. We confirmed that pluripotency of the stem cells is maintained at a high rate irrespective of the skill level of an experimenter's cultural technique when subsequently the above (b) and (c) are repeated successively.SELECTED DRAWING: None

Description

  The present invention relates to a method for culturing human pluripotent stem cells while maintaining an undifferentiated state, and a kit therefor, and more specifically, human pluripotent stem cells are cultured in a first medium that is a medium for pluripotent stem cells containing activin. After the step of culturing, the step of culturing human pluripotent stem cells by replacing the first medium with a second medium which is a medium for pluripotent stem cells not containing activin; The present invention relates to a method for culturing human pluripotent stem cells while maintaining an undifferentiated state by repeating the step of subculturing pluripotent stem cells and a kit for the same.

  Since ES cells can differentiate into various tissues, application to elucidation models of tissue differentiation processes and regenerative medicine is considered. In order to accurately analyze the functions and effects of differentiation-inducing factors, it is important to control cell differentiation.

  Regarding the method for maintaining undifferentiated stem cells, the step of culturing the pluripotent stem cells while suppressing the differentiation of the pluripotent stem cells by n-axis rotation (n is an integer of 2 or more). A pluripotent stem cell culture method provided (for example, see Patent Document 1), a medium for maintaining pluripotency of mesenchymal stem cells characterized by containing TGF-β (for example, see Patent Document 2), stem cells To a basal medium that enables serum-free primary and subculture of 5-10% panexin, 1-100 ng / mL bFGF, 1-100 ng / mL PDGF, A serum-free medium (for example, see Patent Document 3) obtained by mixing 100 ng / mL of EGF and 1-1000 μg / mL of vitamin C, and polyvinyl alcohol (PVA) in the absence of feeder cells, and Movement A method of maintaining embryonic stem cells (see, for example, Patent Document 4) characterized by suspension culture of embryonic stem cells in a serum-free medium containing no product-derived albumin, or a low molecular compound or chloride thereof as an active ingredient A tissue stem cell proliferating agent (for example, see Patent Document 5), a stem cell differentiation inhibitor (for example, see Patent Document 6) containing a low-molecular compound such as an indole derivative as an active ingredient, (a) a standard medium, (b) serum Albumin, (c) transferrin, (d) lipid and fatty acid source, (e) cholesterol, (f) reducing agent, (g) pyruvate, (h) nucleosides for DNA and RNA synthesis, (i) substrate cells, tissues At least one growth factor that stimulates the proliferation and development of cells or organ cells, and (j) an amount of at least one extracellular matrix material effective for culturing the cells Cage, prolonged growth and generating media of cells including embryonic stem cell line is a serum-free or low-serum (for example, see Patent Document 7) have been proposed. In addition, a pluripotent stem cell culture method (see, for example, Patent Document 8) characterized in that pluripotent stem cells are cultured in the presence of decidua-derived cells or extracellular matrix derived from the cells. However, although cell growth was slightly observed on fibronectin, it was reported that the degree was remarkably low compared to cell growth on the extracellular matrix of decidua-derived mesenchymal cells.

  The present inventors have provided a medium for serum-free medium in which ES cells can be cultured for a long period of time while maintaining undifferentiation under serum-free conditions without using feeder cells in a type I collagen-coated flask. Using a basal medium for producing such a medium (see, for example, Patent Document 9) and i) a culture container coated with a basic culture support composed of protein, bovine insulin, human transferrin in the culture container , Sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with bovine albumin from which fatty acids have been removed, and also fibroblast growth factor, heparin, ascorbic acid or phosphorylated ascorbic acid, or derivatives thereof Preparing primate embryonic stem cells with a cell culture medium supplemented with ii), and ii) the primate embryonic stem cells Step of maintaining an undifferentiated state, more composed, in cell culture conditions without feeder cells and serum, we have developed a method for maintaining the primate embryonic stem cells (for example, see Patent Document 10). In developing a serum-free medium, it was confirmed that laminin and fibronectin act in a suppressive manner against the undifferentiated maintenance state of mouse ES cells and promote differentiation in various matrix components (for example, Non-patent document 1).

  Furthermore, the present inventors have confirmed that human ES cells using Shef human ES cells and the HUES-1 strain can be maintained and cultured when coated with type I collagen, and HEPES is added to a serum-free medium. It has been confirmed that, when added, it has an adverse effect on the maintenance of human ES cells (see, for example, Non-Patent Document 2) and assists the action of FGF-2 that promotes the proliferation of ES cells. It has been reported that even when heparin is added in the absence of FGF-2, the proliferative action of ES cells is promoted (see, for example, Non-Patent Document 3). When activin is added, ERK (extracellular signal-regulated kinases) is produced. It is activated, and differentiation induction into nerves or mesendoderm is promoted, but it is confirmed that an undifferentiated state can be maintained when an ERK inhibitor is added (for example, non-patent literature) See), further summarizes the basic culture method for human ES cells (e.g. see Non-Patent Document 5).

JP 2010-193910 A JP 2010-094062 A JP 2008-148643 A JP 2007-228815 A JP 2006-180763 A JP 2005-013152 A Japanese National Patent Publication No. 8-508891 JP 2010-166901 A International Publication WO2005 / 063968 Pamphlet Special table 2009-542247 gazette

The 52nd Matrix Conference 2005 Oita O-07 Functional analysis of matrix components in the differentiation control of mouse ES cells PNAS, vol.105, no.36, 13409-13414 In Vitro Cell.Dev. Biol.- Animal (2010) 46: 573-576 Stem Cell Res. 2010 Tiss. Cult. Res Commun. 27; 139-147 (2008)

  An object of the present invention is to provide a method for culturing human pluripotent stem cells that is more efficient than conventional methods while maintaining an undifferentiated state, and a kit therefor.

  As described above, the development of a serum-free medium or the like that can culture pluripotent stem cells such as ES cells for a long period of time has been promoted in various fields. Proliferating pluripotent stem cells in these media Death may occur at a high frequency or pluripotency may be lost in the culture process, often relying on the intuition and know-how of the experimenter, which is not impossible but requires advanced culture techniques It was.

  Further, conventional media usually contain animal-derived raw materials. For example, the use of bovine serum albumin, porcine-derived type I collagen and the like has a problem in that the supply amount is limited and the cost is high. It was. In addition, since the above animal-derived raw materials are usually non-heated preparations, there is a possibility of prion causing viral bovine spongiform encephalopathy (BSE), pathogens such as viruses, and the presence of foreign components. It has been considered that this is one of the causes of lowering the probability of culturing while maintaining the undifferentiated state, because there is a problem in sex, and variation occurs between lots of the produced medium.

  Based on these findings, the present inventors can replace or delete animal-derived components to culture pluripotent stem cells that are highly safe and that do not vary from lot to lot while maintaining an undifferentiated state. Attempts have been made to prepare For example, when replacing an animal-derived component with a recombinant (recombinant) prepared by genetic recombination technology, the component of the recombinant may have a structure different from that of a conventional animal-derived component, Since it has been known that immunogenicity may be different from conventional animal-derived raw materials, each component has been carefully examined. As described above, it was confirmed that an undifferentiated state can be maintained when an ERK inhibitor is added to a medium supplemented with activin, and therefore, detailed studies were continued on the relationship between activin and other components. However, surprisingly, (a) human pluripotent stem cells are cultured in a first medium which is a medium for pluripotent stem cells containing activin, and (b) the first medium is used as a non-activin-containing medium. The pluripotent stem cells are replaced with a second medium that is a medium for pluripotent stem cells, and human pluripotent stem cells are cultured. (C) The human pluripotent stem cells are subcultured in the first medium, and then sequentially (b) When (c) and (c) were repeated, it was found that the pluripotency of stem cells was maintained at a high rate regardless of the level of proficiency of the experimenter's culture technique. Until now, only 1 out of 5-6 persons in the laboratory has been able to maintain and culture pluripotent stem cells by a conventionally known method, whereas 4-5 out of 5-6 persons using such a method. With the above probability, it was confirmed that pluripotent stem cells could be passaged for a long time while maintaining undifferentiation. The present inventors have continued to study components that further increase the undifferentiated maintenance rate of pluripotent stem cells. When a protein kinase C inhibitor is added to the medium, the cells have a higher probability of being undifferentiated. Maintained, and the need to mechanically remove some differentiated cells is almost eliminated. Based on the above findings, the present inventors have completed the method for culturing human pluripotent stem cells of the present invention.

  That is, the present invention provides [1] (a) a step of culturing human pluripotent stem cells in a first medium which is a medium for pluripotent stem cells containing activin; (b) the first medium comprising activin Culturing human pluripotent stem cells by replacing with a second medium which is a medium for pluripotent stem cells not containing; (c) subculturing human pluripotent stem cells in the first medium; (A) After step, (b) and (c) steps are sequentially repeated, a method for culturing human pluripotent stem cells while maintaining an undifferentiated state, [2] first medium and The second medium is prepared by adding a supplement to the basal medium for pluripotent stem cells, and comprises activin as at least one component of the supplement added for the preparation of the first medium. As described in [1] above And [3] the supplement added for the preparation of the first medium and the supplement added for the preparation of the second medium, each of which contains fibronectin as at least one component Protein kinase C inhibition as at least one component in the method of [2] and [4] the supplement added for the preparation of the first medium and the supplement added for the preparation of the second medium A pluripotent stem cell while maintaining the undifferentiated state according to any one of [2] to [4] above, or [5] the method according to [2] or [3], which comprises an agent The present invention relates to a culture kit comprising a supplement and a basal medium for pluripotent stem cells.

  By using the method of the present invention, human pluripotent stem cells can be cultured and expanded while maintaining an undifferentiated state, and the pluripotent stem cells maintaining such an undifferentiated state are treated under appropriate conditions. The cells can be differentiated into the target cells.

It is the figure which showed the result of having measured the adhesive ability of each cell with respect to collagen I, gelatin, laminin, and fibronectin. The results of human ES cell KhES-1 strain (a), human iPS cell Tic strain (JCRB1331) (b), and embryonic cancer cell PA-1 strain (JCRB9061) (c) are shown. Cells obtained by culturing ES cell strain H9 (WA09) with a medium supplemented with BSA or rHSA and / or oleic acid in hESF9 medium were fluorescently immunostained, and TRA-1-60, SSEA-1, SSEA-4 It is the graph which analyzed the positive rate of Nanog expression cell. The value when culturing under the culture condition (1) was set to “1”, and the rate of change relative to that value was shown. It is a microscope picture at the time of culture | cultivating ES cell H9 strain | stump | stock (WA09) in the culture medium which added oleic acid conjugate rHSA, and immunostaining by TRA-1-60, SSEA-1, SSEA-4, and Nanog. The state of the second passage cell of the H9 strain (WA09) cultured in a medium obtained by removing heparin from hESF-9 is shown. It is a microscope picture of the case where the ES cell strain H9 (WA09) is cultured in an activin-containing medium (a) and the medium is replaced with an activin-free medium (b). It is a figure which shows the microscope picture of the cell of 1st-6th passage of ES cell H9 strain | stump | stock (WA09) cultured by the method of this invention. It is a figure which shows the expression of the undifferentiation / differentiation marker in the 5th passage of ES cell H9 strain | stump | stock (WA09) cultured by the method of this invention. SSEA-3, SSEA-4, TRA-2-54, TRA-1-60, TRA-1-80, CD90 shows expression of undifferentiated markers, and SSEA-1, CD105, CD56, A2B5 expresses differentiation markers Indicates. The state of cells when 0, 20, 40, 80, 160, 320 μg / well of fibronectin is added to the medium and cultured by the method of the present invention is shown. The situation of cells cultured in medium supplemented with 0, 1, 2.5, 5, 10 μM myristoylated protein kinase C peptide inhibitor is shown.

  The method of culturing human pluripotent stem cells while maintaining the undifferentiated state of the present invention includes (a) a step of culturing human pluripotent stem cells in a first medium that is a medium for pluripotent stem cells containing activin. (B) replacing the first medium with a second medium which is a medium for pluripotent stem cells not containing activin, and culturing human pluripotent stem cells; (c) The step of subculturing human pluripotent stem cells is not particularly limited as long as it is a method in which (a) step, (b) and (c) steps are sequentially repeated, and the undifferentiated state of the present invention is maintained. The kit for culturing human pluripotent stem cells is not particularly limited as long as it is a kit comprising a supplement and a basal medium component for pluripotent stem cells. Here, the “medium” can cultivate cells. Add water to the “medium ingredients” It refers to those of the state. In addition, “maintaining an undifferentiated state” here means a state in which the self-replicating ability is maintained and the characteristics of the pluripotent cell are maintained such that it has the ability to differentiate into all types of cells existing in the living body. Say.

In the present invention, human pluripotent stem cells are targeted, and pluripotent stem cells are isolated from embryonic stem cells (embryonic stem cells: ES cells) isolated from early embryos or primordial germ cells in the fetal stage Embryonic germ cells (EG cells) (see, for example, Proc Natl Acad Sci US A. 1998, 95: 13726-31) and germline stem cells (GS) isolated from testis immediately after birth Cells (see Nature. 2008, 456: 344-9), stem cells derived from bone marrow such as iliac bone marrow and jaw bone marrow, mesenchymal stem cells such as stem cells derived from adipose tissue, and somatic cells such as skin cells. Introducing a plurality of genes into the body induces dedifferentiation of the subject's own somatic cells, somatic-derived induced pluripotent stem cells (or induced pluripotent stem cells (induced) pluripotent stem cell; iPS cell)) Specifically, human ES cell H9 strain (WA09), human ES cell H1 (WA01) strain (National Stem Cell bank, WISC Bank), KhES-1, KhES-2 and KhES-3 (all Kyoto University) Research Center for Stem Cell Medicine), HES3, HES4, and HES6 (National Stem Cell bank, Monash University) and other human ES cells, Oct3 / 4 gene, Klf4 gene, C-Myc gene, and Sox2 gene IPS cells (RIKEN BioResource Center, Kyoto University), Tic (JCRB1331 strain), Dotcom (JCRB1327 strain), Squeeky (JCRB13)
29 strains), Toe (JCRB1338 strain), Lollipop (JCRB1336 strain) (National Center for Child Health and Development, Research Institute for Intractable Diseases and Disease Resources, JCRB Cell Bank), UTA-1 strain and UTA-1-SF- Exemplify iPS cells such as 2-2 strains (all from the University of Tokyo) and iPS cells (Nat Biotechnol 2008; 26: 101-106) obtained by introducing Oct3 / 4 gene, Klf4 gene and Sox2 gene Can do.

  The basal medium for pluripotent stem cells used in the present invention is not particularly limited as long as it is a medium capable of culturing human pluripotent stem cells while maintaining the undifferentiated state by adding the supplement of the present invention. A chemically synthesized medium is preferable because it is easy and prevents variation from lot to lot. One or more types of sugar (s), one or more types of inorganic salts (s), one or more types of amino acids ( Class) preferably contains 1 or 2 or more types of vitamin (s) and 1 or 2 or more types of trace component (s), and optionally contains antibiotics such as kanamycin for use in drug susceptibility testing You can also.

  Specific examples of the saccharide include monosaccharides such as glucose, lactose, mannose, fructose, and galactose, and disaccharides such as sucrose, maltose, and lactose, among which glucose is particularly preferable. One or a combination of two or more can also be added.

  Specific examples of the inorganic salts include calcium chloride, calcium nitrate, copper sulfate pentahydrate, iron (III) nitrate nonahydrate, iron (II) sulfate heptahydrate, magnesium chloride hexahydrate. Contains magnesium sulfate, potassium chloride, sodium chloride, disodium hydrogen phosphate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate, sodium dihydrogen phosphate dihydrate, zinc sulfate heptahydrate Although combinations can be mentioned, any inorganic salt or a combination thereof can be used as long as it is a component that advantageously acts to maintain the undifferentiated state of pluripotent stem cells.

  Specific examples of the amino acids include alanine, arginine, asparagine, aspartic acid, cystine, cysteine, glutamine, glycine, histidine, glutamic acid, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine. Preferably L-amino acids such as tryptophan, tyrosine, valine and the like, derivatives thereof and salts thereof, and derivatives thereof such as arginine hydrochloride. Derivatives of arginine such as L-arginine monohydrochloride, and the aspartic acid includes L-aspartic acid sodium salt monohydrate, L-aspartic acid monohydrate, L-aspartic acid potassium , L-aspara Aspartic acid derivatives such as magnesium acid can be included, and as the cysteine, cysteine derivatives such as L-cysteine dihydrochloride and L-cysteine hydrochloride monohydrate, and L-lysine hydrochloride The glutamic acid may include a derivative of glutamine such as L-glutamic acid monosodium salt, and the asparagine may include an asparagine such as L-asparagine monohydrate. The tyrosine may include a tyrosine derivative such as L-tyrosine disodium dihydrate, and the histidine may include histidine hydrochloride, histidine hydrochloride monohydrate. Derivatives of histidine such as L-lysine hydrochloride as the lysine It can be included.

  Specific examples of the vitamins include one or more vitamins selected from ascorbic acid, biotin, choline, folic acid, inositol, niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, vitamin B12, and paraaminobenzoic acid (PABA). And derivatives of each of these components and derivatives thereof such as hydrates thereof. For example, ascorbic acid includes ascorbic acid 2-phosphate (Ascorbic acid 2-phosphate). ), May include derivatives of ascorbic acid such as magnesium ascorbate phosphate, sodium ascorbate sulfate, ascorbylaminopropyl phosphate, sodium ascorbate phosphate, and choline derivatives of choline such as choline chloride Can include niacin and Can include niacin derivatives such as nicotinic acid, nicotinic acid amide, nicotinic alcohol, and pantothenic acid includes pantothenic acid derivatives such as calcium pantothenate, sodium pantothenate, panthenol, etc. Pyridoxine can include derivatives of pyridoxine such as pyridoxine hydrochloride, pyridoxal hydrochloride, pyridoxal phosphate, pyridoxamine, and thiamine includes thiamine hydrochloride, thiamine nitrate, bisthiamine nitrate, thiamine dicetyl sulfate Derivatives of thiamine such as ester salts, fursultiamine hydrochloride, octothiamine, benfotiamine and the like can be included, and the ascorbic acid is preferably added.

The trace component is preferably a component that advantageously acts to maintain the undifferentiated state of pluripotent stem cells, such as glutathione, hypoxanthine, lipoic acid, linolenic acid, phenol red, putrescine, pyruvate, thymidine, NaHCO3. _{3 and the} like, and derivatives such as components and derivatives thereof and salts thereof, and hydrates thereof that are usually used as medium components, such as putrescine dihydrochloride, sodium pyruvate, and the like can be mentioned.

  Specific examples of the above basal medium for human pluripotent stem cells include known chemical synthesis such as commercially available Dulbecco's modified Eagle medium (DMEM), minimum essential medium (MEM), Eagle basal medium (BME), RPMI 1640 medium, F12 medium, etc. A medium such as a medium, a medium in which any two or more of these mediums such as a DMEM / F12 medium (a medium in which DMEM and F12 medium are mixed at a ratio of 1: 1), and the like ascorbine A medium supplemented with an acid, preferably ascorbic acid 2-phosphate ester, can be exemplified. Particularly, as a basal medium free of animal products, a basal medium having the composition shown in the following Table 1 (hereinafter referred to as “hESF-grow medium”). Can also be suitably exemplified.

  The concentration (content) of each of the above-mentioned medium components is, for each component of the hESF-grow medium as an example, when the concentration described in the hESF-grow medium composition is 100, A concentration within the range of 0 to 200, preferably a concentration within the range of 40 to 160, more preferably a concentration within the range of 80 to 120, and even more preferably a concentration within the range of 90 to 110 can be exemplified. . For example, in the case of L-arginine, a concentration of 0-100 mg / L, preferably 20-80 mg / L, more preferably 40-60 mg / L, even more preferably 45-55 mg / L is suitable. Can be exemplified.

  The supplement used in the method of the present invention is not particularly limited as long as it is added to the basal medium for pluripotent stem cells, and specifically, fibronectin, insulin, transferrin, 2-mercaptoethanol, 2-ethanol. Examples include those containing components such as amine, sodium selenate, FGF-2, oleic acid-conjugated albumin, protein kinase C inhibitor and the like. Moreover, as a use form of a supplement, the supplement which consists of 1 or 2 or more of the said component may be fractionated into plurality, and can also be put together as one supplement containing many said components.

  The first medium used in the method of the present invention is prepared by adding a supplement to the basal medium for pluripotent stem cells, comprising activin as at least one component of the supplement, and containing human pluripotency The medium is not particularly limited as long as it is a medium capable of culturing stem cells, and the second medium used in the method of the present invention is by adding a supplement containing no activin to the basal medium for pluripotent stem cells. The medium is not particularly limited as long as it is prepared and can culture human pluripotent stem cells. Hereinafter, supplements containing one or more components added to the first medium are collectively referred to as activin-containing supplements, and supplements containing one or more components added to the second medium are collectively referred to. This is called an activin-free supplement. The activin-containing supplement is not particularly limited as long as it contains activin, but activin and fibronectin, insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, selenium, FGF-2, albumin, oleic acid, protein The thing containing 1 or 2 or more components chosen from a kinase C inhibitor etc. can be mentioned. The activin-free supplement is not particularly limited as long as it does not contain activin. Specifically, fibronectin, insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, selenium, FGF-2, albumin, Examples thereof include those containing one or more components selected from oleic acid and protein kinase C inhibitor.

  In the first medium, fibronectin is used as a component constituting a supplement to be added to the basal medium for pluripotent stem cells so as to coat the inside of the culture container in order to enhance the adhesion between the cells and the culture container. Or can be added and used with other supplements. Specifically, after coating fibronectin on a culture container, 1 or 2 selected from insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, selenium, FGF-2, albumin, oleic acid, protein kinase C inhibitor and the like A first medium (hereinafter also referred to as “first medium I”) can be prepared by adding a combination containing the above components and activin, and fibronectin is insulin, transferrin, 2-mercaptoethanol, By adding together one or two or more components selected from 2-ethanolamine, selenium, FGF-2, albumin, oleic acid, protein kinase C inhibitor and the like, and activin, the first medium (hereinafter referred to as “No. One medium II ") can also be prepared.

  When the first medium I is used, after coating fibronectin on the basal medium for pluripotent stem cells on a culture container, insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, selenium, FGF-2, albumin It is preferable to prepare a second medium (hereinafter also referred to as “second medium I”) by adding one or more components selected from oleic acid, protein kinase C inhibitor, etc., but fibronectin. By adding one or more components selected from insulin, transferrin, 2-mercaptoethanol, 2-ethanolamine, selenium, FGF-2, albumin, oleic acid, protein kinase C inhibitor, etc., the second medium (Hereinafter, also referred to as “second medium II”) can be prepared. When the first medium II is used, it is preferable to use the second medium II.

  Each component constituting the activin-containing supplement and / or activin-free supplement is preferably an animal product-free grade, and the animal product-free grade component is not an animal-derived component but is similar to an animal-derived component. Recombinant (recombinant) proteins, chemically synthesized products, plant-derived components, etc. that are artificially prepared and purified by genetic recombination technology, and have a tag as the above-mentioned recombinant protein. Proteins that are not structurally identical to animal-derived components, such as those, can be included, and chemically synthesized products can include derivatives and their salts and hydrates thereof.

  As the fibronectin, known as a kind of extracellular matrix (ECM), naturally-derived fibronectin such as porcine-derived fibronectin, bovine-derived fibronectin, human-derived fibronectin, and animal product free grade fibronectin, Examples thereof include fibronectin, which is a gene recombinant such as a type, a pig type, and a human type.

When the above fibronectin is coated and used, it is well known that it is physically adsorbed or a reactive functional group is introduced into the cell adhesion region of the culture vessel to immobilize the cell adhesion factor on the vessel surface by chemical bonding. Specifically, a solution containing fibronectin in a culture vessel is, for example, 0.5 to 5 μg / cm ² , preferably 1 to 3 μg / cm ² , more preferably 1.5 to Add to a concentration of 2.5 μg / cm ² and leave for 1 hour to 12 hours, preferably 2 hours to 8 hours, more preferably 3 hours to 5 hours, for example at 37 ° C. without drying. The method of sucking and removing the solution immediately before seeding the pluripotent stem cells can be exemplified, and the activin-containing supplement is added to the basal medium for pluripotent stem cells on the coated fibronectin. By prepared by adding components other than fibronectin cement or activin-free supplement, can be prepared a first medium or the second medium of the present invention.

  When fibronectin is used as a medium component, for example, the concentration is 1.0 to 100 μg / mL, preferably 5 to 75 μg / mL, preferably 10 to 50 μg / mL, more preferably 25 to 45 μg / mL. In addition, as a component of the activin-containing supplement and / or activin-free supplement, the first medium and / or the second medium used in the present invention is added to the basal medium for human pluripotent stem cells. Can be prepared.

  Examples of the insulin include naturally-derived insulin such as porcine-derived insulin, bovine-derived insulin and human-derived insulin, and animal product free grade insulin such as bovine, porcine, or human-type recombinant insulin. In particular, human-type recombinant insulin (recombinant human insulin) can be preferably exemplified, and 1 to 20 μg / mL, preferably 5 to 15 μg / mL, more preferably 7.5 to 12 Add to the basal medium for human pluripotent stem cells as a component of the activin-containing supplement and / or activin-free supplement to a final concentration of 5 μg / mL, more preferably 9-11 μg / mL Thus, the first medium and / or the second medium used in the present invention Seisuru can.

  Examples of the transferrin include naturally occurring transferrin such as porcine-derived transferrin, bovine-derived transferrin, and human-derived transferrin, and animal product-free grade insulin such as bovine, porcine, or human-type recombinant transferrin. In particular, human-type recombinant transferrin (recombinant human transferrin) can be preferably exemplified, and iron-containing apo-type transferrin is more preferable than hol-type transferrin bound to iron. 0.1 to 50 μg / mL, preferably 1 to 20 μg / mL, more preferably 2 to 10 μg / mL, and even more preferably 3 to 7 μg / mL. One component of the bottle-free supplements, and added to the basal medium for the human pluripotent stem cells can be prepared a first medium and / or the second medium used in the present invention.

  As said 2-mercaptoethanol, the chemically synthesized product synthesize | combined by the conventional method can be illustrated suitably as an animal product free grade, 1-20micromol, Preferably it is 5-15micromol, More preferably, 7.5-12. By adding to the basal medium for human pluripotent stem cells as a component of the activin-containing supplement and / or activin-free supplement to a final concentration of 5 μM, more preferably 9-11 μM, the present invention The first medium and / or the second medium used in the above can be prepared.

  As the above-mentioned ethanolamine, a chemically synthesized product synthesized by a conventional method, also called 2-aminoethanol or monoethanolamine, can be suitably exemplified as an animal product free grade, and it is 1 to 20 μM, preferably 5 to 15 μM. As a component of the activin-containing supplement and / or activin-free supplement so as to have a final concentration of 7.5 to 12.5 μM, more preferably 9 to 11 μM, more preferably for the above human pluripotent stem cells By adding to the basal medium, the first medium and / or the second medium used in the present invention can be prepared.

  The selenium can include selenium and its derivatives and their salts and hydrates thereof, and is chemically synthesized by ordinary methods, such as selenate, sodium selenate, sodium selenite, sodium hydrogen selenite, etc. As an animal product free grade, the above activin is adjusted so that the final concentration is 1 to 40 μM, preferably 10 to 30 μM, more preferably 15 to 25 μM, and still more preferably 18 to 22 μM in terms of sodium selenate. Preparing the first medium and / or the second medium used in the present invention by adding to the basal medium for human pluripotent stem cells as a component of the supplement containing and / or the activin-free supplement Can do.

  As the above FGF-2, as a basic fibroblast growth factor-2 (also known as bFGF), natural origin such as porcine-derived FGF-2, bovine-derived FGF-2, human-derived FGF-2, etc. FGF-2 and animal product free grade FGF-2 may include recombinant human FGF-2, such as bovine, porcine or human recombinants, particularly human recombinants FGF-2 (recombinant human FGF-2 (rhFGF-2)) can be suitably exemplified, and is 0.1 to 50 ng / mL, preferably 1 to 20 ng / mL, more preferably 3 to 7 ng / mL, As a constituent of the above activin-containing supplement and / or activin-free supplement, preferably at a final concentration of 4-6 ng / mL, By adding to the basal medium for pluripotent stem cells, the first medium and / or the second medium used in the present invention can be prepared.

  Examples of the oleic acid include plant-derived oleic acid and oleic acid chemically synthesized by a conventional method. 0.1 to 50 μg / mL, preferably 5 to 15 μg / mL, more preferably 7.5 to Added to the basal medium for human pluripotent stem cells as a component of the activin-containing supplement and / or activin-free supplement so as to have a final concentration of 11.5 μg / mL, more preferably 9-10 μg / mL Thus, the first medium and / or the second medium used in the present invention can be prepared.

  Examples of albumin include naturally-derived albumin such as ovalbumin, porcine-derived albumin, bovine-derived albumin, and human-derived albumin, and animal product free grade albumin such as bovine, porcine, or human-type recombinants. In particular, human-type recombinant albumin (recombinant human albumin (rHSA)) can be suitably exemplified, and 0.1-5 mg / mL, preferably 0.5-2. One of the activin-containing supplements and / or activin-free supplements so that the final concentration is 5 mg / mL, more preferably 0.75 to 1.5 mg / mL, and still more preferably 0.9 to 1.1 mg / mL. By adding to the basal medium for human pluripotent stem cells as a component, the present invention In order to increase the solubility of the oleic acid in the medium and increase the action and effect of the present invention, the first medium and / or the second medium used can be prepared. It is desirable to add the body-formed form, ie oleic acid conjugated albumin.

  Examples of the protein kinase C inhibitor include peptides and compounds capable of inhibiting protein kinase C. Specifically, myristoylated protein kinase C peptide inhibitor (manufactured by Promega), staurosporine or The staurosporine derivative calphostin, 4′-N-benzoylstaurosporine, and bisindolylmaleimides such as bisindolylmaleimide I, isoquinolinesulfonyl) -2-methylpiperazine dihydrochloride (H-7), N- [2- (methylamino) ethyl] -5-isoquinolinesulfonamide (H-8), N- (2-aminoethyl) -5-isoquinolinesulfonamide (H-9) and the like can be exemplified, The myristoylated protein kinase C peptide inhibitor (Promega) When used, it is added to the basal medium for human pluripotent stem cells as a component of the activin-containing supplement or activin-free supplement so that the concentration is 0.5 to 5 μM, preferably 1 to 2.5 μM. By doing so, the undifferentiated maintenance rate of human pluripotent stem cells can be further increased.

  As the above activins, high homology is maintained in vertebrates such as humans, rats, mice, Xenopus, zebrafish and the like, and there are families such as activin A, activin B, activin AB, activin C and the like. Although known as an animal product free grade activin, there can be mentioned human-type recombinant activin and the like. Particularly, among human-type recombinant activins, recombinant human albumin A is preferably exemplified. One of the above activin-containing supplements to a final concentration of 1 to 40 ng / mL, preferably 10 to 30 ng / mL, more preferably 15 to 25 ng / mL, and even more preferably 18 to 22 ng / mL. As a component, the above-mentioned human pluripotent stem cell group By adding to the culture medium, it is possible to prepare the first medium to be used in the present invention.

The step (a) is a step of seeding and culturing human pluripotent stem cells in a first medium obtained by adding the activin-containing supplement to the basal medium for pluripotent stem cells, and is cryopreserved at a supply destination. The pluripotent stem cells are preferably cultured after preparation by a known method. Examples of the preparation method include release in EDTA dissolved in Dulbecco's Ca ²⁺ and Mg ²⁺ -free phosphate buffered saline, or trypsin, recombinant trypsin, trypsin / EDTA, collagenase, collagenase / trypsin , Dispase, actase, or mechanically exfoliate and collect the released cells in the first medium (solution) to prepare a cell suspension. The cell suspension is about 300-1000 rpm. After the cells are settled and collected, a method of re-preparing the cell suspension one or more times, or on a gelatin-coated plate with fetal calf serum, DMEM or DM / F12, KSR (KnockOut Serum Replacement, GIBCO), L-glutamine, 2-mercaptoethanol, non-essential amino acid (s), and bFGF Charging the medium, and / or support cells (feeder cells) may be a method of culturing under the coexistence. By using the method of the present invention, there is an effect that impurities that can be contained in animal-derived components such as fetal bovine serum, FGF, feeder cells and the like can be excluded while continuing long-term passage.

  In the step (b), the first medium in which the first passage human pluripotent stem cells are cultured is replaced with a second medium supplemented with an activin-free supplement. It is a step of culturing human pluripotent stem cells, and the timing of the medium exchange from the first medium to the second medium is 36 to 84 hours, preferably 48 to 72, after the start of the culture in the first medium. Examples thereof include those within a time, more preferably 54 to 66 hours, and particularly preferably 57 to 63 hours. In the present invention, medium exchange can be performed by a conventional method.

  Cells of the first passage cultured in the second medium in step (b) can be cultured while changing the medium to the second medium one or more times as necessary. When the time when it is determined that the state of the cell being suitable for passage is reached, in the step (c), the first medium is inoculated and passage culture is performed. Cells that have been cultured by passage become cells at the second passage, and the passage time is 1 to 20 days after the medium change from the first medium to the second medium, preferably 3 to 15 days. Day, more preferably 4 to 6 days can be exemplified, but depending on the state of the cultured cells, it is 55 to 95% confluent, preferably 65 to 85% confluent, more preferably 67 to 73. It can also be judged by confluency, such as when% confluence is reached. In addition, when the central part or the edge part of the colony starts to differentiate, it is desirable to subculture regardless of the confluency, and it can also be determined in the differentiated state of the cells. The timing of medium exchange from the second medium to the first medium is performed every 6 hours to 9 days, preferably every 12 hours to 5 days, more preferably every 18 hours to 2 days, and particularly preferably every 24 hours. Can be illustrated.

  Thereafter, step (b) and step (c) are repeatedly performed. That is, the second passage cell that has been subcultured to the first medium needs to be replaced with the second medium again at the first medium exchange. Human pluripotent stem cells cultured in the above medium need to be subcultured and cultured again in the first medium while substituting the medium in the second medium. The passaged cell becomes the third passage cell. In the method of the present invention, the pluripotent stem cell is obtained by repeating the two steps of the exchange from the first medium to the second medium and the passage to the first medium in order, While increasing one by one, the culture can be continued while maintaining the undifferentiated state.

  As a subculturing method when subculturing the human pluripotent stem cells, a method using trypsin, a method using trypsin / EDTA, a method using a mixed solution of trypsin / collagenase / calcium, or dispase is used. Examples include known methods such as a method, a method using collagenase, and a method of cutting cultured cells into a small population of 100 to several hundred cells using a thin tip such as an injection needle or plastic pasteur under a microscope. Specifically, after removing the medium in the culture container by aspiration, etc., 1 unit / ml dispase is allowed to act at 37 ° C. for 1 to 10 minutes, except for the dispase, the first medium of the present invention is added. After detaching the cell population (colony) and centrifuging once to several times, the cells are collected in the basal medium (solution) for pluripotent stem cells of the present invention. After separation, dispersed in a basal medium solution for pluripotent stem cells again centrifuged, it can be exemplified a method of dispersing the first medium.

  The pluripotent stem cell cultured by the method of the present invention is 33 to 40 ° C., preferably 34 to 39 ° C., particularly preferably 37 ° C., and 1 to 20%, preferably 3 to 3 in any of the above steps. In the presence of 15%, particularly preferably 8 to 10% carbon dioxide, it is preferably cultured under a high humidity of 75% or more, preferably 85% or more, more preferably 95%, particularly preferably 100%. .

  As a method for confirming whether or not the cells cultured by the method of the present invention are cells maintaining undifferentiation, detection of expression of undifferentiation markers using antibodies against various markers (proteins) and / Or a method of judging by non-detection of the expression of differentiation markers, a method of judging by detecting the expression of various markers (genes), a method of observing the morphological characteristics of cells, or a specific differentiation-inducing factor A method for determining whether or not the ability to differentiate into the cells can be demonstrated.

  In the determination method using the marker, SSEA-3, SSEA-4, TRA-2-54, TRA-1-60, TRA-1-80, CD90, Nanog, Oct-3, Oct-4, alkali When an undifferentiated marker such as phosphatase is expressed, it can be determined that cells cultured by the method of the present invention maintain undifferentiated state, and differentiation of SSEA-1, CD105, CD56, A2B5, etc. Even when the marker is not expressed, it can be determined that the cells cultured by the method of the present invention maintain undifferentiation.

  When confirming the expression of the undifferentiated and / or differentiation marker at the protein level, it can be confirmed by flow cytometry, immunostaining, ELISA, etc. using the specific antibody of each marker. Expression of the marker Can be confirmed by RT-PCR using a specific primer pair, Northern blotting using a specific probe, etc. of each marker gene.

  As a method for confirming the presence or absence of marker expression by the flow cytometry method, cells cultured by the method of the present invention were trypsinized in a PBS solution containing trypsin / EDTA, and the cells were placed in 1 ml of 10% goat serum for 30 minutes. After suspension, it is centrifuged and then incubated with an anti-mouse antibody of the marker protein of interest for 30 hours, after which the cultured cells are washed three times with PBS containing 1% goat serum, and AlexaFluor-conjugated goat anti-goat An example is a method of reacting with a mouse IgG antibody for 30 minutes, washing 3 times with PBS containing 1% goat serum, and determining resuspended cells using an appropriate flow cytometry instrument.

  As a method for confirming the presence or absence of marker expression by the above immunostaining method, cells cultured by the method of the present invention are fixed with 4% paraformaldehyde (PFA) in PBS, washed with PBS several times, Triton X, etc. After increasing the permeability of cultured cells with 10% goat serum and blocking, immunostain with the target marker protein mouse antibody, reacted with AlexaFluor-labeled goat anti-mouse IgG, and determined by fluorescence microscopy A method can be illustrated.

  As a method for confirming the presence or absence of the expression of the alkaline phosphatase, cells cultured by the method of the present invention are treated with 4.5 mM citric acid, 2.25 mM sodium citrate, 3 mM sodium chloride, 65% methanol and 4% paraformaldehyde. A method of performing so-called alkaline phosphatase staining, in which alkaline phosphatase is visualized using an appropriate kit such as FastRed substrate kit (manufactured by Sigma), which is fixed for 5 minutes and washed, can be exemplified.

  The method of observing and confirming the morphological characteristics of the cells is that the boundary between the cells is unclear, the cells have almost no nuclei and almost no cytoplasm, and the outline of the cell population (colony) is clear and round. The undifferentiated state is maintained with the indication that there are many colonies with slightly elevated cells and that no fibroblast-like cells or nerve-like cells appear from the margin of the colonies. Can be determined.

  As a method for determining whether or not cells cultured by the method of the present invention maintain undifferentiation depending on whether or not the ability to differentiate into specific cells can be exhibited by stimulation with the specific differentiation-inducing factor. For example, when bone morphogenetic factor 4 (BMP4) is added to the medium, a method for determining that undifferentiation is maintained when the cells differentiate into epithelial cells can be mentioned.

  The culture container used in the maintenance culture method of the present invention is not particularly limited as long as it is a container capable of maintaining and culturing the pluripotent stem cells used in the present invention. For example, flasks, tissue culture flasks, dishes, petri dishes, Examples include tissue culture dishes, multi dishes, microplates, microwell plates, petri dishes, tubes, trays, culture bags, and the like.

  The present invention provides a kit for culturing human pluripotent stem cells while maintaining an undifferentiated state. The kit of the present invention comprises one or two or more supplements and basal medium components for human pluripotent stem cells, and the components constituting each of the above-described compositions may be individually or partially packaged. Including mixing and packaging two or more compositions. The kit supplements for culturing human pluripotent stem cells may be divided into activin-containing supplements and activin-free supplements. The kit for culturing human pluripotent stem cells further comprises an antibody against undifferentiated and / or differentiation marker protein, an undifferentiated and / or differentiation marker gene for determining the undifferentiation of human pluripotent stem cells. It is also possible to include a primer, a probe, and the like for detecting.

  EXAMPLES Next, although an Example is given and this invention is demonstrated further in detail, this invention is not limited at all by these. The ES cell lines and iPS cell lines, which are pluripotent stem cells used in the examples, were distributed according to government guidelines and used for experiments. Other cell lines are also stored at the National Institute of Biomedical Innovation and can be distributed under certain conditions.

[Examination of supplements]
(Basic medium)
We examined the replacement of conventional animal-derived materials with animal-free products. The hESF9 medium shown in Table 2 below was used as the conventional medium.

[Supplement ingredient; examination of fibronectin]
Inventors decided to investigate the substitution from animal-derived materials to animal product-free ingredients for ECMs such as collagen I, gelatin, laminin, fibronectin, etc., which have been used in recent years as cell supports to replace feeder cells. . Human ES cell KhES-1 strain (Kyoto University Institute for Regenerative Research, Stem Cell Medical Research Center), human iPS cell Tic strain (JCRB1331) (National Institute for Pharmaceutical Sciences / JSCB Cell Bank), and embryonic cancer cell PA-1 strain as a comparative example (JCRB9061) (National Institute for Pharmaceutical Sciences / JSCB cell bank) was used for examination.

Using a 96-well microplate (Corning), collagen I (Nitta Gelatin), gelatin (Sigma), laminin (Sigma), and fibronectin (Sigma) at 37 ° C. 3 It was processed so as not to be dried for a period of time, and was coated at 0.01, 0.05, 0.1, 0.5, ¹ , 5, and 10 μg / cm ^2, respectively. After addition of the hESF9 medium, the human ES cell KhES-1 strain, human iPS cell Tic strain (JCRB1331), and embryonic cancer cell PA-1 strain (JCRB9061) were counted 3 × 10 ⁶ cells after counting the number of cells. Seeded at / cm ² . After 2 days of culture (embryonic cancer cell PA-1 strain (JCRB9061) is cultured for 1 hour), cells stained with 0.4% crystal violet (manufactured by Sigma) dissolved in methanol for 30 minutes and adhered The nuclei were fixedly stained. The microplate was washed and dried, solubilized with 0.1 M sodium citrate dissolved in 50% methanol, and the absorbance at 595 nm was measured with a microplate reader (Model 550, manufactured by Bio-Rad). The results are shown in FIGS.

(result)
The human ES cell KhES-1 strain had the strongest adhesion ability for laminin and fibronectin, and the adhesion ability of collagen and ceratin was weak regardless of the concentration (see FIG. 1 (a)). The human iPS cell Tic strain (JCRB1331) has the strongest adhesive ability at a concentration of about 10-30 μg / cm ² for laminin and at a concentration of about 3-9 μg / cm ^{2 for} fibronectin. The adhesion ability was weak regardless of the concentration (see FIG. 1 (b)).

(Comparative example)
The embryonic cancer cell PA-1 strain (JCRB9061) was examined for its adhesion to various ECMs, and collagen and gelatin had strong adhesion at a concentration of about 10 μg / cm ² (FIG. 1 (c) )reference).

(Discussion)
The above results show that pluripotent stem cells have strong adhesion to laminin and fibronectin, but cytokeratin is strongly expressed in cells cultured on laminin, so bovine or human-derived fibronectin is present in the present invention. It was decided to use as a component of the supplement.

[Supplement ingredient; examination of substitute ingredient of oleic acid-conjugated bovine albumin]
In many conventional media such as the above-mentioned hESF9 media, bovine albumin (Bovine serum albumin: BSA) has been used. However, since it is an animal-derived material, an alternative to an animal product-free component was examined. According to previous studies by the inventor, it was confirmed that cell proliferation and undifferentiated maintenance tend to be high at concentrations of albumin 1 mg / mL and oleic acid 9.4 μg / mL (manufactured by Sigma). When undifferentiated markers SSEA-4, TRA-1-60, Nanog and differentiation markers SSEA-1 are used, bovine albumin or recombinant human albumin (manufactured by rHSA Millipore) and / or oleic acid are added, and ES cells We decided to examine how the maintenance of the undifferentiated state is affected. When oleic acid and albumin were added, they were added in the form of oleic acid-conjugated albumin. Of the components of the hESF9 medium, BSA and oleic acid are replaced with oleic acid-conjugated BSA (medium condition 1), rHSA alone (medium condition 2), oleic acid-conjugated rHSA (medium condition 3), and the hESF9 medium. The H9 strain (WA09) was cultured using a medium to which none of BSA, rHSA, and oleic acid was added as a negative control (medium condition 4). The cultured H9 strain (WA09) was subcultured for 1 passage in hESF9 medium, fluorescent immunostaining was performed, and the positive rate of the expressed cells was analyzed. The cells were fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 15 minutes, blocked with bovine serum, then anti-SSEA-4 antibody (Abcam) and AlexaFluor-labeled anti-mouse IgG (Invitrogen) Anti-TRA-1-60 antibody (Abcam) and AlexaFluor-labeled anti-mouse IgM (manufactured by Invitrogen), anti-SSEA-1 antibody (Abcam) and AlexaFluor-labeled anti-mouse IgM (Invitrogen) were used. After immunostaining and blocking with bovine serum containing Triton X, immunostaining was performed with anti-Nanog antibody (manufactured by Cell Signaling) and anti-OCT4 antibody (manufactured by Santa Cruz), and AlexaFluor labeled secondary It reacted with the antibody and observed under a fluorescence microscope. The details of each medium condition are shown in Table 3 below, and the results are shown in FIG.

(result)
There is a significant difference in the positive rates of undifferentiated markers SSEA-4, TRA-1-60, and Nanog expressing cells in H9 strain (WA09) cultured for one passage due to differences in medium conditions (1) to (4). Not (see FIG. 2). On the other hand, it was found that the positive rate of SSEA-1-expressing cells, which are differentiation markers, was significantly increased (*) in the medium condition (2) to which only rHSA was added. The above results showed that although rHSA does not affect the expression of undifferentiated markers, the addition of oleic acid-conjugated rHSA reduces the expression of differentiation markers and suppresses differentiation. .

(Fluorescent immunostaining)
In the medium condition (3), the H9 strain (WA09) cultured cells at the third passage were confirmed by fluorescent immunostaining using undifferentiated markers SSEA-4, Tra1-60, Nanog, and differentiation markers SSEA-1.

(result)
Although the H9 strain (WA09) cultured cells immunostained with the undifferentiated markers SSEA-4, Tra1-60, and Nanog antibodies can confirm that each antibody is highly expressed, the differentiation marker SSEA-1 Was confirmed to be low expression. From the above results, it was confirmed that a supplement containing oleic acid-conjugated rHSA is effective in maintaining the undifferentiated nature of pluripotent stem cells.

[Supplement component; removal of porcine heparin]
In the past, the present inventors have developed a medium containing heparin in order to maintain primate embryonic stem cells (see, for example, the above-mentioned JP 2009-542247). Since it is an animal such as a pig, an attempt was made to culture the H9 strain (WA09) in a medium in which activin was added to a medium in which heparin was deleted from hESF-9. When the H9 strain (WA09) stained with the anti-Tra-1-60 antibody was observed with a microscope for the second-passage cultured cells, the cell-to-cell boundary was unclear and the cell morphology in which the nucleus was mostly occupied, It was confirmed that the round outline of the colony and the state in which the cells were slightly raised could be read, and pluripotency was maintained (see FIG. 4). From these results, porcine-derived heparin was excluded from the medium components.

[Basic medium; Preparation of hESF-grow medium]
The inventors added HEPES to a conventional medium (see, for example, the above-mentioned international publication WO2005 / 063968 pamphlet), but adding HEPES to a serum-free medium greatly adversely affects the maintenance of human ES cells. (See, for example, PNAS, vol.105, no.36, 13409-13414) The finding that undifferentiation is maintained when ascorbic acid is added except for HEPES (Japanese Patent Publication No. 2009-542247) Based on the above, a basal medium (hESF-grow medium) having the composition shown in Table 4 below was prepared as an animal product-free basal medium, and sterilized according to a conventional method.

  Based on the findings of the inventors and the above examination, as supplements used for undifferentiated maintenance culture of human pluripotent stem cells, bovine fibronectin, recombinant human insulin, recombinant human transferrin (apo), 2-mercaptoethanol, 2-ethanol Culture of pluripotent stem cells using a medium prepared by appropriately combining amine, sodium selenate, recombinant human FGF-2, oleate-conjugated recombinant human albumin, and recombinant human activin and adding to the hESF-grow medium I decided to try.

  In the following study, cryopreserved H9 strain (WA09) is a 0.1% gelatin (Embryomax, Millipore) coated flask (Corning) and Dulbecco's modified Eagle medium containing 10% fetal calf serum. Then, mouse embryo-derived fibroblasts (Millipore) whose cell growth was stopped with mitomycin C were seeded as feeder cells, 1 mM L-glutamine (21-51-016, GIBCO), 0. 1 mM 2-mercaptoethanol, 1% non-essential amino acid (11140-035, manufactured by GIBCO), 20% KSR (10828-028, manufactured by GIBCO) and 4 ng / ml bFGF (13256-029, manufactured by Katayama Scientific) ) Supplemented with DM / F12 (manufactured by GIBCO) and maintained in a KSR-added medium, preparation before sowing was performed.

(Preparation of medium 1)
In a 25 cm ² flask (manufactured by Corning), (1) human plasma fibronectin (F0895, manufactured by Sigma) was dissolved in PBS to ² μg / cm ² and treated at 37 ° C. for 3 hours to prevent drying. Then, the solution was removed by suction immediately before cell seeding, and (2) 10 μg / ml recombinant human insulin (19278-5ML, Sigma), (3) 5 μg / ml recombinant human transferrin (apo) ( (T 2252, manufactured by Sigma), (4) 10 μM 2-mercaptoethanol (M 7522, manufactured by Sigma), (5) 10 μM 2-ethanolamine (E 0135, manufactured by Sigma), (6) 20 nM Sodium selenate (S 9133, Sigma), (7) 9.4 μg / ml oleic acid (O-1383-5G, Sigma) in 1 mg / mL recombinant human albumin Combined rHSA-conjugated oleic acid, (8) 8 ng / ml basicFGF (manufactured by Katayama Science Co., Ltd.) and activin (N-338 AC / CF, manufactured by R & D) as activin-containing supplements The medium added to the hESF-grow medium was prepared as an activin-containing supplement + hESF-grow medium.

(Preparation of medium 2)
A medium in which a supplement consisting of the above eight factors (1) to (8) was added to the hESF-grow medium as an activin-free supplement was prepared as an activin-free supplement + hESF-grow medium.

(Culture in activin-containing medium)
Sixty hours after the start of culturing of the H9 strain (WA09) in the activin-containing supplement + hESF-grow medium, the medium was changed to the activin-containing supplement + hESF-grow medium. Cells were observed 7 days after the start of culture. A micrograph of the cells is shown in FIG. The shape of the colony was flat and collapsed, and it was confirmed that there was a high possibility that undifferentiated properties were lost.

(Change of medium to activin-free medium)
Sixty hours after the start of culturing of the H9 strain (WA09) in the activin-containing supplement + hESF-grow medium, the medium was replaced with the activin-free supplement + hESF-grow medium. Cells were observed 5 days after the start of culture. A micrograph of the cells is shown in FIG. The shape of the colony was raised in a round shape, and it was confirmed that there was a high possibility that undifferentiation was not lost.

[Undifferentiated maintenance culture of human pluripotent stem cells]
(Fibronectin coating)
Thereafter, the study was continued using the activin-containing supplement + hESF-grow medium as the first medium and the activin-free supplement + hESF-grow medium as the second medium.

  The H9 strain (WA09) maintained in the KSR-supplemented medium is removed, and after the medium is allowed to act, 1 unit / ml dispase is allowed to act at 37 ° C. for 2 minutes, and then the dispase is removed. In the medium solution, collect the cell population using a scraper or the like, centrifuge at 300 rpm for 1 minute, disperse in the solution in the first medium, centrifuge again, disperse in the first medium, The first passage culture was started.

  60 hours after the start of cultivation of the H9 strain (WA09) in the first medium, the medium was replaced with the second medium. Further, every 24 hours, the medium was changed to the second medium three times, and the medium of the cultured H9 strain (WA09) was cultured on the third day after the medium change from the first medium to the second medium. 1 unit / ml dispase was allowed to act at 37 ° C. for 5 minutes, and then the dispase was removed, and the cell population was collected with the above-mentioned first medium solution of the present invention using a scraper or the like at 300 rpm. After centrifugation, the mixture was dispersed with the first medium solution, centrifuged again, and then dispersed into the first medium to transfer (passage), and the second passage culture was performed. When the use timing of the cells used at the first passage is optimal, the passage at the second passage is on the 3rd and 4th days, and after that, the passage is on the 5th day.

  The second passage H9 strain (WA09) subcultured to the first medium was replaced with the second medium 60 hours after the start of the culture in the first medium. After the medium was changed 5 times with the second medium, on the 5th day after the medium change from the first medium to the second medium, the medium was subcultured to the first medium again, and the third culture was performed. Thereafter, the steps of subculture to the first medium and medium exchange to the second medium were repeated, and the culture was continued.

(Morphological observation)
FIG. 6 is a photograph of the cells in the 1st to 6th passages of the H9 strain (WA09) cultured cells cultured by the above method, observed under a phase contrast microscope. At any stage, the cell-cell boundary, which is a characteristic of undifferentiated cells, is unclear, the cells have almost no nucleus and almost no cytoplasm, and the outline of the cell population (colony) is round and clear. However, it is possible to read the fact that there are many colonies that are slightly raised and that no fibroblast-like cells or nerve-like cells appear from the edge of the colony, and pluripotency is maintained. Confirmed that.

(Analysis by flow cytometry method)
For the H9 strain (WA09) cultured cells obtained by culturing the H9 strain (WA09) by the above method, SSEA-3, SSEA-4, TRA-2-54, TRA-1-60, TRA-1-80, And CD90 (manufactured by BD), SSEA-1, CD105 (manufactured by BD), CD56 (manufactured by BD), and A2B5 (manufactured by Millipore) as differentiation markers, and flow cytometry for undifferentiation. Analysis. HLA-abc (manufactured by BD) was used to confirm whether the cells were derived from human. For the above-mentioned H9 strain (WA09) cultured cells at the fifth passage, the cells on the fifth day from the last passage were dispersed in a PBS solution containing trypsin / EDTA, and the cells were treated with 1 ml of 10% bovine serum (high clone). And then centrifuged for 30 minutes, and then incubated with each marker mouse antibody (Abcam) for 30 minutes. The cultured cells were washed 3 times with PBS containing 1% bovine serum and reacted with AlexaFluor-conjugated anti-rat IgM, goat anti-mouse IgG, IgM antibody (Invitrogen) for 15 minutes. The cultured cells were washed three times with PBS containing 1% bovine serum, and the resuspended cells were analyzed using a BD FACSCanto ^™ flow cytometer (BD, NJ USA). The results are shown in FIG.

(result)
As is clear from FIG. 7, cells that are highly expressed for the undifferentiated markers SSEA-3, SSEA-4, TRA-2-54, TRA-1-60, TRA-1-80, and CD90 Of 66.3% to 99.5%. The differentiation markers SSEA-1, CD105, CD56, and A2B5 were only expressed at 1.12% to 12.5%. HLA-abc is 99%, indicating that it is a human-derived cell. These results showed that when fibronectin was coated, the H9 strain (WA09) cultured cells were pluripotent stem cells that remained undifferentiated even after 5 passages.

[Undifferentiated maintenance culture of human pluripotent stem cells]
(Preparation of fibronectin-containing first medium)
In a 6-well plate (BD), 10 μg / ml recombinant human insulin, 5 μg / ml recombinant human transferrin (apo), 10 μM 2-mercaptoethanol, 10 μM 2-ethanolamine, 20 nM in the hESF-grow medium. Of sodium selenite, 9.4 μg / ml oleic acid complexed with 1 mg / mL recombinant human albumin, and 2 ng / mL recombinant human activin, and 0, 20, 40, 80, 160, A medium in which 320 μg / well of bovine fibronectin was added to each well was prepared as a first medium containing fibronectin.

(Preparation of fibronectin-containing second medium)
In a 6-well plate (manufactured by BD), 5 μg / ml recombinant human transferrin (apo), 10 μM 2-mercaptoethanol, 10 μM 2-ethanolamine, 20 nM sodium selenate, and 1 mg / ml in the hESF-grow medium. 9.4 μg / ml oleic acid complexed with mL of recombinant albumin was added, and 0, 20, 40, 80, 160, 320 μg / well of bovine fibronectin was added to each well. Prepared as a second medium.

For the H9 strain (WA09), the fibronectin-containing first medium and the fibronectin-containing second medium are replaced with the fibronectin-containing first medium and the second medium described in the fibronectin coating section, and the fibronectin concentration is changed. The cells were cultured in the same manner as the fibronectin coating except that the cells were cultured in each well. The area of 1 well of the used plate was 9.6 cm ² , and 3 ml of the medium was added.

(result)
FIG. 8 shows a photomicrograph after passage of the cultured cells of the strain H9 (WA09) to a serum-free medium. From the appearance of the cells, it was judged that undifferentiation was most maintained when 80 μg / well of fibronectin was added. It was confirmed that fibronectin is effective in the method of the present invention even if it is added to the basal medium together with other supplement components instead of coating the culture vessel. From these results, it was confirmed that the human pluripotent stem cells maintained undifferentiated state even after repeated passage by using the culture method of the present invention.

[Undifferentiated maintenance culture of human ips cells]
(Culture in medium containing protein kinase C peptide inhibitor)
The fibronectin-coated supplements described in Example 3 containing activin + hESF-grow medium and activin-free supplements + hESF-grow medium were added with 0, 1, 2.5, 5, 10 μM myristoylated protein kinase C peptide inhibitor (Myr. RFARKGALRQKNV, respectively). ) (Manufactured by Promega) was prepared and used as a first medium containing a protein kinase C inhibitor and a second medium containing a protein kinase C inhibitor.

  The human iPS cell Tic strain (JCRB1331) was dispersed in the first medium containing the protein kinase C inhibitor and culture was started. Sixty hours after the start of cultivation of the Tic strain, the medium was replaced with the second medium containing the protein kinase C inhibitor. Furthermore, the medium was changed twice every 24 hours, and fixed with 4.5 mM citric acid, 2.25 mM sodium citrate, 3 mM sodium chloride, 65% methanol and 4% paraformaldehyde for 5 minutes on the 4th day after the start of the culture. Thereafter, alkaline phosphatase staining was performed using a FastRed substrate kit (manufactured by Sigma) according to the manufacturer's guidelines. The results are shown in FIG.

(result)
A photograph on the fourth day after the start of the cultivation of the Tic strain is shown in FIG. When cultured in a medium supplemented with 1 to 5 μM myristoylated protein kinase C (PKC) peptide inhibitor, it was observed that differentiation was further suppressed as compared to the case where the addition concentration was 0, and 1-2. In the medium added with 5 μM, it was observed that the growth was increased.

  Recently, regenerative medicine that artificially induces differentiation of stem cells that are thought to have the ability to differentiate in multiple directions, creates tissues and organs, and compensates for defective tissues compensates for the drawbacks of conventional organ transplantation It is attracting attention as a treatment method. By the method of the present invention, it becomes possible to supply pluripotent stem cells that maintain undifferentiated properties, and it can be applied to regenerative medicine for constructing a target tissue or organ in vitro or in vivo. Can solve various problems of transplantation therapy.

Claims (1)

Human pluripotent stem cells are cultured while maintaining an undifferentiated state characterized by repeating the following steps (a) to (c), step (b) and step (c) after step (a) Method.
(A) a medium for pluripotent stem cells containing activin and one or more supplements selected from the group consisting of fibronectin, insulin, transferrin, selenium, and FGF-2 (provided that knockout serum replacement and / or laminin 111 is added) Culturing human pluripotent stem cells in a first medium that is excluding one);
(B) The first medium contains one or more supplements selected from the group consisting of fibronectin, insulin, transferrin, selenium, and FGF-2, and does not contain activin (provided that the first medium is a medium for pluripotent stem cells) Culturing human pluripotent stem cells by replacing with a second medium, excluding those containing knockout serum replacement and / or laminin 111);
(C) subculturing human pluripotent stem cells in the first medium;