Method for producing iPS cell-derived keratinocytes

Abstract

The present invention aims to provide a method for producing iPS cell-derived keratinocytes that can induce keratinocytes that highly express keratinocyte markers in a feeder-free and serum-free environment. [Solution] A method for producing iPS cell-derived keratinocytes, comprising an induction step of inducing iPS cells into keratinocytes using one or more induction media in a feeder-free and serum-free environment, wherein for at least a portion of the induction step, a medium containing fibroblast growth factor 7 (FGF7) is used as the induction medium.

Description

[Technical Field]

[0001] This invention relates to a method for producing iPS cell-derived keratinocytes. [Background technology]

[0002] Human skin has a three-layered structure consisting of the epidermis, dermis, and subcutaneous tissue, in that order from the outside. The epidermis performs the function of transpiration and acts as an important barrier protecting the body from microorganisms, chemical substances, and physical stimuli. The epidermis is mainly composed of cells called keratinocytes. Keratinocytes are born as basal cells with the ability to divide in the basal layer, the innermost layer of the epidermis, and are gradually pushed up to the outside of the epidermis, changing shape into spinous cells, granular cells, and corneocytes, until they finally peel off the skin as dead skin cells.

[0003] iPS cells (induced pluripotent stem cells), developed in 2006, are stem cells established by introducing specific factors (reprogramming factors) into somatic cells, and they exhibit pluripotency similar to ES cells (embryonic stem cells). It is expected that utilizing iPS cells will enable the creation of transplantable tissues and organs without rejection reactions, while avoiding the ethical issues associated with ES cells, which are stem cells derived from fertilized eggs. One of the various applications of iPS cells is the induction of keratinocytes. If keratinocytes derived from iPS cells can be produced, they are thought to have potential applications in fields such as artificial skin, wound healing, and patch testing to examine skin reactivity to chemical substances.

[0004] As a method for producing iPS cell-derived keratinocytes, an ex vivo method for obtaining a population of human keratinocytes derived from human pluripotent stem cells is known, which includes a step of co-culturing human pluripotent stem cells with cells that support ectoderm differentiation in the presence of a drug that stimulates epidermal induction and a drug that stimulates terminal differentiation of keratinocytes (see Japanese Patent Publication No. 2011-525370). As an inducer for inducing iPS cells into keratinocytes, for example, a combination of BMP4 (bone morphogenetic protein 4) and retinoic acid (a type of vitamin A derivative) is known.

[0005] However, existing methods for producing iPS cell-derived keratinocytes have difficulty effectively inducing iPS cells into keratinocytes, and tend to include a certain amount of cell populations that do not express keratinocyte markers (undifferentiated cells or cells differentiated into cells other than keratinocytes). Furthermore, existing methods for producing iPS cell-derived keratinocytes commonly use biological materials such as feeder cells and serum, which means that these biological materials may contaminate the produced keratinocytes. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Special Publication No. 2011-525370 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a method for producing iPS cell-derived keratinocytes that can be manufactured from iPS cells in a feeder-free and serum-free environment, under the circumstances described above. [Means for solving the problem]

[0008] To solve the above problems, the inventors conducted various studies and found that by adding fibroblast growth factor 7 (FGF7) to the culture medium in the early stages of inducing iPS cell-derived keratinocytes, keratinocytes could be effectively induced even in a feeder-free and serum-free environment, resulting in high-quality keratinocytes that highly express keratinocyte markers. While FGF7 was known to act as a keratinocyte growth factor, the inventors were the first to demonstrate that it also acts as a keratinocyte inducer for iPS cells. The present invention provides a method for producing iPS cell-derived keratinocytes based on these findings, and is a method for producing iPS cell-derived keratinocytes using FGF7 in a feeder-free and serum-free environment. In other words, the gist of the present invention is as follows.

[0009] [1] The induction step comprises inducing iPS cells into keratinocytes using one or more induction media in a feeder-free and serum-free environment, A method for producing iPS cell-derived keratinocytes, wherein a medium containing fibroblast growth factor 7 (FGF7) is used as the induction medium for at least a portion of the induction step. [2] The method for producing iPS cell-derived keratinocytes according to [1], wherein the FGF7-containing medium is Essential 6 medium. [3] The method for producing iPS cell-derived keratinocytes according to [1] or [2], wherein the FGF7-containing medium is an epithelial cell proliferation medium. [4] The method for producing iPS cell-derived keratinocytes according to [1] or [2], wherein the concentration of FGF7 in the FGF7-containing medium is 1 ng / mL or more and 100 ng / mL or less. [5] A method for producing iPS cell-derived keratinocytes according to [1] or [2], wherein the induction medium contains at least one selected from the group consisting of bone morphogenetic factor 4 (BMP4), retinoic acid, and epidermal growth factor (EGF) for at least a portion of the induction step. [6] A method for producing iPS cell-derived keratinocytes according to [1] or [2], wherein the period for which the FGF7-containing medium is used in the induction step is 5 days or more and 20 days or less. [7] The process further comprises a maturation step for maturing the keratinocytes after the induction step, A method for producing iPS cell-derived keratinocytes according to [1] or [2], wherein the maturation step involves using a culture medium that does not contain FGF7. [8] The iPS cells are on-feeder iPS cells, The induction process is as follows: A suspension culture step in which the iPS cells are cultured in suspension to form embryoid bodies, The system comprises an adhesion culture step for adhering and culturing the aforementioned embryoid bodies, The induction medium used in the suspension culture step does not contain FGF7. A method for producing iPS cell-derived keratinocytes according to [1] or [2], using the FGF7-containing medium in the adhesion culture step. [9] The iPS cells are feeder-free iPS cells, The induction process is as follows: A first induction step involves adhering culture in the induction medium that does not contain FGF7, A method for producing iPS cell-derived keratinocytes according to [1] or [2], comprising a second induction step of adhering culture in the FGF7-containing medium. [Effects of the Invention]

[0010] The present invention provides a method for producing iPS cell-derived keratinocytes that allows for the production of high-quality keratinocytes from iPS cells in a feeder-free and serum-free environment. [Brief explanation of the drawing]

[0011] [Figure 1] This figure illustrates a specific scheme for the method of producing keratinocytes according to the present invention. [Figure 2] This figure illustrates a specific scheme for the method of producing keratinocytes according to the present invention. [Figure 3-1] This figure shows the flow cytometry analysis results of iPS cell-derived keratinocytes produced in Test Examples 1-4. [Figure 3-2]It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 5 to 7 by flow cytometry. [Figure 3-3] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 8 to 11 by flow cytometry. [Figure 3-4] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 12 to 14 by flow cytometry. [Figure 4] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 1 to 14 by flow cytometry. [Figure 5] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Example 13 by immunocytochemistry. [Figure 6-1] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 1 to 4 by flow cytometry. [Figure 6-2] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 5 to 7 by flow cytometry. [Figure 6-3] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 8 to 11 by flow cytometry. [Figure 6-4] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 12 to 14 by flow cytometry. [Figure 7] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 1 to 14 by flow cytometry. [Figure 8-1] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 1 to 4 by flow cytometry. [Figure 8-2] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 5 to 7 by flow cytometry. [Figure 8-3] It is a figure showing the analysis results of iPS cell-derived keratinocytes produced in Test Examples 8 to 11 by flow cytometry. [Figure 8-4] This figure shows the flow cytometry analysis results of iPS cell-derived keratinocytes produced in Test Examples 12-14. [Figure 9] This figure shows the flow cytometry analysis results of iPS cell-derived keratinocytes produced in Test Examples 1-14. [Figure 10] This figure shows the flow cytometry analysis results of iPS cell-derived keratinocytes produced in Test Examples 1-14. [Figure 11] This figure shows the results of immunocytochemistry analysis of iPS cell-derived keratinocytes produced in Test Example 13. [Figure 12] This figure shows the flow cytometry analysis results of iPS cell-derived keratinocytes produced in Test Examples 15-18. [Figure 13] This figure shows the flow cytometry analysis results of iPS cell-derived keratinocytes produced in Test Examples 19-22. [Figure 14] This figure shows the flow cytometry analysis results of iPS cell-derived keratinocytes produced in Test Examples 19-22. [Modes for carrying out the invention]

[0012] The present invention will be described in detail below.

[0013] [Method for producing iPS cell-derived keratinocytes] The present invention provides a method for producing iPS cell-derived keratinocytes (hereinafter sometimes referred to as "method for producing keratinocytes"), which comprises an induction step of inducing iPS cells into keratinocytes using one or more induction media in a feeder-free and serum-free environment. Preferably, the method for producing keratinocytes of the present invention further comprises a preparation step of seeding and adhering iPS cells to a culture vessel before the induction step, and / or a maturation step of maturing the keratinocytes.

[0014] The method for producing keratinocytes according to the present invention is typically carried out at a temperature of 30-37°C, under a 2-7% CO2 environment, and under a 5-21% O2 environment. Culture conditions not explicitly stated herein can be the same as those used in known iPS cell or keratinocyte culture methods. First, the iPS cells used in the present invention will be described.

[0015] (iPS cells) Methods for producing iPS cells are well known in the field and can be produced by introducing reprogramming factors into any somatic cell. Examples of reprogramming factors include genes or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1. These reprogramming factors may be used individually or in combination. The combinations of initialization factors are WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, etal. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, etal. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al.(2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat. Cell Biol. 11:197-203, RL Judson et al., (2009), Nat. Biotechnol., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci US A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. Examples of combinations are given in the following publications: 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, and Maekawa M, et al. (2011), Nature. 474:225-9.

[0016] Somatic cells are not particularly limited and include somatic cells of fetuses (offspring), somatic cells of newborns (offspring), and mature, healthy or diseased somatic cells. Furthermore, somatic cells include primary cultured cells, passaged cells, and established cell lines. Specifically, examples of somatic cells include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) differentiated cells such as blood cells (peripheral blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0017] The species from which iPS cells are derived is not particularly limited, but mammals are preferred, and humans are more preferred. Furthermore, when keratinocytes obtained by the keratinocyte production method of the present invention are used for treatment or examination, it is preferable that the individual from which the iPS cells are derived is the subject of treatment or examination themselves.

[0018] iPS cells may be cultured using either adherent culture or suspension culture. In the case of adherent culture, culture vessels coated with extracellular matrix components may be used, or the cells may be co-cultured with feeder cells. Feeder cells are not particularly limited, but examples include fibroblasts (mouse fetal fibroblasts (MEF), mouse fibroblasts (STO), etc.). It is preferable that the feeder cells are inactivated by known methods, such as irradiation with radiation (gamma rays, etc.) or treatment with anticancer agents (mitomycin C, etc.). Examples of extracellular matrix components include fibrous proteins such as Matrigel (Niwa A, et al. PLoS One.6(7):e22261, 2011), gelatin, collagen, and elastin, as well as glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, and cell adhesion proteins such as proteoglycans, fibronectin, vitronectin, laminin, and entactin.

[0019] In addition, iPS cells co-cultured with feeder cells are sometimes referred to as "on-feeder iPS cells," and iPS cells cultured without feeder cells are sometimes referred to as "feeder-free iPS cells." Feeder-free iPS cells are preferred as the iPS cells used in the induction step from the viewpoint of suppressing contamination of keratinocytes by feeder cells. However, although the method for producing keratinocytes of the present invention is carried out in a feeder-free and serum-free manner at least from the induction step onward, iPS cells may be cultured on feeders before the induction step.

[0020] <Induction process> In the induction step, iPS cells are induced into keratinocytes using one or more induction media in a feeder-free and serum-free environment. The induction step refers to the period during which iPS cells are cultured using keratinocyte-inducing factors (e.g., FGF7, all-trans retinoic acid (ATRA), bone morphogenetic factor 4 (BMP4), etc.) in a method for producing iPS cell-derived keratinocytes. The induction medium refers to a medium containing keratinocyte-inducing factors.

[0021] The phrase "using one or more induction media" means either using only one induction medium throughout the entire induction process, or switching induction media at some point during the induction process. It is preferable to use multiple induction media during the induction process and switch between them as needed. "Feeder-free and serum-free environment" means not using feeder cells or serum throughout the entire induction process.

[0022] The present invention's method for producing keratinocytes suppresses contamination of the produced keratinocytes with feeder cells or serum by performing the induction step in a feeder-free and serum-free environment.

[0023] The duration of the induction process is not particularly limited, but is preferably 7 to 25 days, and more preferably 13 to 19 days. After the induction process, iPS cells are induced into keratinocytes (including immature keratinocytes).

[0024] In the induction process, FGF7-containing medium may be used for at least a portion of the time, or it may be used for the entire period. The period for which FGF7-containing medium is used in the induction process is preferably 5 to 20 days, and more preferably 8 to 17 days. By using FGF7-containing medium within this range, iPS cells can be sufficiently induced into keratinocytes.

[0025] (FGF7-containing culture medium) An FGF7-containing medium is a serum-free medium containing at least FGF7. An FGF7-containing medium can be prepared, for example, by adding FGF7 to a base medium (hereinafter sometimes referred to as "base medium"), and other components other than FGF7 may be added further. Examples of base media for FGF7-containing media include basal media and media to which additives have been added to basal media. Here, "base medium for FGF7-containing medium" refers to, for example, a single medium or mixed medium containing 90% by mass or more (preferably 99% by mass or more) of FGF7 in the FGF7-containing medium. It is preferable that the FGF7-containing medium does not contain any animal-derived components other than serum as much as possible.

[0026] A basal medium refers to a culture medium containing essential carbon sources, nitrogen sources, and inorganic salts for the culture of animal cells. The basal medium may also contain trace amounts of beneficial substances such as micronutrient enhancers and precursors, as needed. While known basal media can be used, specific examples include minimal essential media (MEM) such as Eagle medium, Dulbecco's modified Eagle medium (DMEM), minimal essential medium α (MEM-α), mesenchymal cell basal medium (MSCBM), Ham's F-12 and F-10 media, DMEM / F12 medium, Williams medium E, RPMI-1640 medium, MCDB medium, 199 medium, Fisher medium, Iscove modified Dulbecco's medium (IMDM), McCoy modified medium, and mixtures thereof. While the basal medium is not particularly limited, DMEM, F12 medium, or mixtures thereof are preferred.

[0027] Additives that can be used in FGF7-containing culture media include amino acids, inorganic salts, vitamins, growth factors, antibiotics, carbon sources, and other additives. The concentrations of these additives are not particularly limited and can be used at concentrations commonly used in animal cell culture media. Additives may also be included in the basal culture medium.

[0028] Examples of amino acids include glycine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0029] Examples of inorganic salts include calcium chloride, copper sulfate, iron(III) nitrate, iron sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

[0030] Examples of vitamins include choline, vitamin A (especially all-trans retinoic acid (ATRA)), vitamin B1, vitamin B2, vitamin B3, vitamin B4, vitamin B5, vitamin B6, vitamin B7, vitamin B12, vitamin B13, vitamin B15, vitamin B17, vitamin Bh, vitamin Bt, vitamin Bx, vitamin C (ascorbic acid), vitamin D, vitamin E, vitamin F, vitamin K, vitamin M, and vitamin P.

[0031] Examples of growth factors or proliferation factors include FGF family members other than FGF7 (e.g., FGF1, FGF2, etc.), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), thrombopoietin (TPO), hepatocyte growth factor (HGF), and others.

[0032] Examples of antibiotics include penicillin, streptomycin, neomycin sulfate, amphotericin B, blastocydin, chloramphenicol, amoxicillin, bacitracin, bleomycin, cephalosporins, chlortetracycline, zeosin, and puromycin. Examples of carbon sources include glucose, galactose, fructose, and sucrose. Other additives include, for example, trace metals such as magnesium, iron, zinc, calcium, potassium, sodium, copper, selenium, cobalt, tin, molybdenum, nickel, and silicon; stem cell differentiation inducers such as β-glycerophosphate, dexamethasone, rosiglitazone, isobutylmethylxanthine, and 5-azacitidine; antioxidants such as 2-mercaptoethanol, catalase, superoxide dismutase, and N-acetylcysteine; and adenosine 5'-monophosphate, corticosterone, ethanolamine, insulin, reduced glutathione, lipoic acid, melatonin, hypoxanthine, phenol red, progesterone, putrescine, pyruvate, thymidine, triiodothyronine, transferrin, and lactoferrin. All culture media used in this invention (FGF7-containing media and other media) may contain antibiotics as needed.

[0033] Examples of base media for FGF7-containing media include "Essential 6," "Essential 8," epidermal cell proliferation medium, and other stem cell culture media.

[0034] "Essential 6" and "Essential 8" are xeno-free culture media with clearly defined compositions that can support the spontaneous or induced differentiation of pluripotent stem cells (PSCs). The components of "Essential 6" and "Essential 8" are shown below (Reference: Chen G, Gulbranson DR, Hou Zet al. (2011) Chemically defined conditions for human iPSC derivation and culture. Nat Methods 8 (5):424-429.). In the following, "DMEM / F-12" refers to a mixed medium of DMEM and F-12 (e.g., a mixing ratio of 1:1). "Essential 6" and "Essential 8" may be those sold under the same names by Thermo Fisher Scientific, for example.

[0035] • "Essential 6": DMEM / F-12, L-ascorbic acid, selenium, transferrin, sodium bicarbonate, insulin • "Essential 8": DMEM / F-12, L-ascorbic acid, selenium, transferrin, sodium bicarbonate, insulin, FGF2, TGFβ1

[0036] For epithelial cell growth medium, commercially available media for epithelial cells from various manufacturers can be used. As epithelial cell growth medium, for example, a low-calcium medium (e.g., calcium concentration: 0.01~0.20 mM, preferably calcium concentration: 0.07 mM) can be used. As epithelial cell growth medium, the "CnT-Prime" series from CELLnTEC is preferred, and "CnT-Prime Epithelial Culture Medium" from CELLnTEC (indicated as "CnT-PR" in the catalog) is more preferred. Hereafter, "CnT-Prime Epithelial Culture Medium" from CELLnTEC may be referred to as "CnT-PR".

[0037] Other stem cell culture media can be those commercially available from various manufacturers for use with ES cells or iPS cells. Preferred other stem cell culture media include "Primate ES Cell Medium" (hereinafter sometimes referred to as "Primate ESC") manufactured by Reprocell Co., Ltd., which is a culture medium for primate ES / iPS cells, and the "StemFIT®" series manufactured by Ajinomoto Healthy Supply Co., Ltd., which is commercially available as an iPS / ES cell proliferation medium free of animal and human-derived components (for example, "StemFIT® AK02N" for basic research and "StemFIT® AK03N" for clinical research). Note that "StemFIT®" is sold as a set consisting of base solutions A and B and a supplement solution C. When using "StemFIT®" as an FGF7-containing medium, it is preferable to use only solutions A and B mixed together, without using solution C.

[0038] The FGF7-containing medium preferably contains "Essential 6". In this case, "Essential 6" is preferably the base medium of the FGF7-containing medium, either alone or in combination with other media. When "Essential 6" and other media are used in combination, the mixing ratio is preferably 4:6 to 6:4, and more preferably 5:5. The FGF7-containing medium also preferably contains epidermal cell proliferation medium (especially "CnT-PR"). In this case, the epidermal cell proliferation medium is preferably the base medium of the FGF7-containing medium, in combination with other media (especially "Essential 6" or other stem cell culture media). When epidermal cell proliferation medium and other media are used in combination, the mixing ratio is preferably 4:6 to 6:4, and more preferably 5:5. By including these media in the FGF7-containing medium, iPS cells can be more effectively induced into keratinocytes.

[0039] (FGF7) FGF7 is a fibroblast growth factor also known as "keratinocyte growth factor (KGF)". FGF7 generally has heparin-binding properties and exhibits cell proliferation activity in keratin-producing cells, but does not exhibit cell proliferation activity in fibroblasts or endothelial cells. The FGF7 used in the induction step is not particularly limited, and various animal-derived FGF7 such as human FGF7, mouse FGF7, and rat FGF7 can be used, but human FGF7 is preferred. Furthermore, FGF7 may be genetically modified or chemically treated as long as it retains FGF7 activity. FGF7 may be expressed in prokaryotes such as E. coli, expressed in mammalian cultured cells such as human, mouse, or rat, or obtained by other methods.

[0040] The FGF7 concentration in the FGF7-containing medium is not particularly limited, but is preferably 1 to 100 ng / mL, more preferably 3 to 40 ng / mL, and even more preferably 10 to 20 ng / mL. For example, the FGF7 concentration in the FGF7-containing medium can be 10 ng / mL.

[0041] (Suitable optional components of FGF7-containing culture medium) The FGF7-containing medium may contain not only the base medium and FGF7, but also other components. The FGF7-containing medium preferably further contains at least one of the known keratinocyte-inducing factors (particularly BMP4 or ATRA) and EGF, and more preferably further contains only EGF, or further contains all of BMP4, ATRA, and EGF.

[0042] When the FGF7-containing medium contains BMP4, the BMP4 concentration in the FGF7-containing medium is not particularly limited, but is preferably 1 to 50 ng / mL, more preferably 2 to 25 ng / mL, and even more preferably 4 to 15 ng / mL. For example, the BMP4 concentration in the FGF7-containing medium can be 10 ng / mL.

[0043] When the FGF7-containing medium contains ATRA, the ATRA concentration in the FGF7-containing medium is not particularly limited, but is preferably 0.01 to 10 μM, more preferably 0.1 to 5 μM, and particularly preferably 1 to 4 μM. The ATRA concentration in the FGF7-containing medium can be, for example, 1 μM or 2 μM.

[0044] When the FGF7-containing medium also contains EGF, the EGF concentration in the FGF7-containing medium is not particularly limited, but is preferably 1 to 500 ng / mL, more preferably 2 to 100 ng / mL, and even more preferably 4 to 30 ng / mL. For example, the EGF concentration in the FGF7-containing medium can be 10 ng / mL.

[0045] Specific compositions of FGF7-containing culture media include, for example, the following compositions. In the following compositions, it is preferable to use only solutions A and B of the included solutions A to C, without using solution C, as "StemFIT®". " / " indicates that the two culture media are used in a mixture (for example, a mixing ratio of 4:6 to 6:4, preferably a mixing ratio of 1:1). Composition a: "Essential 6", BMP4, ATRA, EGF, FGF7 Composition b: "Essential 6" / "CnT-PR", EGF, FGF7 Composition c: "StemFIT (registered trademark)", BMP4, ATRA, EGF, FGF7 Composition d: "StemFIT (registered trademark)" / "CnT-PR", EGF, FGF7

[0046] The culture medium may be changed as needed during the induction process. Unless otherwise specified, it is preferable not to subculture during the induction process.

[0047] (Induction medium that does not contain FGF7) In the induction step, at least an FGF7-containing medium is used, but one or more induction media that do not contain FGF7 may be used in combination. In this case, in the induction step, an FGF7-containing medium is used for a certain period, and an induction medium that does not contain FGF7 is used for another period. An example of the composition of an induction medium that does not contain FGF7 is a composition obtained by removing FGF7 from the preferred composition of the FGF7-containing medium described above.

[0048] During at least a portion of the induction process, the induction medium (which may be either an FGF7-containing medium or an induction medium without FGF7) preferably further contains at least one of BMP4, ATRA, and EGF, and more preferably further contains EGF, or further contains all of BMP4, ATRA, and EGF.

[0049] The induction process may differ depending on whether on-feeder iPS cells or feeder-free iPS cells are used. First, we will describe the details of the preferred induction process when using on-feeder iPS cells.

[0050] (Induction process when using on-feeder iPS cells) When the iPS cells are on-feeder iPS cells, the induction process preferably comprises a suspension culture step in which the iPS cells are cultured in suspension to form embryoid bodies, and an adherent culture step in which the embryoid bodies are cultured in an adherent culture. In this case, it is preferable that the induction medium used in the suspension culture step does not contain FGF7, and that an FGF7-containing medium is used in the adherent culture step.

[0051] (Suspension culture process) In the suspension culture process, iPS cells are cultured in suspension to form embryoid bodies, which are cell aggregates with a three-dimensional structure. Embryoid body formation is performed, for example, by adding an iPS cell suspension to a low-adhesion multiple-well plate. In preparing the iPS cell suspension, for example, on-feeder iPS cells are first detached and removed by treating them with CTK solution (a solution containing collagenase, trypsin, and KSR). Next, the iPS cells are washed with a buffer and detached with a cell detachment solution, and then the iPS cells are suspended in a culture medium to prepare the iPS cell suspension. Examples of cell detachment solutions for iPS cells include TrypLE (Thermo Fisher Scientific "TrypLE Select Enzyme (1X), no phenol red (Gibco, 12563011)") and Accumax (Innovative Cell Technologies AM105). The duration of the suspension culture process is not particularly limited, but is preferably 2 to 10 days, more preferably 4 to 6 days, and even more preferably 5 days.

[0052] Examples of base media used for preparing cell suspensions in the suspension culture process include the aforementioned "Essential 6," "Essential 8," and other stem cell culture media. Other stem cell culture media are preferred as the base media used for preparing cell suspensions, and "Primate ESC" is more preferred.

[0053] In the suspension culture process, the culture medium used to prepare the cell suspension preferably contains a ROCK-specific inhibitor (such as Y27632) as a component that suppresses cell death. The concentration of the ROCK-specific inhibitor in the culture medium used to prepare the cell suspension is not particularly limited, but is preferably 1 to 500 nM, more preferably 2 to 100 nM, and even more preferably 4 to 30 nM. For example, the concentration of the ROCK-specific inhibitor in the culture medium used to prepare the cell suspension can be 10 nM.

[0054] The culture medium used to prepare the cell suspension in the suspension culture process preferably contains keratinocyte-inducing factors other than FGF7, and more preferably contains BMP4. The BMP4 concentration in the culture medium used to prepare the cell suspension is not particularly limited, but is preferably 1 to 500 ng / mL, more preferably 2 to 100 ng / mL, and even more preferably 4 to 30 ng / mL. For example, the BMP4 concentration in the culture medium used to prepare the cell suspension can be 10 ng / mL.

[0055] The culture medium used to prepare the cell suspension in the suspension culture process preferably contains extracellular matrix components, more preferably laminin, collagen IV and / or entactin, and even more preferably contains products from Corning's "Matrigel®" series. Examples of "Matrigel®" series products include "Matrigel® Basement Membrane Matrix Growth Factor Reduced (Corning 354230)," which contains a mixture of 61% laminin, 30% collagen IV, and 7% entactin. Other products in the "Matrigel®" series may also be used for coating culture vessels, although these are not particularly limited.

[0056] The concentration of extracellular matrix components in the culture medium used to prepare the cell suspension is not particularly limited, but is preferably 0.05 to 4% by mass, more preferably 0.1 to 2% by mass, and even more preferably 0.3 to 1% by mass. For example, the concentration of extracellular matrix components in the culture medium used to prepare the cell suspension can be 0.5% by mass.

[0057] For the culture medium used to prepare the cell suspension in the suspension culture process, a medium is preferred in which Y27632, "Matrigel®", and BMP4 are added to "Primate ESC" as the base medium.

[0058] In the suspension culture process, the seeding rate of iPS cells is 1 × 10⁶ when using a 96-well plate. 3 ~1 × 10 5A cell density of 50 μL / well is preferred, and 5 × 10 3 ~2×10 4 A cell density of 50 μL / well is more preferable. When using culture vessels other than 96-well plates, the seeding rate of iPS cells can be appropriately adjusted based on the above numerical range.

[0059] In the suspension culture process, it is preferable to add a volume of culture medium approximately equal to the volume of the cell suspension after a certain period of time following the addition of the cell suspension to the culture vessel. The number of times the culture medium is added during the suspension culture process is, for example, 1 to 3 times, with 2 times being preferable. The interval between adding the culture medium during the suspension culture process is preferably 20 to 60 hours. If the culture medium is added twice during the suspension culture process, it is preferable to do so, for example, 1 day (24 hours later) and 3 days (72 hours later) after the cell suspension is added to the culture vessel.

[0060] For the base medium used to add culture medium in the suspension culture process, it is preferable to use the same base medium as the one used to prepare the cell suspension, and "Primate ESC" is more preferable.

[0061] The culture medium used for adding to the culture medium in the suspension culture process preferably contains keratinocyte-inducing factors other than FGF7, and more preferably contains BMP4 and ATRA.

[0062] The appropriate ATRA concentration in the culture medium used for adding culture medium during the suspension culture process can be the same as the ATRA concentration in the FGF7-containing medium described above, for example, 1 μM or 2 μM. The appropriate BMP4 concentration in the culture medium used for adding culture medium during the suspension culture process can be the same as the BMP4 concentration in the FGF7-containing medium described above, for example, 10 ng / mL.

[0063] For the culture medium used to add to the culture medium in the suspension culture process, a medium prepared by adding ATRA and BMP4 to the base medium "Primate ESC" is preferred.

[0064] (Adhesion culture process) In the adherent culture step, embryoid bodies obtained in the suspension culture step are collected and seeded in a culture vessel for adherent culture. When seeding the embryoid bodies in the culture vessel, no dissociation treatment such as trypsin treatment is performed; they should be seeded directly into the culture vessel. As the culture vessel, a dish coated with an extracellular matrix component is preferred, and a dish coated with collagen (particularly a combination of collagen I and collagen IV) is more preferred. The duration of the adherent culture step is not particularly limited, but 5 to 20 days is preferred, 8 to 12 days is more preferred, and 10 days is even more preferred.

[0065] In the adhesion culture process, it is sufficient to use FGF7-containing medium for at least a portion of the period, but it is preferable to use FGF7-containing medium for the entire period. The FGF7-containing medium used in the adhesion culture process has already been explained above, so a redundant explanation will be omitted.

[0066] In the adhesion culture step, it is preferable to culture in a medium using only "Essential 6" as the base medium (for example, the medium with composition a described above), and then to culture in a medium using a mixed medium of "Essential 6" and epidermal cell growth medium (for example, "CnT-PR") as the base medium (for example, the medium with composition b described above). The period for culturing in a medium using only "Essential 6" as the base medium is preferably 1 to 7 days, more preferably 2 to 4 days, and even more preferably 3 days. Similarly, the period for culturing in a medium using a mixed medium of "Essential 6" and epidermal cell growth medium as the base medium is preferably 3 to 15 days, more preferably 5 to 9 days, and even more preferably 7 days.

[0067] In the induction step when using on-feeder iPS cells, it is preferable to culture them for 3 to 15 days in a medium containing at least BMP4 and ATRA, more preferably for 5 to 9 days, and even more preferably for 7 days. It is preferable that these periods are continuous.

[0068] (Induction process when using feeder-free iPS cells) If the iPS cells are feeder-free iPS cells, the induction process preferably comprises a first induction step of adherent culture in an induction medium that does not contain FGF7, and a second induction step of adherent culture in an FGF7-containing medium. In this case, it is preferable to perform the preparation steps described later beforehand, followed by the first induction step.

[0069] As the culture vessel used in the induction step, a dish coated with an extracellular matrix component is preferred, and a dish coated with laminin (particularly LN511) is more preferred.

[0070] (First induction process) In the first induction step, adherent culture is performed using an induction medium that does not contain FGF7. The duration of the first induction step is not particularly limited, but is preferably 10 hours to 3 days, more preferably 1 to 2 days, and even more preferably 1 day. In the first induction step, it is sufficient to use an induction medium that does not contain FGF7 for at least a portion of the period, but it is preferable to use an induction medium that does not contain FGF7 for the entire period.

[0071] As an induction medium that does not contain FGF7, a medium can be used in which a keratinocyte-inducing factor other than FGF7 (BMP4, ATRA, etc.) is added to a base medium (for example, "Essential 6" and / or other stem cell culture medium). Preferably, this medium is "StemFIT®" with BMP4 added, or a medium is a mixed medium of "StemFIT®" and "Essential 6" with BMP4 added. The mixing ratio of the mixed medium is preferably 4:6 to 6:4, and more preferably 5:5. In this induction medium, it is preferable to use only solutions A and B of the attached solutions A to C of "StemFIT®", without using solution C.

[0072] A suitable BMP4 concentration in an induction medium that does not contain FGF7 can be the same as the BMP4 concentration in the FGF7-containing medium described above, for example, 10 ng / mL.

[0073] (Second induction process) In the second induction step, adherent culture is performed in an FGF7-containing medium after the first induction step. For example, the second induction step can be started by changing the culture medium with an FGF7-containing medium after the first induction step. The duration of the second induction step is not particularly limited, but is preferably 7 to 25 days, more preferably 12 to 18 days, and even more preferably 15 days.

[0074] In the second induction step, it is sufficient to use FGF7-containing medium for at least a portion of the period, but it is preferable to use FGF7-containing medium for the entire period. The FGF7-containing medium used in the second induction step has already been explained above, so a redundant explanation will be omitted.

[0075] In the second induction step, it is preferable to culture the cells in an FGF7-containing medium using "Essential 6" or other stem cell culture medium as the base medium (for example, a medium with composition a or c as described above) (hereinafter sometimes referred to as the "initial stage of the second induction step"), and then to culture the cells in an FGF7-containing medium using a mixed medium of "Essential 6" or other stem cell culture medium and epidermal cell proliferation medium as the base medium (for example, a medium with composition b or d as described above) (hereinafter sometimes referred to as the "late stage of the second induction step"). As other stem cell culture media, "StemFIT®" (in particular, a medium using only solutions A and B mixed together, without using solution C) is preferred. As epidermal cell proliferation medium, "CnT-PR" is preferred.

[0076] The FGF7-containing medium used in the initial stage of the second induction process preferably contains BMP4, ATRA, and EGF. The FGF7-containing medium used in the later stage of the second induction process preferably does not contain BMP4 and ATRA, but contains EGF. The initial stage of the second induction process is preferably 3 to 15 days, more preferably 5 to 9 days, and even more preferably 7 days. Similarly, the initial stage of the second induction process is preferably 4 to 16 days, more preferably 6 to 10 days, and even more preferably 8 days.

[0077] <Preparation process> In the preparation step, iPS cells are seeded and adhered to a culture vessel before the induction step. When feeder-free iPS cells are used as the iPS cells in the keratinocyte production method of the present invention, it is preferable to further include this step. As the culture vessel, a dish coated with an extracellular matrix component is preferred, and a dish coated with laminin (particularly LN511) is more preferred.

[0078] Known methods can be used to seed iPS cells into a culture vessel. For example, one method involves suspending iPS cells that have been dissociated using a cell detachment solution (e.g., TrypLE, Accumax, etc.) in a culture medium and adding the resulting cell suspension to the culture vessel.

[0079] Examples of base media used for preparing the cell suspension in the preparation step include the aforementioned "Essential 6", "Essential 8", and other stem cell culture media. Preferably, the base media used for preparing the cell suspension is "Essential 8", "StemFIT®", or a mixture thereof, and more preferably, "StemFIT®", or a mixture of "Essential 8" and "StemFIT®" (with a mixing ratio of, for example, 4:6 to 6:4). When used in the preparation step, it is preferable to mix all of the included solutions A to C with "StemFIT®".

[0080] The culture medium used to prepare the cell suspension in the preparation step preferably contains a ROCK-specific inhibitor (such as Y27632). The concentration of the ROCK-specific inhibitor in the culture medium used to prepare the cell suspension in the preparation step can be the same as the concentration exemplified in the culture medium used to prepare the cell suspension in the suspension culture step, for example, it can be 10 nM.

[0081] There is no particular limit to the amount of cells to be seeded, but when using a 6cm dish as a culture vessel, 0.1 to 100 × 10⁶ cells is a good guideline. 3 Cells / dishes are preferred, 0.5-20 × 10 3Cells / dishes are more preferred, 1-8 × 10 3 Cells / dishes are even more preferable. If a culture vessel other than a 6cm dish is used, the amount of seeded cells can be appropriately adjusted according to the size of the culture vessel.

[0082] The preparation period (from seeding to induction) is not particularly limited, but is preferably 1 to 10 days, more preferably 1 to 3 days, and even more preferably 2 days. In the preparation period, it is preferable to change the culture medium with a medium that does not contain a ROCK-specific inhibitor on the day after seeding. It is preferable that this medium be the same medium used for preparing the cell suspension.

[0083] <Maturity process> In the maturation process, keratinocytes are matured after the induction process. In the maturation process, a culture medium that does not contain FGF7 (hereinafter sometimes referred to as the "maturation process medium") is used. Preferably, the maturation process medium does not contain keratinocyte-inducing factors other than FGF7. The duration of the maturation process is not particularly limited, but is preferably 7 to 25 days, more preferably 12 to 18 days, and even more preferably 15 days.

[0084] The present invention's method for producing keratinocytes, by incorporating a maturation step, allows for the production of keratinocytes more suitable for transplantation and testing. Furthermore, the inventors' research has yielded new findings that FGF7 is effective in inducing keratinocytes from iPS cells (early induction), but is not necessary for keratinocyte maturation (late induction). Therefore, the present invention's method for producing keratinocytes, by using FGF7 in the early induction stage and not using FGF7 in the late induction stage, can effectively induce keratinocytes from iPS cells.

[0085] In the maturation process, it is preferable to use only one type of maturation medium. The maturation medium is preferably an epithelial cell proliferation medium (such as "CnT-PR") to which EGF and a ROCK-specific inhibitor (such as Y27632) have been added. As for EGF, human EGF or recombinant human EGF (rhEGF) is preferred.

[0086] The EGF concentration in the culture medium for the maturation process can be the same as the EGF concentration in the FGF7-containing medium described above, for example, it can be 10 ng / mL.

[0087] The concentration of the ROCK-specific inhibitor in the culture medium for the maturation process is not particularly limited, but is preferably 1 to 500 nM, more preferably 2 to 100 nM, and even more preferably 4 to 30 nM. For example, the concentration of the ROCK-specific inhibitor in the culture medium for the maturation process can be 10 nM.

[0088] As the culture vessel used in the maturation process, a dish coated with an extracellular matrix component is preferred, and a dish coated with collagen (particularly collagen IV) is more preferred.

[0089] It is preferable to perform subculturing between the induction step and the maturation step. Subculturing can be carried out by known methods. The maturation step can be started by subculturing the keratinocytes cultured in the induction step and using the maturation step medium as the subculturing medium. During the maturation step, subculturing may be performed at predetermined intervals (e.g., 5 to 10 days) to prevent the keratinocytes from reaching confluence.

[0090] <Other processes> The method for producing keratinocytes of the present invention may further include other steps. For example, the method for producing keratinocytes of the present invention may further include steps for post-treatment such as purification, washing, and processing of the obtained keratinocytes.

[0091] <Application> The keratinocytes obtained by this invention can be widely used not only for research purposes but also for medical and / or cosmetic applications. In particular, by creating iPS cells from tissue derived from a subject (human or animal) and then manufacturing keratinocytes using these iPS cells as material, subject-derived keratinocytes can be obtained. Subject-derived keratinocytes are expected to be used, for example, as a material when developing artificial skin that does not cause rejection reactions. Furthermore, by transplanting subject-derived keratinocytes into subjects with wounds, burns, keloids, scars, etc., skin regeneration at the transplant site can be promoted, and effects such as improved healing speed and improved aesthetics after healing can be expected. In addition, by testing the reactivity to chemical substances using subject-derived keratinocytes, the reactivity of the subject's skin can be safely and efficiently tested, which can be used to develop customized skin pharmaceuticals and cosmetic compositions optimized for the subject's skin.

[0092] <Specific Scheme> The specific schemes of the present invention are shown in Figures 1 and 2 below. Figure 1 is a scheme when using on-feeder iPS cells. Figure 2 is a scheme (two types) when using feeder-free iPS cells. In Figures 1 and 2, "D" represents the number of days, with Day 0 being the day the main process started. "Passage" indicates that subculturing is performed. Descriptions such as "[D0 Medium]" indicate the composition of the culture medium used. For example, "[D0 Medium]" indicates that on Day 0, a culture medium was used in which 10 nM Y27632, 0.5% "Matrigel® Basement Membrane Matrix Growth Factor Reduced (Corning 354230) (manufactured by Corning)" and 10 nM BMP4 were added to "Primate ESC" as the base medium.

[0093] In the scheme shown in Figure 1, the induction process is performed from day 0 to day 15, and the maturation process is performed from day 15 to day 30. Specifically, within the induction process, the suspension culture process is performed from day 0 to day 5, and the adherent culture process is performed from day 5 to day 15.

[0094] The scheme shown in Figure 2 has two versions, A and B, which use different culture media. In both versions, the preparation process is performed from day -3 to day -1, the induction process is performed from day -1 to day 15, and the maturation process is performed from day 15 to day 30. In the induction process, the first induction process is performed from day -1 to day 0, and the second induction process is performed from day 0 to day 15. Note that "StemFIT" in Figure 2 indicates that "StemFIT®" manufactured by Ajinomoto Healthy Supply Co., Ltd. is used according to the normal usage method (mixing solutions A, B, and C). "StemFIT B only" in Figure 2 indicates that "StemFIT®" manufactured by Ajinomoto Healthy Supply Co., Ltd. is used by mixing only solutions A and B. [Examples]

[0095] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples. Unless otherwise specified, the units of the numerical values ​​in each table are units of percent. Each culture medium used in the examples was used with 50 units / 50 mg / mL of penicillin / streptomycin (Thermo Fisher Scientific, Gibco brand) added.

[0096] <Manufacturing of keratinocytes using on-feeder iPS cells as material> Tests 1-14 were conducted to produce keratinocytes from on-feeder iPS cells according to the scheme shown in Figure 1. However, in Tests 1-14, the "D5 Medium" and "D8 Medium" in the scheme shown in Figure 1 were modified as appropriate, as shown in Table 1 below.

[0097] [Table 1]

[0098] The terms used in Table 1 are explained below. "Primate" refers to "Primate ES Cell Medium (product number: RCHEMD001)" manufactured by Reprocell Corporation.

[0099] "Primate+Supp" is a medium obtained by adding the supplement of composition (X) shown below to the aforementioned "Primate". · Adenine 24 μg / mL ("014-11511" manufactured by FUJIFILM Wako Pure Chemical Corporation) · Ascorbic acid 0.3 mM ("012-04802" manufactured by FUJIFILM Wako Pure Chemical Corporation) · Hydrocortisone 0.5 μg / mL (""082-10191" manufactured by FUJIFILM Wako Pure Chemical Corporation) · Insulin 5 μg / mL ("090-06481" manufactured by FUJIFILM Wako Pure Chemical Corporation) · Triiodothyronine 1.37 ng / mL ("T5516-1MG" manufactured by Sigma-Aldrich)

[0100] "nFAD S" is a medium obtained by adding the supplement of the aforementioned composition (X) and 20% FBS (fetal bovine serum) ("35-010-CV" manufactured by Corning) to a mixed medium of Ham's F-12 medium ("087-08335" manufactured by FUJIFILM Wako Pure Chemical Corporation) and DMEM medium ("08459-64" manufactured by Nacalai Tesque) (DMEM:Ham's F-12 = 3:1).

[0101] "fFAD S" is Ca 2+ A medium obtained by adding the supplement of the aforementioned composition (X) and 20% FBS (fetal bovine serum) ("35-010-CV" manufactured by Corning) to a mixed medium of free Ham's F-12 medium (a medium obtained by decalcifying "087-08335" manufactured by FUJIFILM Wako Pure Chemical Corporation by Cosmo Bio Co., Ltd.) and Ca 2+ Free DMEM medium ("16972" manufactured by Nacalai Tesque) (DMEM:Ham's F-12 = 3:1).

[0102] "nFAD KSR" is a serum-free medium obtained by substituting 20% FBS with 20% serum-free supplement ("KnockOut Serum Replacement" manufactured by Thermo Fisher Scientific) based on the composition of the aforementioned "fFAD S".

[0103] "E6" refers to the "Essential 6" manufactured by Thermo Fisher Scientific.

[0104] "O E6" is "Essential 6" prepared by the inventor himself based on the composition of the disclosed "Essential 6" ("O" stands for Original).

[0105] In Table 1, the " / " in the base medium indicates a medium in which two types of media are mixed in a 1:1 ratio. For example, "Primate / CnT-PR" in "D8 medium" in Test Example 1 means a base medium in which the aforementioned "Primate" and "CnT-PR" are mixed in a 1:1 ratio.

[0106] In Table 1, if the "FGF7" item is "Present," it indicates that the D5 and D8 media in Test Examples 8-14 had FGF7 (Proteintech) added to the base medium at a concentration of 10 ng / mL. In Test Examples 1-7, if the "FGF7" item is "Absent," it indicates that FGF7 was not used in the D5 and D8 media. In Table 1, under "Other Components," "BMP4" indicates that BMP4 (R&D Systems) was added to the base medium at a concentration of 10 ng / mL. "ATRA" indicates that ATRA (Fujifilm Wako Pure Chemical Industries) was added to the base medium at a concentration of 1 μM. "EGF" indicates that EGF (R&D Systems) was added to the base medium at a concentration of 10 ng / mL.

[0107] To explain in more detail, for example, in Test Example 1, "D5 Medium" in the scheme of Figure 1 was "D5 Medium" and "D8 Medium" in Table 1 was "D8 Medium". The "D5 Medium" used in Test Example 1 is a medium with "Primate" as the base medium, to which BMP4 10 ng / mL, ATRA 1 μM, and EGF 10 ng / mL were added. The "D8 Medium" used in Test Example 1 is a medium with a 1:1 mixed medium of "Primate" and "CnT-PR" as the base medium, to which EGF 10 ng / mL was added. The same applies to Test Examples 2 to 14.

[0108] (Preparation of on-feeder iPS cells) 585B1 cell line (human iPS cells) was cultured in primate ES / iPS cell medium (ReproCELL "Primate ES Cell Medium") supplemented with 4 ng / mL bFGF (Fujifilm Wako Pure Chemical Industries, Ltd.) on an SNL feeder cell layer treated with mitomycin C (MMC) (Fujifilm Wako Pure Chemical Industries, Ltd.).

[0109] (Suspension culture process) The on-feeder iPS cells described above were dissociated using the following method. First, the iPS cells and feeder cells in the culture vessel were treated with CTK solution (composition below) for 2-3 minutes to detach the feeder cells from the culture vessel. Then, the feeder cells were removed from the culture vessel by washing it 2-3 times with PBS. Subsequently, the iPS cells in the culture vessel were treated with a cell detachment enzyme for 5 minutes to detach and dissociate the iPS cells. TrypLE (Thermo Fisher Scientific "TrypLE Select Enzyme (1X), no phenol red (Gibco, 12563011)") was used as the cell detachment enzyme.

[0110] (CTK solution) 0.1 mg / mL Collagenase IV (Gibco 17104019, manufactured by Thermo Fisher Scientific) 0.25% trypsin (Gibco 15090046, manufactured by Thermo Fisher Scientific) 20% KSR (Gibco 10828028, manufactured by Thermo Fisher Scientific) 1mM CaCl2 (Nacalai tesque, 06729-55)

[0111] The obtained single-cell human iPS cells were suspended in culture medium. The culture medium used was the aforementioned "Primate ES Cell Medium," which contained 10 μM Y27632 (Nacalai Tesque), 0.5% "Matrigel® Basement Membrane Matrix Growth Factor Reduced (Corning 354230) (Corning Corporation)," and 10 ng / mL BMP4 (R&D Systems). The human iPS cell suspension was then placed in a low-adhesion 96-well plate in a 1 × 10⁶ layer. 4 Embryoid bodies were formed by seeding cells at 50 μL / well and culturing them (Day 0). 50 μL of medium (A), containing 10 ng / mL of BMP4 (R&D Systems) and 2 μM of retinoic acid (all-trans retinoic acid: ATRA) (Fujifilm Wako Pure Chemical Industries), was added to the wells 24 hours after seeding (Day 1). 50 μL of medium (B), which had the same composition as medium (A) except that the retinoic acid concentration was changed to 1 μM, was added to the wells 72 hours after seeding (Day 3).

[0112] (Adhesion culture process) On day 5, embryoid bodies were collected from each well. Embryoid bodies were cultured on dishes coated with Collagen IV (Nitta Gelatin Co., Ltd.) using one of the "D5 mediums" from Table 1, which contained at least 10 ng / mL BMP4, 1 μM ATRA, and 10 ng / mL EGF. On days 8, 11, and 13, the culture medium was changed to one of the "D8 mediums" from Table 1, which did not contain BMP4 or ATRA.

[0113] (maturation process) On day 15, the cells after the adherent culture process were detached using the aforementioned cell-detaching enzyme, and the detached cells were passed through a 100 μm cell strainer (Corning) and collected in wash buffer. The wash buffer consisted of 20% Gibco KSR (Thermo Fisher Scientific), 1% sodium pyruvate, and 1% Gibco GlutaMAX (Thermo Fisher Scientific) added to Ca 2+ Free DMEM (Nacalai Tesque) was used. The harvested cells were resuspended and cultured on dishes coated with Collagen IV (Nitta Gelatin Co., Ltd.) in "CnT-PR" containing 10 ng / mL EGF and 10 μg Y27632 until each measurement was performed. During culture, the cells were subcultured before they reached confluence (performed on day 22).

[0114] On day 30, matured cells (keratinocytes) were collected, and the expression of keratinocyte markers was analyzed by flow cytometry and immunocytochemistry.

[0115] The keratinocyte markers whose expression was examined in this example are as follows: • ΔNp63: Keratinocyte master gene (transcription factor) • KRT14 (Keratin 14): A keratinocyte marker in cells • KRT18 (Keratin 18): A keratinocyte marker in cells • CD49f (Integrin α6 (ITGA6)): Epithelial cell marker of the cell membrane • CD104 (Integrin β4 (ITGB4)): Cell membrane epithelial cell marker

[0116] <Flow cytometry (ΔNp63)> The cells (keratinocytes) obtained in Test Examples 1-14 were pre-stained using Biolegend's "Zombie Red dye" at room temperature. Next, the pre-stained cells were fixed at room temperature for 30 minutes using Invitrogen's "IC Fixation Buffer". Then, the fixed cells were permeabilized at 4°C for 30 minutes using methanol (Nacalai Tesque) at -20°C. Next, the permeabilized cells were washed twice with Invitrogen's "Permeabilization Buffer". Next, the cells were stained at room temperature for 30 minutes using "Permeabilization Buffer" to which the primary antibody described later was added. Next, the cells were washed twice with "Permeabilization Buffer" and then stained at room temperature for 30 minutes using "Permeabilization Buffer" to which the secondary antibody described later was added.

[0117] (Primary antibody and isotype control) • Anti-ΔNp63 (Abcam, "#ab203826", rabbit IgG antibody) (dilution ratio 400x) • Rabbit IgG monoclonal Isotype Control (Abcam, "#ab172730") (Dilution ratio 1600x) (Secondary antibody) • Goat anti-rabbit IgG Alexa Fluor 488 (Jackson ImmunoResearch, "#111-546-144") (Dilution ratio 1500x)

[0118] Cell analysis was performed using the multicolor cell sorter "FACS Aria II (BD Biosciences)," and data analysis was performed using the analysis software "FlowJo (BD Biosciences)."

[0119] The results are shown in Figures 3-1 to 3-4. In Figures 3-1 to 3-4, the white histograms represent cell populations using the ΔNp63 antibody as the primary antibody, and the gray histograms represent cell populations using the isotype control as the primary antibody. In Figures 3-1 to 3-4, the vertical axis represents cell number (Modal), and the horizontal axis represents fluorescence intensity (ΔNp63). In Figures 3-1 to 3-4, the parenthetical notes below each test example indicate the type of base medium used for D5 culture and the presence or absence of FGF7. For example, "Test Example 1 (Primate)" indicates that in Test Example 1, "Primate" was used as the base medium for D5 culture, and FGF7 was not added (the same applies to Figures 6-1 to 6-4 and Figures 8-1 to 8-4). In Figures 3-1 to 3-4, cells showing a stronger fluorescence intensity than the cell population using the isotype control were considered ΔNp63-positive cells. The proportion of ΔNp63-positive cells in the cell population is shown in Table 2 below.

[0120] [Table 2]

[0121] Except for changing the timing of keratinocyte harvesting to 15 days after induction (D15, timing of Passage #2 in Figure 1), the same keratinocyte production and flow cytometry procedures as described above were performed 6 times (n=6) for test examples 1-7 (without FGF7) and 5 times (n=5) for test examples 8-14 (with FGF7). The percentage of ΔNp63-positive cells was measured in each test, and the mean and standard error were calculated. The results are shown in Table 3 and Figure 4 below.

[0122] [Table 3]

[0123] As is clear from the comparison between Test Examples 1-7 and Test Examples 8-14, regardless of the type of base medium used in the induction medium, the proportion of ΔNp63-positive cells increased when FGF7 was used in the induction process. Furthermore, as shown in Test Examples 13 and 14, the proportion of ΔNp63-positive cells was particularly high when "Essential 6" was used as the base medium for the induction medium.

[0124] <Immunocytological staining (keratin 18 and ΔNp63)> The expression of keratin 18 and ΔNp63, keratinocyte markers, was confirmed by immunohistochemistry in the cells (keratinocytes) obtained in Test Example 13 using the following method. 4% PFA solution (Nacalai Tesque) was added to the culture vessel in which the keratinocytes were cultured, and the cells were fixed for 15 minutes. Next, the cells were permeabilized with 0.5% Triton-X solution (Nacalai Tesque) for 15 minutes. Then, blocking was performed using a blocking buffer consisting of PBS (Nacalai Tesque) supplemented with 1% BSA (Nacalai Tesque), 0.1% Tween 20 (Nacalai Tesque), and 0.1M glycine (Nacalai Tesque). After blocking, the cells were washed twice with PBS. Next, the cells were treated overnight at 4°C with a primary antibody solution prepared by adding the primary antibody described below to a staining buffer (the blocking buffer from which glycine was removed).

[0125] The following day, the cells were washed twice with PBS, and then stained at room temperature for 3 hours using a secondary antibody solution, which was the aforementioned staining buffer with the secondary antibody described below.

[0126] (Primary antibody) • Anti-ΔNp63 (CST Corporation "#67825", rabbit IgG antibody) (dilution ratio 1000x) • Anti-keratin 18 (CST Corporation "#4548", mouse IgG1 antibody) (dilution ratio 1000x) (Secondary antibody) • Goat anti-mouse IgG Alexa Fluor Plus 488 (Invitrogen, "#A32723") (Dilution ratio 1000x) • Goat anti-rabbit IgG Alexa Fluor Plus 647 (Invitrogen, "#A32733") (Dilution ratio 1000x)

[0127] For nuclear staining, Hoechst 33342 (Invitrogen, "#H3570") (dilution ratio 10,000x) was added to the secondary antibody solution. Observation and image processing were performed using a fluorescence microscope "BZ-X710" (Keyence Corporation). The results are shown in Figure 5.

[0128] The four images in Figure 5 (4x magnification) show the same region of the keratinocyte culture vessel obtained in Experiment Example 13. The upper left image shows fluorescence derived from the nucleus (Hoechst 33342), the upper center image shows fluorescence derived from keratin 18, and the upper right image shows fluorescence derived from ΔNp63. The lower left image shows the cells without fluorescence excitation. The cell clumps located slightly below the center of each image are embryoid body residues. As shown in Figure 5, it was confirmed that almost all cells stained with Hoechst 33342 express keratin 18 and ΔNp63.

[0129] <Flow cytometry (CD49f and CD104)> Flow cytometry was performed on the cells (keratinocytes) obtained in Test Examples 1-14 using CD49f and CD104. To stain the cell surface molecules, the cells were detached and dissociated using the aforementioned "TrypLE Select" and washed twice with FACS buffer (2% FBS PBS). The primary antibody and isotype control described below were added to the FACS buffer and stained at 4°C for 30 minutes.

[0130] (Primary antibody and isotype control) • Anti-CD49f PE-Cy7 (Biolegend, "#313622", rat IgG2a antibody) (dilution ratio 500x) • Anti-CD104 PE (Biolegend "#327808", mouse IgG2a antibody) (dilution ratio 20x) • Rat IgG2a Isotype PE-Cy7 (Biolegend "#400522") (Dilution ratio 500x) • Mouse IgG2a Isotype PE (Biolegend "#400214") (Dilution ratio 20x)

[0131] After washing twice with FACS buffer, the cells were resuspended in FACS buffer containing 7-AAD (Biolegend "#420404") (100-fold dilution) (nuclear stain for dead cells) and stained at room temperature for 10 minutes.

[0132] The results are shown in Figures 6-1 to 6-4. In Figures 6-1 to 6-4, the vertical axis represents the expression of CD104 (fluorescence intensity of PE), and the horizontal axis represents the expression of CD49f (fluorescence intensity of PE-Cy7). The percentage of cells expressing both CD104 and CD49f in the cell population is shown in Table 4 below.

[0133] [Table 4]

[0134] The same keratinocyte preparation and flow cytometry procedures described above were performed four times (n=4) (recovery timing: D30). In each test, the percentage of cells expressing both CD104 and CD49f was measured, and the mean and standard error were calculated. The results are shown in Table 5 and Figure 7 below.

[0135] [Table 5]

[0136] Examples 1 and 8, 2 and 9, 3 and 10, 4 and 11, 5 and 12, 6 and 13, and 7 and 14 are pairs of experiments conducted under identical conditions except for the presence or absence of FGF7 in the induction process (initial induction stage). As is clear from the comparison of each pair, regardless of the type of base medium used in the induction process, the use of FGF7 in the induction process increased the proportion of CD104 and CD49f-positive cells. In other words, by using FGF7 in the initial induction stage, keratinocytes could be efficiently induced regardless of the type of base medium. This effect was also confirmed when serum-free medium was used as the induction medium.

[0137] <Flow cytometry (keratin 14)> Flow cytometry was performed on the cells (keratinocytes) obtained in Test Examples 1-14 using the same method as for ΔNp63 flow cytometry, except that the primary and secondary antibodies were changed as follows.

[0138] (Primary antibody and isotype control) • Anti-keratin 14 (Biolegend "#905304", rabbit IgG antibody) (dilution ratio 200x) • Purified Rabbit Polyclonal Isotype Ctrl Antibody (Biolegend "#910801") (Dilution ratio 400x) (Secondary antibody) • Goat anti-rabbit IgG Alexa Fluor 488 (Jackson ImmunoResearch, "#111-546-144") (Dilution ratio 1500x)

[0139] The results are shown in Figures 8-1 to 8-4. In Figures 8-1 to 8-4, the white histograms represent cell populations using keratin 14 antibody as the primary antibody, and the gray histograms represent cell populations using isotype control as the primary antibody. In Figures 8-1 to 8-4, the vertical axis represents cell number (Modal), and the horizontal axis represents fluorescence intensity (keratin 14). In Figures 8-1 to 8-4, cells showing stronger fluorescence intensity than the cell population using isotype control were considered keratin 14-positive cells. The proportion of keratin 14-positive cells in the cell population is shown in Table 6 below.

[0140] [Table 6]

[0141] The same keratinocyte preparation and flow cytometry procedures described above were performed five times (n=5) (recovery timing: D30). In each test, the percentage of keratin 14-positive cells was measured, and the mean and standard error were calculated. The results are shown in Table 7 and Figure 9 below. In addition, the Mean Fluorescence Intensity (MFI) of keratin 14 was also measured in each test, and the mean and standard error were calculated. The results are shown in Table 8 and Figure 10 below.

[0142] [Table 7]

[0143] [Table 8]

[0144] Examples 1 and 8, 2 and 9, 3 and 10, 4 and 11, 5 and 12, 6 and 13, and 7 and 14 are pairs of experiments conducted under identical conditions except for the presence or absence of FGF7 in the induction process (initial induction stage). As is clear from the comparison of each pair, regardless of the type of base medium used in the induction process, the use of FGF7 in the induction process increased the proportion of keratin 14-positive cells and MFI. In other words, by using FGF7 in the initial induction stage, keratinocytes could be efficiently induced regardless of the type of base medium. This effect was also confirmed when serum-free medium was used as the induction medium. Furthermore, as shown in Examples 13 and 14, the proportion of keratin 14-positive cells and MFI were particularly high when "Essential 6 (both commercially available and in-house prepared)" was used as the base medium for the induction medium.

[0145] <Immunocytochemistry (Keratin 14)> For the keratinocytes obtained in Test Example 13, the expression of keratin 14, a keratinocyte marker, was confirmed by immunohistochemistry using the same method as described above for immunohistochemistry (keratin 18 and ΔNp63), except that the following primary and secondary antibodies were used. For comparison, images obtained by staining primary cultured keratinocytes (HPEK: human primary epidermal keratinocytes) using the same method are shown. The HPEK cells were purchased from CELLnTEC and cultured in "CnT-PR".

[0146] (Primary antibody) • Anti-keratin 14 (Biolegend "#905304", rabbit IgG antibody) (dilution ratio 2000x) (Secondary antibody) • Goat anti-rabbit IgG Alexa Fluor Plus 647 (Invitrogen, "#A32733") (Dilution ratio 1000x)

[0147] The results are shown in Figure 11 (magnification 4x). As shown in Figure 11, it was confirmed that almost all keratinocytes obtained in test example 13 expressed keratin 14 across the entire field of view.

[0148] <Manufacturing of keratinocytes using feeder-free iPS cells as material> Test examples 15-18 were conducted to produce keratinocytes from feeder-free iPS cells, following the scheme shown in "Version B" of Figure 2. However, in test examples 15-18, the "D0-7 Medium (D0-7 medium)" and "D7-15 Medium (D7-15 medium)" in the scheme shown in Figure 2 were modified as appropriate, as shown in Table 9 below.

[0149] [Table 9]

[0150] The terms used in Table 9 are explained below. "StemFIT" refers to "StemFIT(registered trademark) AK02N" manufactured by Ajinomoto Healthy Supply Co., Ltd. "CnT-PR", " / ", "E6", "BMP4", "ATRA", "EGF", and "FGF7" are equivalent to the terms used in Table 1 mentioned above.

[0151] To explain in more detail, for example, in Test Example 15, "D0-7 Medium" from Table 9 was used as "D0-7 Medium" in the scheme of Figure 2, and "D7-15 Medium" from Table 9 was used as "D7-15 Medium". "D0-7 Medium 15" in Test Example 15 is a medium prepared by using "StemFIT (registered trademark)" as the base medium and adding BMP4 10 ng / mL, ATRA 1 μM, and EGF 10 ng / mL. "D7-15 Medium" in Test Example 15 is a medium prepared by using a 1:1 mixed medium of "Primate" and "CnT-PR" as the base medium and adding EGF 10 ng / mL. The same applies to Test Examples 16-18.

[0152] (Preparation of feeder-free iPS cells) Feeder-free 585B1 cell line (human iPS cells) was cultured in human ES / iPS cell medium (Ajinomoto Healthy Supply Co., Ltd. "StemFIT® AK02N") on laminin-coated petri dishes (Matrixome Inc. "iMatrix-511 silk", coating concentration: 0.5 μg / mL).

[0153] (preparation process) The aforementioned iPS cells were divided into 0.5-1 × 10⁻⁶ cells. 4 Cells were seeded at a density of one cell per 6 cm dish on 6 cm dishes (Corning) coated with laminin (iMatrix-511 silk, Matrixosome). The culture medium used was a 1:1 mixture of human ES / iPS cell medium (StemFIT® AK02N, Ajinomoto Healthy Supply Co., Ltd.) and Essential 8, supplemented with Y27632. 24 hours after seeding, the medium was changed to a 1:1 mixture of StemFIT® AK02N and Essential 8 without Y27632, and the cells were cultured for another 24 hours (total 48 hours).

[0154] (Induction process) The culture medium was changed to a 1:1 mixed medium of "StemFIT(registered trademark) AK02N" (mixing only solutions A and B, without solution C) and "Essential 6" supplemented with 10 ng / mL of BMP4, and pretreated for 24 hours (first induction step). Next, the cells were cultured for 7 days using one of the "D0-7 mediums" in Table 9, which contained at least 10 ng / mL of BMP4, 1 μM ATRA, and 10 ng / mL of EGF (first induction step). The start of the first induction step was considered day 0, and the medium was changed on days 3 and 5. On day 7, the cells were cultured until day 15 using one of the "D7-15 mediums" in Table 9, which contained at least 10 ng / mL of EGF and "CnT-PR" (the medium was changed on days 10 and 13).

[0155] (maturation process) A 15-day maturation process was carried out using the same method as described in the aforementioned <Production of Keratinocytes Using On-Feeder iPS Cells> (passaging was performed on the 22nd day).

[0156] On day 30, cells (keratinocytes) were collected, and the expression of keratinocyte markers was analyzed by flow cytometry.

[0157] <Flow cytometry (ΔNp63)> Flow cytometry using ΔNp63 antibody was performed on keratinocytes derived from feeder-free iPS cells obtained in Test Examples 15-18, using the same method as performed on keratinocytes derived from on-feeder iPS cells obtained in Test Examples 1-14. This series of keratinocyte production and flow cytometry was performed four times (n=4) (recovery timing: D15), and the mean and standard error of the proportion of ΔNp63-positive cells were calculated. The results are shown in Figure 12 and Table 10 below. In Figure 12, the parenthetical notes below each test example indicate the type of base medium for "D0-7 medium" and "D7-15 medium," and the presence or absence of FGF7. For example, "Test Example 15 (FIT FIT / PR)" indicates that in Test Example 15, "StemFIT (registered trademark) (a medium mixed with only solutions A and B)" was used as the base medium for "D0-7 medium," and a 1:1 mixed medium of "StemFIT (registered trademark) (a medium mixed with solutions A to C)" and "CnT-PR" was used as the base medium for "D7-15 medium," with no FGF7 added to either medium (the same applies to Figure 13).

[0158] [Table 10]

[0159] As is clear from the comparison between Test Example 15 and Test Example 16, or between Test Example 17 and Test Example 18, the proportion of ΔNp63-positive cells increased when FGF7 was used in the induction process, regardless of the type of base medium. In other words, keratinocytes could be efficiently induced by using FGF7 in the early stages of induction. This effect was also confirmed when serum-free medium was used as the induction medium. Furthermore, as shown in Test Example 18, the proportion of ΔNp63-positive cells was particularly high when "Essential 6" was used as the base medium for the induction medium.

[0160] <Manufacturing of keratinocytes using feeder-free iPS cells as material> Tests 19-22 were conducted in the same manner as in Tests 15-18, except that the culture medium used was changed as follows, to produce keratinocytes from feeder-free iPS cells.

[0161] [Table 11]

[0162] The terms in Table 11 are explained below. "StemFIT B" refers to a culture medium prepared by mixing only solutions A and B from the solutions A to C included with "StemFIT(registered trademark) AK02N" manufactured by Ajinomoto Healthy Supply Co., Ltd., without using solution C. "E6" refers to "Essential 6" manufactured by Thermo Fisher Scientific. Other terms are the same as in Table 9.

[0163] <Flow cytometry (keratin 14)> Keratin 14 flow cytometry was performed on keratinocytes obtained in Test Examples 19-22 using the same method as that performed on keratinocytes obtained in Test Examples 1-14.

[0164] The results are shown in Figure 13. In Figure 13, the white histograms represent cell populations using keratin 14 antibody as the primary antibody, and the gray histograms represent cell populations using isotype control as the primary antibody. The horizontal axis of the histograms represents fluorescence intensity (keratin 14). In Figure 13, cells showing stronger fluorescence intensity than the isotype control cell population were considered keratin 14-positive cells. The proportion of keratin 14-positive cells in the cell population is shown in Table 12 below.

[0165] [Table 12]

[0166] The same keratinocyte preparation and flow cytometry procedures described above were performed four times (n=4) (recovery timing: D30). In each test, the percentage of keratin 14-positive cells was measured, and the mean and standard error were calculated. The results are shown in Table 13 below. In addition, the MFI of keratin 14 was measured in each test, and the mean and standard error were calculated. The results are shown in Table 14 and Figure 14 below.

[0167] [Table 13]

[0168] [Table 14]

[0169] As is evident from the comparison between Test Example 19 and Test Example 20, or between Test Example 21 and Test Example 22, regardless of the type of base medium, the addition of FGF7 to the induction medium increased the MFI of keratin 14. In other words, using FGF7 in the early stages of induction allowed for efficient induction of keratinocytes. This effect was also confirmed when serum-free medium was used as the induction medium.

[0170] As the above test examples demonstrate, it has been confirmed that by using FGF7 in the early stages of induction when producing keratinocytes from iPS cells, it is possible to produce keratinocytes that highly express keratinocyte markers even in a feeder-free and serum-free environment.

Claims

1. The system includes an induction step in which iPS cells are induced into keratinocytes using one or more induction media in a feeder-free and serum-free environment. A method for producing iPS cell-derived keratinocytes, wherein a medium containing fibroblast growth factor 7 (FGF7) is used as the induction medium for at least a portion of the induction step.

2. The method for producing iPS cell-derived keratinocytes according to claim 1, wherein the FGF7-containing medium contains Essential 6 medium.

3. The method for producing iPS cell-derived keratinocytes according to claim 1 or 2, wherein the FGF7-containing medium contains an epithelial cell proliferation medium.

4. A method for producing iPS cell-derived keratinocytes according to claim 1 or 2, wherein the concentration of FGF7 in the FGF7-containing medium is 1 ng / mL or more and 50 ng / mL or less.

5. A method for producing iPS cell-derived keratinocytes according to claim 1 or 2, wherein for at least a portion of the induction step, the induction medium contains at least one selected from the group consisting of bone morphogenetic factor 4 (BMP4), retinoic acid, and epidermal growth factor (EGF).

6. A method for producing iPS cell-derived keratinocytes according to claim 1 or 2, wherein the period for which the FGF7-containing medium is used in the induction step is 5 days or more and 20 days or less.

7. The process further comprises a maturation step for maturing the keratinocytes after the induction step, A method for producing iPS cell-derived keratinocytes according to claim 1 or 2, wherein a culture medium that does not contain FGF7 is used in the maturation step.

8. The iPS cells mentioned above are on-feeder iPS cells, The induction process is as follows: A suspension culture step in which the iPS cells are cultured in suspension to form embryoid bodies, The system comprises an adhesion culture step for adhering and culturing the aforementioned embryoid bodies, The induction medium used in the suspension culture step does not contain FGF7. A method for producing iPS cell-derived keratinocytes according to claim 1 or 2, wherein the FGF7-containing medium is used in the adhesion culture step.

9. The iPS cells mentioned above are feeder-free iPS cells, The induction process is as follows: A first induction step involves adhesion culture in the induction medium that does not contain FGF7, A method for producing iPS cell-derived keratinocytes according to claim 1 or 2, comprising a second induction step of adhering culture in the FGF7-containing medium.

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