Method for producing artificial pluripotent stem cells using epithelial cells

Abstract

The first problem of the present invention is to provide a novel technique with which it is possible to produce iPS cells using epithelial cells such as keratinocytes. The present invention for solving the problem is a method for producing an artificial pluripotent stem cell using an epithelial cell, the method comprising: an initialization step for introducing a reprogramming gene or a translation product thereof, which has been subjected to nucleus initialization, into the epithelial cell; and an adhesion culture step in which the cells that have undergone the initialization step are subjected to adhesion culture on a culture surface coated with laminin or a fragment thereof.

Description

Technical Field

[0001] The present invention relates to a method for producing artificial pluripotent stem cells by utilizing epithelial cells. Background Art

[0002] Induced pluripotent stem cells (iPS cells) and other pluripotent stem cells have been widely studied as drugs or pharmaceutical raw materials for regenerative medicine. In 2006, iPS cells were first established using mouse somatic cells (Non-Patent Document 1), and the establishment of human iPS was also reported the following year (Non-Patent Document 2).

[0003] Currently, a method has been developed to establish iPS cells using blood cells such as monocytes from peripheral blood or umbilical cord blood, and somatic cells such as fibroblasts. Institutions such as Kyoto University have also made public related plans.

[0004] In addition, methods for efficiently producing iPS cells using somatic cells that can be obtained non-invasively are also being studied. For example, Patent Document 1 discloses a method for inducing iPS cells using urine-derived cells such as human exfoliated proximal tubular epithelial cells (HEPTEC) recovered from urine.

[0005] In addition, a method for inducing iPS cells from keratinocytes derived from the outer root sheath of hair follicles is disclosed (Non-Patent Document 3).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Publication No. 2017-169574

[0009] Non-patent literature

[0010] Non-patent literature 1: Cell. 2006; 126(4): 663-676

[0011] Non-patent document 2: Cell. 2007; 131(5): 861-872

[0012] Non-patent document 3: Nat Protoc. 2010 Feb; 5(2): 371-82. Summary of the invention

[0013] Problems to be Solved by the Invention

[0014] In the context of the above-mentioned prior art, a first object of the present invention is to provide a new technology capable of producing iPS cells using epithelial cells such as keratinocytes.

[0015] In addition, a second object of the present invention is to provide a new culture method for obtaining epithelial cells from a tissue containing epithelial cells such as hair follicles.

[0016] Means for solving problems

[0017] The present invention for solving the above-mentioned first problem is a method for producing artificial pluripotent stem cells using epithelial cells, the method comprising:

[0018] The initialization step includes introducing a reprogramming gene or its translation product for performing nuclear initialization into epithelial cells;

[0019] The adhesion culture step comprises the step of subjecting the cells subjected to the initialization step to adhesion culture on a culture surface coated with laminin or a fragment thereof.

[0020] The production method of the present invention has the following advantages: by performing adhesion culture of cells on a culture surface coated with laminin or a fragment thereof in the initialization step, the efficiency of establishing iPS cells is high.

[0021] In a preferred embodiment of the present invention, the epithelial cells are derived from the outer root sheath.

[0022] Since epithelial lineage cells derived from the outer hair root sheath that can be obtained by a non-invasive method are used, iPS donor cells can be easily obtained from a donor individual.

[0023] In a preferred embodiment of the present invention, the epithelial cells are derived from tissue obtained by plucking hair.

[0024] iPS cells can be efficiently produced by using epithelial cells derived from tissue obtained by plucking hair.

[0025] In a preferred embodiment of the present invention, the method further comprises a step of culturing the epithelial cells used in the initialization step.

[0026] The culturing step includes the step of culturing tissue containing the outer hair root sheath and proliferating epithelial cells.

[0027] By carrying out the above-mentioned culture step, epithelial cells derived from the outer hair root sheath can be obtained by a non-invasive method, thereby easily obtaining iPS donor cells from a donor individual.

[0028] In a preferred embodiment of the present invention, the tissue containing the outer hair root sheath is cultured in a state of being attached to the hair.

[0029] According to the present invention, the trouble of taking out a processing sample such as a tissue containing the outer root sheath from the hair can be omitted, thereby simplifying the process.

[0030] In a preferred embodiment of the present invention, the tissue containing the outer root sheath is obtained from hair obtained by plucking.

[0031] Since culture samples can be easily obtained by plucking hair as a non-invasive method, the burden on the donor individual in obtaining the sample can be reduced.

[0032] In a preferred embodiment of the present invention, the initialization step includes inhibiting the function of p53.

[0033] The efficiency of iPS cell establishment can be further improved by inhibiting the function of p53 during cell reprogramming.

[0034] In a preferred embodiment of the present invention, the functional inhibition of p53 is induced by one or more substances selected from the following (a) to (c).

[0035] (a) Chemical inhibitors of p53;

[0036] (b) dominant negative mutants of p53 and nucleic acids encoding them; and

[0037] (c) siRNA, shRNA and their encoding DNA targeting p53.

[0038] In a preferred embodiment of the present invention, the method comprises culturing the cells into which the reprogramming gene or its translation product has been introduced in the resetting step in a medium containing a ROCK inhibitor.

[0039] By using the above method, the efficiency of establishing iPS cells can be further improved.

[0040] In a preferred embodiment of the present invention, the culturing step includes a step of contacting the obtained tissue containing the outer root sheath with a disinfectant for less than 1 minute before culturing.

[0041] By adopting the above-mentioned steps, the adhesion of the sample hair to the culture medium can be improved, and epithelial cells can be efficiently obtained from the outer hair root sheath.

[0042] In a preferred embodiment of the present invention, the culturing step comprises:

[0043] A step of seeding the obtained tissue containing the outer hair root sheath on a plate coated with an extracellular matrix; and

[0044] The process of culturing the tissue containing the outer root sheath seeded on the plate for 1 to 3 days while replenishing fresh culture medium without replacing the entire amount of the culture medium, and then statically culturing the tissue containing the outer root sheath for 2 to 7 days without replacing or replenishing the culture medium to allow epithelial cells to proliferate.

[0045] By carrying out the culture step having the above characteristics, a large number of epithelial cells can be obtained efficiently from a small amount of hair.

[0046] In a preferred embodiment of the present invention, the laminin is laminin 511.

[0047] By using laminin 511, the efficiency of establishing iPS cells can be improved.

[0048] In a preferred embodiment of the present invention, the reprogramming genes include Oct3 / 4 gene, Klf4 gene and Sox2 gene.

[0049] In a preferred embodiment of the present invention, the reprogramming gene includes c-Myc gene or L-Myc gene.

[0050] In a preferred embodiment of the present invention, the epithelial cells are keratinocytes.

[0051] In a preferred embodiment of the present invention, the culture medium used in the culturing step is a serum-free and / or xeno-free culture medium.

[0052] In a preferred embodiment of the present invention, the culturing step includes a step of culturing in a stem cell culture medium.

[0053] By including a step of culturing in a stem cell culture medium, epithelial cells can be efficiently proliferated even under serum-free and / or xeno-free conditions.

[0054] In a preferred embodiment of the present invention, the culturing step includes a step of culturing in an epithelial cell culture medium after culturing in the stem cell culture medium.

[0055] By using the above-mentioned method, epithelial cells can be preferentially proliferated.

[0056] In a preferred embodiment of the present invention, the present invention comprises:

[0057] A step of culturing in a high calcium medium having a calcium concentration of 0.2 to 4.0 mM; and

[0058] A step of culturing the cells in the high calcium medium and then culturing the cells in a low calcium medium having a calcium concentration of 40 to 80 μM.

[0059] By using the above-described method, epithelial cells can be efficiently proliferated even under serum-free and / or xeno-free conditions.

[0060] In a preferred embodiment of the present invention, the stem cell culture medium and / or the high calcium culture medium contains epithelial cell growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone, transferrin, basic fibroblast growth factor (bFGF), TGFβ1 (Transforming Growth Factor-β1), L-ascorbic acid, selenium and insulin.

[0061] By using the above-mentioned method, epithelial cells can be efficiently obtained from epithelial tissues under serum-free and / or xeno-free conditions.

[0062] In a preferred embodiment of the present invention, the epithelial cell culture medium and / or the low calcium culture medium contains epithelial cell growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone and transferrin.

[0063] By using the above method, epithelial cells can be efficiently obtained from tissues containing the outer root sheath under serum-free and / or xeno-free conditions.

[0064] In a preferred embodiment of the present invention, the step of degrading components of the extracellular matrix in the tissue containing the outer root sheath is not included before the tissue containing the outer root sheath is seeded.

[0065] By using the above method, the trouble of handling the sample can be omitted and the damage to the sample can be minimized, so that epithelial cells can be efficiently proliferated from the tissue containing the outer root sheath.

[0066] In a preferred embodiment of the present invention, the degradation step is a step of treating the sample with one or more selected from the group consisting of trypsin, collagenase and dispase.

[0067] The present invention for solving the above-mentioned first problem is as follows.

[0068] [1] A method for producing artificial pluripotent stem cells using epithelial cells, the method comprising:

[0069] The initialization step includes introducing a reprogramming gene or its translation product for performing nuclear initialization into epithelial cells;

[0070] The adhesion culture step comprises the step of subjecting the cells subjected to the initialization step to adhesion culture on a culture surface coated with laminin or a fragment thereof.

[0071] [2] The production method according to [1], wherein the epithelial cells are derived from the outer root sheath.

[0072] [3] The production method according to [1] or [2], wherein the epithelial cells are derived from a tissue obtained by plucking hair.

[0073] [4] The manufacturing method according to any one of [1] to [3], further comprising a culturing step of culturing the epithelial cells used in the initialization step, wherein the culturing step includes culturing a tissue containing an outer root sheath and causing the epithelial cells to proliferate.

[0074] [5] The production method according to [4], wherein the tissue containing the outer hair root sheath is cultured in a state of being attached to the hair.

[0075] [6] The production method according to [4] or [5], wherein the tissue containing the outer hair root sheath is obtained from hair obtained by plucking.

[0076] [7] The production method according to any one of [1] to [6], wherein the initialization step includes inhibiting the function of p53.

[0077] [8] The method according to [7], wherein the functional inhibition of p53 is induced by one or more substances selected from the following (a) to (c):

[0078] (a) Chemical inhibitors of p53;

[0079] (b) dominant negative mutants of p53 and nucleic acids encoding them; and

[0080] (c) siRNA, shRNA and their encoding DNA targeting p53.

[0081] [9] The production method according to any one of [1] to [8], comprising culturing the cells into which the reprogramming gene or its translation product has been introduced in the resetting step in a medium containing a ROCK inhibitor.

[0082]

[10] The production method according to any one of [4] to [9], wherein the culturing step includes a step of contacting the obtained tissue containing the outer hair root sheath with a disinfectant for less than 1 minute before culturing.

[0083]

[11] The production method according to any one of [4] to

[10] , wherein the culturing step comprises:

[0084] The process of sowing the tissue containing the obtained outer root sheath on a plate coated with an extracellular matrix; and the process of culturing the tissue containing the outer root sheath sown on the plate for 1 to 3 days while supplementing with fresh culture medium without replacing the entire amount of the culture medium, and then statically culturing the tissue containing the outer root sheath for 2 to 7 days without replacing or supplementing the culture medium, thereby allowing epithelial cells to proliferate.

[0085]

[12] The production method according to any one of [1] to

[11] , wherein the laminin is laminin 511.

[0086]

[13] The production method according to any one of [1] to

[12] , wherein the reprogramming genes include Oct3 / 4 gene, Klf4 gene and Sox2 gene.

[0087]

[14] The production method according to any one of [1] to

[13] , wherein the reprogramming gene includes c-Myc gene or L-Myc gene.

[0088]

[15] The production method according to any one of [1] to

[14] , wherein the epithelial cells are keratinocytes.

[0089]

[16] The production method according to any one of [4] to

[15] , wherein the culture medium used in the culturing step is a serum-free and / or xeno-free culture medium.

[0090]

[17] The production method according to

[16] , wherein the culturing step includes a culturing step in a stem cell culture medium.

[0091]

[18] The production method according to

[17] , wherein the culturing step includes a step of culturing in an epithelial cell culture medium after culturing in the stem cell culture medium.

[0092]

[19] The production method according to any one of

[16] to

[18] , wherein the culturing step comprises:

[0093] A step of culturing in a high calcium medium having a calcium concentration of 0.2 to 4.0 mM; and

[0094] A step of culturing the cells in the high calcium medium and then culturing the cells in a low calcium medium having a calcium concentration of 40 to 80 μM.

[0095]

[20] The production method according to any one of

[17] to

[19] , wherein the stem cell culture medium and / or the high calcium culture medium contains epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone, transferrin, basic fibroblast growth factor (bFGF), TGFβ1 (transforming growth factor-β1), L-ascorbic acid, selenium and insulin.

[0096]

[21] The production method according to any one of

[18] to

[20] , wherein the epithelial cell culture medium and / or the low calcium culture medium contains epithelial growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone and transferrin.

[0097]

[22] The production method according to any one of [4] to

[21] , which does not include: before sowing the tissue containing the outer root sheath, a degradation step of degrading the components of the extracellular matrix in the tissue containing the outer root sheath.

[0098]

[23] The production method according to

[22] , wherein the degradation step is a step of treating the sample with one or more selected from trypsin, collagenase and dispase.

[0099] The present invention for solving the above-mentioned second problem is a method for growing epithelial cells from tissues under serum-free and / or xeno-free conditions.

[0100] Preferred embodiments of the present invention described above are as follows.

[0101] [1] A method for culturing epithelial cells, comprising culturing a sample containing epithelial tissue under serum-free and / or xeno-free conditions.

[0102] [2] The culture method according to [1], wherein the epithelial cells are keratinocytes.

[0103] [3] The culture method according to [2], wherein the keratinocytes are hair follicle keratinocytes.

[0104] [4] The culture method according to any one of [1] to [3], wherein the sample contains an outer root sheath.

[0105] [5] The culture method according to [4], wherein the outer root sheath is in a state of being attached to the hair.

[0106] [6] The culture method according to any one of [1] to [5], wherein the sample is obtained from hair plucked out.

[0107] [7] The culture method according to any one of [1] to [6], comprising the step of culturing the sample in a stem cell culture medium.

[0108] [8] The culture method according to [7], wherein the calcium concentration of the stem cell culture medium is 0.2 to 4.0 mM.

[0109] [9] The culture method according to [7] or [8], comprising the step of culturing the sample in an epithelial cell culture medium after culturing the sample in the stem cell culture medium.

[0110]

[10] The culture method according to any one of [1] to [9], comprising:

[0111] The step of culturing the sample in a high calcium medium with a calcium concentration of 0.2 to 4.0 mM; and

[0112] A step of culturing the sample in a low calcium medium having a calcium concentration of 40 to 80 μM after culturing in the high calcium medium.

[0113]

[11] The culture method according to

[10] , wherein the high calcium culture medium is a stem cell culture medium.

[0114]

[12] The culture method according to

[10] or

[11] , wherein the low calcium culture medium is an epithelial cell culture medium.

[0115]

[13] The culture method according to any one of [7] to

[12] , wherein the stem cell culture medium and / or the high calcium culture medium contains epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone, transferrin, basic fibroblast growth factor (bFGF), TGFβ1, L-ascorbic acid, selenium and insulin.

[0116]

[14] The culture method according to any one of [9] to

[13] , wherein the epithelial cell culture medium and / or the low calcium culture medium contains epithelial growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone and transferrin.

[0117]

[15] The culture method according to any one of [1] to

[14] , wherein the sample is cultured under serum-free and xeno-free conditions.

[0118]

[16] The culture method according to any one of [1] to

[15] , wherein the culture method is a primary culture method.

[0119]

[17] The culture method according to any one of [1] to

[16] , which does not include a degradation step of degrading components of the extracellular matrix in the sample before seeding the sample.

[0120]

[18] The culture method according to

[17] , wherein the degradation step is a step of treating the sample with one or more selected from trypsin, collagenase and dispase.

[0121]

[19] The culture method according to any one of [1] to

[18] , comprising a step of contacting the sample with a disinfectant for less than 1 minute before culture.

[0122]

[20] The culture method according to any one of [1] to

[19] , comprising:

[0123] A step of seeding the obtained sample on a plate coated with an extracellular matrix; and

[0124] A static culture step, wherein the sample seeded on the plate is cultured for 1 to 3 days while replenishing fresh culture medium without replacing the entire amount of the culture medium, and then the sample is statically cultured for 2 to 7 days without replacing or replenishing the culture medium, thereby allowing epithelial cells to proliferate.

[0125]

[21] The culture method according to

[20] , wherein the culture medium used in the static culture step is the stem cell culture medium.

[0126]

[22] The culture method according to

[21] , wherein the calcium concentration of the stem cell culture medium is 0.2 to 4.0 mM.

[0127] Effects of the Invention

[0128] According to the production method of the present invention, iPS cells derived from epithelial cells can be produced efficiently.

[0129] Furthermore, according to the production method of the present invention, epithelial cells can be obtained from tissues containing epithelial cells such as hair follicles under serum-free and / or xeno-free conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] [ Figure 1 ] is a process diagram for illustrating a production process of iPS cells using epithelial cells according to one embodiment of the present invention.

[0131] [ Figure 2 ] is a bright field photograph showing the state of epithelial cells proliferating from the outer hair root sheath in Example 1. The scale bar is 1 mm.

[0132] [ Figure 3] is a bright field photograph showing the proliferation of iPS cells 20 days after the morphological transformation in Example 1. The scale bar is 200 μm.

[0133] [ Figure 4 ] is a fluorescent photograph showing the results of cell immunostaining for iPS cell markers (OCT4, SOX2, NANOG, and TRA1-81) of the iPS cells induced in Example 1. The scale bar is 200 μm.

[0134] [ Figure 5 ] is a bright field photograph showing the state of epithelial cells proliferating from the outer root sheath in Example 2. The scale bar is 300 μm.

[0135] [ Figure 6 ] is a bright field photograph showing the proliferation of iPS cells induced from epithelial cells in Example 2. The scale bar is 100 μm.

[0136] [ Figure 7 ] is a fluorescent photograph showing the results of cell immunostaining for iPS cell markers (OCT4, SOX2, NANOG, and TRA1-81) of iPS cells induced in Example 2. The scale bar is 100 μm.

[0137] [ Figure 8 ] is a fluorescent photograph showing the results of immunostaining of epithelial cell markers (KRT14 and CD200) of epithelial cells obtained in Example 1 in Example 3. The scale bar is 500 μm. DETAILED DESCRIPTION

[0138] <Method for producing artificial pluripotent stem cells using epithelial cells>

[0139] Next, we will refer to Figure 1 A preferred embodiment of the method for producing artificial pluripotent stem cells using epithelial cells of the present invention will be described.

[0140] It should be noted that the present invention is not limited to the present embodiment, and the design may be modified as appropriate.

[0141] according to Figure 1 The manufacturing method comprises: a culture step S1 of culturing epithelial cells, an initialization step S2 of initializing the obtained epithelial cells, and an adhesion culture step S3 of culturing the epithelial cells on a culture surface coated with laminin or a fragment thereof and inducing artificial pluripotent stem cells (iPS cells).

[0142] Here, in the present invention, artificial pluripotent stem cells (iPS cells) refer to cells having differentiation pluripotency and proliferation ability artificially produced by allowing reprogramming factors described later to act on somatic cells.

[0143] Hereinafter, each process of the present invention will be described in detail.

[0144] (1) Cultivation step S1

[0145] The culturing step S1 is a step of culturing epithelial cells used in the initialization step S2.

[0146] The epithelial cells used in the present invention may be cells derived from mammals (eg, humans, etc.). In the present invention, it is preferred to culture human-derived epithelial cells.

[0147] In the present invention, epithelial cells refer to cells derived from epithelial tissue, which can be obtained by culturing a sample containing epithelial tissue. The epithelial cells in the present invention can be a cell population obtained by culturing epithelial tissue, and can include epithelial cells expressing epithelial cell markers (e.g., keratinocytes) and stem cells expressing epithelial stem cell markers (e.g., hair follicle stem cells (HFSC)).

[0148] In the present invention, keratinocytes can be used as epithelial cells, and hair follicle keratinocytes can be preferably used among them.

[0149] The epithelial cells used in the present invention can be obtained by culturing the skin and skin appendages, which are specimens having epithelial cells. In the present invention, cells of the outer root sheath of hair are preferably cultured for reasons of invasiveness.

[0150] In a preferred embodiment of the present invention, since epithelial cells derived from the outer root sheath of hair that can be obtained by a non-invasive method are used, the burden of collecting samples from donor individuals for inducing iPS cells can be reduced.

[0151] There is no particular limitation on the individual (specifically, a mammalian individual) from whom the tissue for obtaining epithelial cells is collected. When iPS cells are used as a source of screening cells for evaluating the cosmetic effect, drug sensitivity, or presence or absence of side effects of the donor individual, epithelial cells are preferably collected from the donor himself or another person who has the same gene polymorphism related to drug sensitivity, etc. as the donor.

[0152] In addition, when used for transplantation into patients, epithelial cells are preferably collected from the patient or other person whose HLA type is the same or substantially the same as that of the donor. This can avoid the risk of rejection, and the iPS cells produced can be used for regenerative medicine.

[0153] Here, the "substantially identical" HLA type means that when cells obtained by differentiation induction from iPS cells derived from the somatic cells using immunosuppressants, etc. are transplanted into a donor individual, the HLA type is consistent with the degree to which the transplanted cells can survive. For example, the HLA (e.g., three loci of HLA-A, HLA-B, and HLA-DR) are identical.

[0154] The medium used to culture epithelial cells is not particularly limited, and a known medium suitable for culturing epithelial cells such as keratinocytes can be used for culture. Such medium includes but is not limited to EpiLife TM Medium (Gibco), Keratinocyte Growth Medium, Minimum Essential Medium (MEM) containing about 5-20% fetal bovine serum, Dulbecco's Modified Eagle Medium (DMEM), RPMI1640 medium, 199 medium, F12 medium, etc. In addition, components suitable for keratinocyte culture (calcium, growth factors, etc.) may be appropriately added.

[0155] In the present invention, it is preferred to use a culture medium containing 40 to 400 μM of a calcium salt.

[0156] In order to prevent a decrease in introduction efficiency, it is preferred that the culture medium be replaced with a serum-free culture medium before the initialization step S2.

[0157] The epithelial cells are preferably cultured by adhesion using a culture vessel to which the cells can adhere.

[0158] The culture vessel used for adhesion culture can be appropriately selected according to the culture scale, culture conditions and culture period. Cell adhesion culture vessels include culture vessels whose surface is artificially treated to improve cell adhesion, specifically, culture vessels with surface processing or culture vessels coated with a coating agent.

[0159] In the present invention, epithelial cells are preferably cultured using a culture vessel coated with a coating agent.

[0160] The coating agent includes: extracellular matrix, such as laminin, entactin, collagen, gelatin, vitronectin (Vitronectin), Synthmax (Corning), matrigel, etc.; polymer, such as polylysine, polyornithine, etc. In the present invention, the extracellular matrix is ​​preferably used, and collagen I is preferably used.

[0161] In the present invention, laminin or a fragment thereof is also preferably used as a coating agent. Preferred forms of laminin or a fragment thereof are as described in the adhesion culture step S3 described later.

[0162] In addition, specific forms of culture vessels include, for example, flasks, tissue culture flasks, culture dishes (plates), tissue culture plates, multi-well culture dishes, microplates, microwell plates, multi-well plates, chamber slides, culture dishes, test tubes, trays, culture bags, microcarriers, microbeads, stacked plates, spinner flasks or roller bottles.

[0163] Next, the specific steps of the method for isolating and culturing epithelial cells using hair, that is, the culturing step S1 will be described.

[0164] First, a tissue containing an outer root sheath is obtained. The outer root sheath is a layer of epithelial root sheath that constitutes the hair follicle, and the tissue containing the outer root sheath can be obtained by plucking the hair of a donor individual. The number of hairs used in the present invention only needs to be at least 1, preferably 2 or more, and more preferably 5 or more. Specifically, it is preferred to use 1 to 10 hairs. Then, the lower part of the hair collected from the donor individual is cut to obtain the tissue containing the outer root sheath. The size of the tissue to be obtained is preferably 2 to 3 mm or more. In addition, the tissue to be obtained may also be in a form in which the outer root sheath remains attached to the hair. That is, it may also be a hair follicle containing the outer root sheath attached to the hair obtained by cutting the part containing the outer root sheath of the plucked hair.

[0165] The outer root sheath of the hair obtained by plucking is attached to the area above the hair bulb. The tissue obtained by plucking and cultured in the present invention may include cells present in the bulge area of ​​the hair follicle.

[0166] Then, the obtained tissue (sample) containing the outer root sheath is brought into contact with a disinfectant. The method of bringing the sample into contact with the disinfectant is not particularly limited, but it is preferred that the sample be immersed in the disinfectant.

[0167] The contact time with the disinfectant is preferably less than 1 minute, more preferably less than 30 seconds, further preferably less than 10 seconds, and particularly preferably less than 5 seconds.

[0168] By setting the contact time with the disinfectant within the above range, damage to the hair can be reduced, and the adhesion of the hair to the culture medium can be improved, so that epithelial cells can be efficiently obtained from the tissue containing the outer root sheath.

[0169] The disinfectant used in the present invention is preferably a solution to which an antibiotic is added. The preferred antibiotic is one or more selected from a cell wall synthesis inhibitor, a protein synthesis inhibitor, and a cell membrane function inhibitor. More preferably, one or more antibiotics are selected from a cell wall synthesis inhibitor, a protein synthesis inhibitor, and a cell membrane function inhibitor.

[0170] The cell wall synthesis inhibitor is preferably a β-lactam antibiotic. Examples of the β-lactam cell wall synthesis inhibitor include penicillin, ampicillin, carbenicillin sodium, and the like. In the present invention, penicillin is preferably used.

[0171] As the protein synthesis inhibitor, aminoglycoside antibiotics are preferred.

[0172] Examples of the aminoglycoside protein synthesis inhibitor include kanamycin, gentamicin, streptomycin, neomycin, hygromycin, and tobramycin. In the present invention, streptomycin is preferably used.

[0173] As the cell membrane function inhibitor, polyene antibiotics are preferred. Examples of polyene cell membrane function inhibitors include amphotericin B, nystatin, natamycin, and the like. In the present invention, amphotericin B is particularly preferably used.

[0174] The disinfectant solution according to the present invention is preferably a solution containing the antibiotics penicillin, streptomycin and amphotericin B.

[0175] Next, the obtained sterilized sample is seeded on a plate coated with a coating agent and cultured for a period of time to allow proliferation of epithelial cells.

[0176] The plate used is preferably a 12-well plate or a 24-well plate for cell culture, more preferably a 24-well plate. In addition, it is preferred to seed one sterilized sample per well.

[0177] The amount of the culture medium during seeding is preferably such that the entire tissue of the excised sample is submerged and the liquid level is 1 mm or less above the upper part of the entire tissue. By setting the culture medium during seeding to the above amount, the hair follicle tissue can be efficiently adhered to the plate.

[0178] In a preferred embodiment, the culture of the sample containing the outer root sheath is carried out in the following three steps in sequence.

[0179] In the first step S11, the seeded sample is cultured while replenishing the fresh culture medium without replacing the entire amount of the culture medium. The number of times the culture medium is replenished is preferably 1 to 3 times, more preferably 2 times.

[0180] In the first step S11, the amount of supplemented culture medium is preferably enough to immerse the entire tissue of the excised sample, and the liquid level is 2 mm or less above the upper part of the entire tissue, and more preferably the liquid level is 1.5 mm or less above the upper part of the entire tissue.

[0181] In addition, the amount of culture medium supplemented is preferably 0.5 to 3 times the amount of culture medium when the sample is sown, more preferably 1 to 3 times, and further preferably 1 to 2 times. When the culture medium is supplemented more than twice, it is preferred that the amount of culture medium supplemented after the second time is more than the amount of culture medium supplemented for the first time. In this case, the amount of culture medium supplemented after the second time is preferably 1.2 to 3 times the amount of culture medium supplemented for the first time, more preferably 1.5 to 2.5 times, and further preferably 2 to 2.5 times. In addition, the amount of culture medium supplemented after the second time is preferably an amount that allows the entire excised tissue to be immersed and the liquid level to be 1 to 2 mm higher than the upper part of the entire tissue, and more preferably an amount that allows the liquid level to be 1 to 1.5 mm higher than the upper part of the entire tissue.

[0182] In addition, the culture period in the first step S11 is preferably 1 to 3 days.

[0183] As a specific method of the first step S11, the following method is preferred: the culture medium is supplemented twice and the culture period is 3 days; between 24 and 48 hours after the sample is sown, the culture medium is supplemented once with an amount equal to (1 times) the culture medium at the time of sowing; between 48 and 72 hours after the sample is sown, the culture medium is supplemented once with an amount twice that of the culture medium at the time of sowing.

[0184] In the second step S12, after the medium is supplemented in the first step S11, the epithelial cells are cultured statically without replacing or supplementing the medium to proliferate. The culture period is preferably 2 to 7 days, more preferably 3 to 5 days, and even more preferably 3 to 4 days. Specifically, 3 days is preferred.

[0185] Such static culture can promote the proliferation of epithelial cells derived from the outer hair root sheath.

[0186] In the second step S12, it can be confirmed that the epithelial cell population begins to spread from the seeded outer root sheath. The spread distance of the epithelial cells proliferating from the outer root sheath is preferably 0.8 mm or more, more preferably 1 mm or more, and particularly preferably 1 to 2 mm from the hair shaft.

[0187] It should be noted that the step of sequentially performing the first step S11 and the second step S12 is also referred to as a static culture step in the present invention.

[0188] In the third step S13, the culture medium is replaced, and the cells extending from the outer root sheath are continuously cultured. Preferably, the culture medium is replaced in full when the culture medium is replaced for the first time after the second step S12, and then the culture medium is repeatedly replenished and replaced. The replenishment and replacement of the culture medium are preferably performed alternately at intervals of 45 to 55 hours, more preferably at intervals of 48 to 50 hours, and specifically, preferably alternately every other day.

[0189] In the third step S13, the amount of the medium added when replacing the medium is preferably 0.5 to 3 times, more preferably 1 to 2 times, and even more preferably 1.5 to 2 times the amount of the medium added when the sample was sown before the first step S11.

[0190] The amount of the culture medium to be supplemented is preferably 0.5 to 2 times, more preferably 1 to 1.5 times, and even more preferably the same amount (1 time) as the amount of the culture medium added when the culture medium is replaced in the third step S13.

[0191] As a specific method of the third step S13, the following method is preferred: completely replace the culture medium, add twice the amount of culture medium as when the sample was sown, and after 2 days, add the same amount of culture medium (1 times) as the amount of culture medium added when the culture medium was replaced and culture.

[0192] The third step S13 is preferably continued until the confluence of the proliferating cells reaches 50-80%. The culture period after supplementation and replacement of the culture medium is preferably 7-28 days, more preferably 14-21 days.

[0193] In addition, in the third step S13, it is preferred to perform subculture in the development area of ​​cells proliferating from the outer root sheath before the cells become too dense. For example, in the case of culturing using a 12-well plate (well bottom diameter of 21 mm to 22.3 mm), it is preferred to perform subculture at a stage where the confluence of the proliferating cells reaches 50 to 80%. In the subsequent initialization step S2, cells with a confluence of 50 to 80% after subculture can be appropriately used.

[0194] The culture period after subculture is preferably 3 to 7 days, more preferably 4 to 6 days. In addition, it is preferred that the culture medium is replaced every day after subculture, and the amount of culture medium added during replacement is preferably 0.5 to 2 times the amount of culture medium added during the culture medium replacement in the third step S13, more preferably 1 to 1.5 times, and even more preferably the same amount (1 times).

[0195] By adopting the above three steps, epithelial cells of sufficient quantity to induce iPS cells can be obtained from a small amount of hair. Therefore, if the present invention is used, the burden of collecting samples from donor individuals can be reduced.

[0196] In one embodiment, in the first step S11 and the second step S12, it is preferred to mix the calcium-containing medium and the MEF acclimation medium, and specifically, EpiLife TM A mixed medium of MEF medium (containing 60 μM calcium, Gibco) and EmbryoMax MEF acclimation medium (Merck). In addition, the calcium salt concentration in the medium used in the first step S11 and the second step S12 is preferably 60 to 400 μM.

[0197] In the third step S13, it is preferred to use a calcium-containing culture medium, specifically EpiLife TM Medium (containing 60 μM calcium, Gibco) The calcium salt concentration in the medium used in the third step S13 is preferably 40 to 80 μM.

[0198] In addition, the culture medium used in the first step S11 to the third step S13 preferably contains bovine pituitary extract (BPE), recombinant human insulin-like growth factor-I (Recombinant humaninsulin-like growth factor-I), hydrocortisone, bovine transferrin and human epidermal growth factor (Humanepidermal growth factor).

[0199] In addition, when the size of the cells extending from the outer root sheath is less than a specified value, for example, less than 1 mm, it is preferred to include a step of culturing using MEF-cis medium before turning to the third step S13. In this case, the MEF-cis medium preferably contains bovine pituitary extract (BPE), recombinant human insulin-like growth factor-I (Recombinant human insulin-like growth factor-I), hydrocortisone, bovine transferrin and human epidermal growth factor (Human epidermal growth factor).

[0200] In another embodiment, the culture medium used in the culture step S1 is preferably a serum-free or xeno-free culture medium. More preferably, the culture medium used in the culture step S1 is preferably a serum-free and xeno-free culture medium.

[0201] The serum-free and / or xeno-free culture medium preferably contains one or more supplements selected from the group consisting of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone and transferrin, and more preferably contains all four of the above supplements.

[0202] By containing these components, epithelial cells can be efficiently proliferated.

[0203] The concentration of EGF is preferably 5 to 50 ng / ml, more preferably 10 to 30 ng / ml, further preferably 15 to 25 ng / ml, and particularly preferably 20 ng / ml.

[0204] In addition, EGF is preferably human epithelial cell growth factor.

[0205] The concentration of IGF-I is preferably 5 to 50 ng / ml, more preferably 10 to 30 ng / ml, further preferably 15 to 25 ng / ml, and particularly preferably 20 ng / ml.

[0206] In addition, IGF-I is preferably recombinant human insulin-like growth factor.

[0207] The concentration of hydrocortisone is preferably 50 to 500 ng / ml, more preferably 100 to 300 ng / ml, further preferably 150 to 250 ng / ml, and particularly preferably 200 ng / ml.

[0208] The concentration of transferrin is preferably 1 to 50 μg / ml, more preferably 3 to 30 μg / ml, further preferably 5 to 20 μg / ml, and particularly preferably 10 μg / ml.

[0209] In addition, the transferrin is preferably human transferrin.

[0210] In addition, the serum-free and / or xeno-free medium may contain any epithelial cell proliferation supplement. The epithelial cell supplement is preferably xeno-free, preferably Supplement S7 (Gibco).

[0211] When a serum-free and / or xeno-free medium is used, the culturing step S1 preferably includes a step of culturing in a stem cell culture medium.

[0212] Preferred stem cell culture media to be used include Essential 8, StemPro (manufactured by Life Technologies), mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), or StemFit (manufactured by Ajinomoto Co., Ltd.), among which Essential 8 is preferably used.

[0213] In the stem cell culture medium, supplements such as antibiotics, serum, growth factors and hormones may be optionally added.

[0214] More preferably, the step includes a step of culturing in an epithelial cell culture medium after culturing in a stem cell culture medium.

[0215] As the epithelial cell culture medium to be used, the medium suitable for epithelial cell culture described in the culture step S1 can be used as appropriate, and EpiLife TM Medium (Gibco), HuMedia-KG2 (Kurabo), preferably EpiLife TM Medium. In the epithelial cell culture medium, antibiotics, serum, growth factors, hormones, calcium and other additives can be optionally added.

[0216] When a serum-free and / or xeno-free medium is used, the culturing step S1 preferably includes a step of culturing in a medium having a high calcium concentration (high calcium medium).

[0217] In addition, when using a serum-free and / or xeno-free medium, it is preferred to use two mediums with different calcium concentrations. In one embodiment, it is preferred to culture in a high calcium medium and then culture in a medium with a low calcium concentration (low calcium medium).

[0218] The calcium concentration of the high calcium medium is preferably 0.2 mM or more, more preferably 0.5 mM or more, further preferably 0.8 mM or more, and even more preferably 1.0 mM or more.

[0219] The calcium concentration of the high calcium medium is preferably 4.0 mM or less, more preferably 3.5 mM or less, and even more preferably 3.0 mM or less.

[0220] By setting the calcium concentration of the high calcium medium within the above range, epithelial cells can be efficiently proliferated from the outer root sheath after seeding.

[0221] The concentration of the high calcium medium can be specifically set to 0.2 to 4.0 mM, more preferably 0.5 to 3.5 mM, further preferably 1.0 to 3.5 mM, and even more preferably 1.0 to 3.0 mM.

[0222] The calcium concentration of the low calcium medium is preferably 40 μM or more, more preferably 45 μM or more, and even more preferably 50 μM or more.

[0223] The calcium concentration of the low calcium medium is preferably 80 μM or less, more preferably 75 μM or less, and even more preferably 70 μM or less.

[0224] By setting the calcium concentration of the low calcium medium within the above range, target epithelial cells can be efficiently proliferated from cells proliferated by culturing in a high calcium medium.

[0225] The concentration of the low calcium medium can be specifically set to 40 to 80 μM, more preferably 45 to 75 μM, and even more preferably 50 to 70 μM.

[0226] Next, a specific example is given to explain the preferred implementation of the serum-free and xeno-free culture medium. In this embodiment, the culture step S1 includes: a primary culture step using a high calcium culture medium and a stem cell culture medium for culture, and a secondary culture step using a low calcium culture medium and a secondary culture medium for culture.

[0227] The primary culture medium preferably has DMEM / F12 as the base medium. In addition, the primary culture medium preferably contains basic fibroblast growth factor (bFGF) and / or TGFβ1 (transforming growth factor-β1). By containing these components, cells can be efficiently proliferated from the outer root sheath of hair.

[0228] In addition, the primary culture medium preferably contains L-ascorbic acid, selenium, transferrin and insulin. Culture media containing these ingredients include Essential 8.

[0229] The primary culture medium preferably contains one or more additives selected from the group consisting of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone and transferrin in the above culture medium, and more preferably contains all four of the above. The preferred embodiment of the additive is as described above.

[0230] In the present invention, the primary culture medium may be a culture medium to which the above four additives are added to Essential 8. The preferred calcium concentration is as described in the conditions of the high calcium culture medium.

[0231] EpiLife TMMedium (containing 60 μM calcium, Gibco) was used as the epithelial cell culture medium.

[0232] The secondary culture medium preferably contains one or more additives selected from the group consisting of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone and transferrin in the above culture medium, and more preferably contains all four of the above. The preferred embodiment of the additive is as described above.

[0233] In the primary culture step, the first step S11 and the second step S12 are preferably performed using the primary culture medium. Preferred embodiments of the first step S11 and the second step S12 are as described above.

[0234] In the primary culture process, preferably after the second step S12 is implemented, the culture medium is replaced in the primary culture medium and the culture is continued. When the culture medium is replaced in the primary culture medium, the replacement amount of the culture medium is preferably 0.5 to 2 times the amount of the culture medium added during sowing, more preferably 1 to 1.5 times, and further preferably the same amount (1 times).

[0235] The culture period of the primary culture step is preferably 10 days or more, more preferably 14 days or more, and further preferably 18 days or more. In addition, the culture period of the primary culture step is preferably 30 days or less, more preferably 25 days or less, and further preferably 22 days or less.

[0236] By setting the culture period of the primary culture step within the above range, epithelial cells can be efficiently obtained from tissues containing the outer root sheath.

[0237] For example, the culture period of one culture step can be set to 10 to 30 days, more preferably 14 to 25 days, and even more preferably 18 to 22 days.

[0238] In the secondary culture step, culture is performed using a secondary culture medium after the primary culture step.

[0239] Preferably, in the primary culture step, after the cells extending from the outer root sheath grow to a predetermined size, for example, 1 mm or more, preferably 1 mm to 2 mm, the secondary culture step is performed.

[0240] In the secondary culture step, the third step S13 is preferably performed using a secondary culture medium.

[0241] The culture period of the secondary culture step is preferably 5 days or more, more preferably 7 days or more, and further preferably 10 days or more. In addition, the culture period of the secondary culture step is preferably 20 days or less, more preferably 18 days or less, and further preferably 16 days or less.

[0242] By setting the culture period of the secondary culture step within the above range, keratinocytes derived from the outer hair root sheath can be obtained efficiently.

[0243] For example, the culture period of the secondary culture step can be set to 5 to 20 days, more preferably 7 to 18 days, and even more preferably 10 to 16 days.

[0244] In the secondary culture step, it is preferred to replace and supplement the culture medium according to the cell proliferation status.

[0245] In addition, the secondary culture process may include a step of performing subculturing.

[0246] The preferred embodiment of the replacement and supplementation of the culture medium and the subculture is as described in the third step S13. It should be noted that the third step S13 in the secondary culture step is also preferably a method of replacing the culture medium in its entirety without supplementing the culture medium.

[0247] Furthermore, the present invention preferably does not include a degradation step of degrading components of the extracellular matrix of the sample before seeding the tissue containing the outer root sheath.

[0248] In the present invention, it is preferred that the method does not include a step of treating the sample with one or more selected from trypsin, collagenase and dispase before seeding the sample containing epithelial tissue, and it is more preferred that the method does not include a step of treating the sample with trypsin, collagenase and dispase.

[0249] By not degrading the extracellular matrix, the trouble of handling the sample can be saved and the damage to the sample can be minimized, thereby allowing the epithelial cells of the present invention to proliferate efficiently from the tissue containing the outer root sheath.

[0250] It should be noted that, in the above-mentioned embodiment, a method including a culture step S1 for culturing epithelial cells is exemplified, but the present invention can also be implemented from the initialization step S2 of (2) described later. In this case, commercially available epithelial cells such as keratinocytes can be used to induce iPS cells. Such keratinocytes include normal human epidermal keratinocytes (Normal Human Epidermal Keratinocytes: NHEK) (manufactured by PromoCell), hair follicular keratinocytes (Hair Follicular Keratinocytes: HHFK) (manufactured by ScienCell Research Laboratories), etc.

[0251] (2) Initialization step S2

[0252] The reprogramming step S2 includes the steps of introducing a reprogramming gene or a translation product thereof that performs cell nucleus reprogramming into epithelial cells and culturing the cells into which the reprogramming gene and the like have been introduced.

[0253] The reprogramming gene in the present invention refers to a gene encoding a protein factor (reprogramming factor) that can induce iPS cells from somatic cells. Such reprogramming genes are, for example, one or more genes selected from the group consisting of members of the Oct family, Klf family, Sox family, Myc family, Lin family, and Glis family.

[0254] The reprogramming gene of the present invention preferably contains at least Oct3 / 4, Klf4 and Sox2. In addition, it preferably contains one or more selected from c-Myc, L-Myc, Lin28 and Lin28b, and more preferably further combines c-Myc or L-Myc with Lin28 or Lin28b.

[0255] In a preferred embodiment of the present invention, the combination of reprogramming genes is Oct3 / 4, Klf4, Sox2, L-Myc and Lin28.

[0256] In the present invention, nucleic acid encoding reprogramming genes can be introduced into epithelial cells, or the translation product (protein) of reprogramming genes can be introduced into epithelial cells. In addition, nucleic acid encoding reprogramming genes can be in any form of DNA or RNA.

[0257] There is no particular limitation on the method of introducing reprogramming genes or their translation products into epithelial cells. The introduction methods include infection with viral vectors such as retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, and adeno-associated virus vectors; gene introduction by calcium phosphate method, liposome transfection method, electroporation method, etc. using various vectors (plasmid vectors, free vectors, RNA vectors, etc.); direct introduction by microinjection, etc.

[0258] In the present invention, it is preferred to use an episomal vector to introduce a DNA encoding a reprogramming gene. In addition, the introduction of the episomal vector is preferably performed by liposome transfection.

[0259] In the initialization step S2, it is preferred that the function of p53 be suppressed while introducing the reprogramming gene or the like.

[0260] In the present invention, "inhibiting the function of p53" means inhibiting the function of p53 protein by directly acting on the p53 protein, or inhibiting the function of the p53 protein or the expression of the p53 gene by acting on a factor involved in the signal transduction of the p53 protein.

[0261] Inhibition of p53 function includes, but is not limited to, contacting cells with chemical inhibitors of p53, dominant negative mutants of p53, anti-p53 antagonist antibodies, decoy nucleic acids containing consensus sequences of p53 response elements, substances that inhibit p53 pathways, and the like.

[0262] In the present invention, the inhibition of p53 function can be induced by introducing one or more substances selected from the following substances: (a) chemical inhibitors of p53; (b) dominant negative mutants of p53 and their encoding nucleic acids; (c) siRNA, shRNA and their encoding DNA targeting p53.

[0263] For specific examples of p53 function inhibitory substances, methods for obtaining them, and methods for contacting them with somatic cells, reference can be made to WO 2009 / 157593 and the like.

[0264] (a) Chemical inhibitors of p53 include low molecular weight inhibitors such as Pifithrin-α (PFT-α).

[0265] (b) The dominant negative mutant of p53 is preferably p53DD (Bowman, T., Genes Develop., 10, 826-835 (1996)) in which the amino acids 14 to 301 of mouse p53 (corresponding to 11 to 304 in human p53) are deleted.

[0266] (c) siRNA and shRNA targeting p53 can be designed using existing methods based on mouse or human p53 cDNA sequence information.

[0267] In the present invention, the function of p53 is preferably inhibited by introducing one or more substances selected from the group consisting of (b) dominant negative mutants of p53 and nucleic acids encoding them, and (c) siRNA, shRNA and DNA encoding them for p53.

[0268] In a more preferred embodiment, the function of p53 is inhibited by introducing into cells a vector containing a DNA encoding the (b) dominant negative mutant of p53 or a DNA encoding the (c) siRNA or shRNA targeting p53.

[0269] It should be noted that, in the present invention, the p53-related gene sequence and the above-mentioned reprogramming gene sequence can be encoded in one vector, or the p53-related gene sequence and the reprogramming gene sequence can be encoded in different vectors. In the latter case, it is preferred that the vector encoding the p53-related gene sequence and the vector encoding the reprogramming gene sequence are mixed and introduced into the cell at the same time.

[0270] In addition, in the present invention, it is preferable to use an episomal vector having a DNA sequence encoding a dominant negative mutant of p53 (p53DD).

[0271] Here, in a preferred embodiment, the episomal vector used contains the EBNA-1 gene sequence. In addition, the reprogramming factors and p53DD are preferably controlled by the CAG promoter.

[0272] In addition, the episomal vector used in the present invention is preferably a vector carrying an expression cassette containing a reprogramming gene or a combination of multiple reprogramming genes. The expression cassette is preferably: (i) an expression cassette containing a nucleic acid encoding Oct3 / 4; (ii) an expression cassette containing a nucleic acid encoding Sox2 and a nucleic acid encoding Klf4; (iii) an expression cassette containing a nucleic acid encoding L-Myc and a nucleic acid encoding Lin28; (iv) an expression cassette containing a nucleic acid encoding mMp53DD. The vector carrying the expression cassette is preferably: pCE-hOCT3 / 4 or pCE-hOCT3 / 4-shp53-F encoding human OCT3 / 4, pCE-hSK encoding human Sox2 and human Klf4, pCE-hUL encoding human L-Myc and human LIN28, pCE-mp53DD encoding mouse p53DD, etc. In the present invention, it is preferred that all four vectors encoding the expression cassettes of (i) to (iv) above are introduced into cells.

[0273] In addition, the episomal vector used in the present invention preferably includes a vector encoding the EBNA-1 gene sequence instead of the reprogramming factor and p53DD, for example, pCXB-EBNA1.

[0274] This vector is preferably Human iPS Cell Generation TM Episomal Vector Mix (manufactured by TakaraBio, Inc.).

[0275] In the initialization step S2, the introduction of the reprogramming gene or its translation product into the epithelial cells and / or the subsequent culture of the epithelial cells are preferably carried out in the presence of a ROCK inhibitor. The ROCK inhibitor used is not particularly limited, as long as it can inhibit the function of Rho kinase (ROCK), such as Y-27632, Fasudil / HA1077, H-1152, Wf-536 and derivatives thereof, and antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), dominant negative mutants and their expression vectors.

[0276] In the present invention, one or more ROCK inhibitors may be used.

[0277] In the present invention, Y-27632 is preferably used as a ROCK inhibitor.

[0278] The concentration of Y-27632 used can be set, for example, in the range of 100 nM to 50 μM, preferably in the range of 1 to 30 μM, but is not limited thereto. In the present invention, it is preferably 10 μM.

[0279] Furthermore, the culture from the introduction of the reprogramming gene or its translation product into the epithelial cells in the initialization step S2 to the adhesion culture step S3 is preferably an adhesion culture performed on a culture surface coated with a coating agent.

[0280] The coating agent used is not particularly limited, and the method used in the above-mentioned culture step S1 can be adopted. In the present invention, collagen I is preferably used.

[0281] (3) Adhesion culture step S3

[0282] The adhesion culture step S3 is a step of adhesion-culturing the epithelial cells that have undergone the initialization step S2 on the culture surface coated with the extracellular matrix.

[0283] In the adhesion culture step S3, first, epithelial cells are allowed to adhere to a culture vessel whose surface is coated with an extracellular matrix.

[0284] The extracellular matrix used is laminin or its fragment. Preferred laminins used include laminin α5β1γ1 (laminin 511), laminin α5β2γ1 (laminin 521), laminin α4β1γ1 (laminin 411), laminin α2β1γ1 (laminin 211) and laminin fragments thereof, and laminin 511 or laminin 521 is more preferably used. Recombinant laminins and laminin fragments may also be used.

[0285] The laminin or laminin fragment described above preferably has an integrin binding site.

[0286] Furthermore, the laminin fragment more preferably forms a heterotrimer.

[0287] The type of integrin to which laminin or a laminin fragment binds is preferably integrin α6β1, integrin α6β4, integrin α3β1, integrin α7β1, and more preferably integrin α6β1. The use of laminin or a laminin fragment that binds to integrin α6β1 can promote the proliferation of iPS cells.

[0288] As the laminin fragment having binding activity to these integrins, preferably, a laminin fragment derived from at least one selected from laminin 511, laminin 521, laminin 411 and laminin 211 is used, more preferably a laminin fragment derived from laminin 511 or laminin 521, and further preferably a laminin fragment derived from laminin 511. Such a laminin fragment is preferably an E8 fragment obtained by digesting laminin with elastase and a recombinant E8 fragment.

[0289] In the present invention, laminin 511E8 is more preferably used. As laminin 511E8, commercially available products such as iMatrix-511 (manufactured by Nippi) can be used.

[0290] The culture vessel used for adhesion culture is not particularly limited as long as cells can adhere, and the same culture vessel as that used in the above-mentioned culture step S1 can be used.

[0291] Adhesion culture is preferably carried out using a serum-free medium after the first subculture. As a serum-free medium, a serum-free medium containing KSR or B27, or a medium under xeno-free conditions is preferred. Here, "xeno-free" refers to a condition that excludes components derived from biological species different from the biological species of the cells to be cultured.

[0292] On the other hand, it is preferred that after the initialization step S2 and before the first subculture, the cells are cultured in a culture medium containing bovine pituitary extract (BPE), recombinant human insulin-like growth factor-I (Recombinant humaninsulin-like growth factor-I), hydrocortisone, bovine transferrin and human epidermal growth factor (Human epidermal growth factor).

[0293] When epithelial cells obtained under serum-free and xeno-free conditions are used, it is preferred that the cells be cultured using a secondary culture medium after the initialization step S2 and before the first subculture.

[0294] The culture in the adhesion culture step S3 is preferably carried out in the absence of feeder cells (feeder-free). The feeder-free culture medium used as the undifferentiated maintenance medium can use commercially available products, such as Essential 8 (manufactured by Life Technologies), S-medium (manufactured by DS PHARMA BIOMEDICAL CO., LTD.), StemPro (manufactured by Life Technologies), mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), or StemFit (manufactured by Ajinomoto Co., Ltd.).

[0295] In addition, in the absence of feeder cells (feeder cell-free) means a condition in which feeder cells are substantially not contained (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less).

[0296] In the adhesion culture step S3, Essential 8 is preferably used.

[0297] Essential 8 Medium contains DMEM / F12, and as additives, L-ascorbic acid-2-phosphate magnesium, sodium selenium, insulin, NaHCO 3, transferrin, bFGF, and a TGFβ family signaling pathway activator (TGFβ1 or Nodal).

[0298] The adhesion culture is preferably performed in the presence of a ROCK inhibitor. The ROCK inhibitor used in the adhesion culture and its preferred form are the same as those in the initialization step S2.

[0299] In the adhesion culture step S3, it is also preferred to inhibit the function of p53, as in the initialization step S2. The method for inhibiting the function of p53 can be appropriately adopted from the method described in the initialization step S2.

[0300] The adhesion culture period is preferably 5 days or longer, more preferably 10 days or longer, and even more preferably 15 days or longer.

[0301] By continuing the adherence culture, the emergence of cell colonies can be confirmed. Then, candidate colonies of iPS cells are preferably selected using existing methods.

[0302] Selection of candidate iPS cell colonies can also be carried out by visual morphological observation, confirmation of the expression of iPS cell-specific surface antigens such as TRA1-60 and SSEA4, and the like.

[0303] In order to further improve the establishment efficiency of iPS cells in the initialization step S2 and the adhesion culture step S3, a known substance having an effect of improving the establishment efficiency may be used.

[0304] These substances include, for example, histone deacetylase (HDAC) inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC 1293, M344, etc., DNA methyltransferase inhibitors such as 5'-azacytidine, G9a histone methyltransferase inhibitors such as BIX-01294, L-calcium channel agonists such as Bayk8644, p38 inhibitors, UTF1, Wnt signal activators, 3'-phosphoinositide-dependent kinase-1 (PDK1) activators such as PS48, etc., but are not limited to these.

[0305] The iPS cells produced according to the present invention can be used in a variety of ways, such as as pharmaceutical raw materials or in the screening of materials. For example, using existing differentiation induction methods, various types of cells such as muscle cells, blood cells, nerve cells, vascular endothelial cells, insulin-secreting cells, and epithelial cells can be produced from iPS cells. In addition, the differentiated cells can also be used to perform in vitro screening of the efficacy or toxicity of candidate pharmaceutical compounds.

[0306] Furthermore, by establishing iPS cells using a sample (eg, hair) of a subject and using skin tissues differentiated from iPS cells inherent to the subject, the effectiveness of drugs, cosmetics, etc. corresponding to each subject can be confirmed.

[0307] In one embodiment of the present invention, any one of the culture step S1, the initialization step S2, and the adhesion culture step S3 can be performed under serum-free and xeno-free conditions. That is, the present invention can perform a series of production steps under serum-free and xeno-free conditions.

[0308] <Method for culturing epithelial cells>

[0309] The present invention also relates to a method for culturing epithelial cells, which comprises culturing a sample containing epithelial tissue under serum-free and / or xeno-free conditions.

[0310] The method for culturing epithelial cells of the present invention (hereinafter referred to as the culturing method of the present invention) is to obtain epithelial cells by culturing under serum-free and / or xeno-free conditions. Furthermore, the culturing method of the present invention relates to the culturing method under serum-free and / or xeno-free conditions of the culturing step S1 in the above-mentioned <Method for producing artificial pluripotent stem cells using epithelial cells>.

[0311] The "epithelial cells" cultured in the culture method of the present invention are a cell population obtained by culturing a sample containing epithelial tissue, including epithelial cells expressing epithelial cell markers (e.g., keratinocytes) and stem cells expressing epithelial stem cell markers (e.g., hair follicle stem cells (HFSC)).

[0312] In the present invention, the epithelial cells may include keratinocytes, and preferably include hair follicle keratinocytes.

[0313] In addition, the culture method of the present invention is preferably a culture method for primary culture.

[0314] The primary culture in the present invention refers to the culture performed after sowing the collected sample.

[0315] The epithelial tissue of the present invention is preferably a tissue containing epithelial cells to be cultured by the method of the present invention.

[0316] The sample containing epithelial tissue used in the present invention is not particularly limited as long as it is obtained from an epithelial tissue, and for example, epithelial tissue of hair follicles can be used. In the present invention, as a sample containing epithelial tissue, it is preferred to use a tissue containing the outer hair root sheath. In addition, the sample containing epithelial tissue used in the present invention may contain cells present in the bulge area of ​​the hair follicle.

[0317] As the sample containing epithelial tissue, a sample collected from a mammal (eg, human, etc.) can be used. In the culture method of the present invention, it is preferable to use a tissue collected from a human.

[0318] The method for obtaining the tissue to be used can adopt the existing tissue collection method. When collecting a sample containing the outer root sheath, it can be performed by biopsy or plucking the hair. From the perspective of non-invasiveness, it is preferred to obtain the sample by plucking the hair.

[0319] The outer root sheath of the hair obtained by plucking is attached to the upper part of the hair bulb. In the present invention, the sample containing the outer root sheath is preferably cultured in a state where it is attached to the hair.

[0320] Preferred embodiments of the culture method of the present invention (the optional disinfection step, the culture from the first step S11 to the third step S13, etc.) refer to the description of the culture step S1 in the above-mentioned <Method for producing artificial pluripotent stem cells using epithelial cells>. In this case, "tissue (sample) containing the outer root sheath" can also be appropriately understood as "sample containing epithelial tissue".

[0321] Example

[0322] Hereinafter, the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0323] <Example 1>

[0324] (1) Reagents used

[0325] (a) EpiLife medium

[0326] EpiLife 500ml TM A total of 5 mL of Human Keratinocyte Growth Supplement (HKGS, Gibco), 5 mL of a penicillin-streptomycin mixed solution (nacalai tesque, inc.), and 500 μL of Amphotericin-B were added to Medium with 60 μM calcium (Gibco) and mixed.

[0327] (b) MEF-Conditioned Medium

[0328] To 10 mL of EmbryoMax (registered trademark) MEF cis medium (Merck), 0.1 mL of a penicillin-streptomycin mixed solution (nacalai tesque, inc.), 10 μL of Amphotericin-B, and 0.1 mL of HKGS (Gibco) were added and mixed.

[0329] (c)Antibiotic Mix

[0330] Transfer 247 ml of HBSS into a sterile container, and add 2.5 ml of penicillin-streptomycin mixed solution (final concentration of 100 U / ml penicillin, 100 μg / ml streptomycin) and 250 μL of Amphotericin B solution (final concentration of 250 ng / ml).

[0331] (d) Shape-conversion solution

[0332] Human iPS Cell GenerationTM Episomal Vector Mix (Takara) and Opti-MEM (Thermo Fisher Scientific) were mixed, and then FuGENE HD Transfection (Promega) was added and allowed to stand at room temperature for 20 minutes. Before the transformation, EpiLife medium was added.

[0333] (e) Collagen coating

[0334] The collagen acid solution I-PC (telopeptide collagen) (5 mg / mL) was diluted 10 times with 1 mM hydrochloric acid, and the diluted collagen solution was added at 400 μL / well in the case of a 12-well cell culture plate, and at 1000 μL / well in the case of a 6-well cell culture plate, and allowed to stand at 37°C for more than 1 hour, and then removed with an aspirator. Then, it was washed three times with PBS before use.

[0335] (f) iMatrix coating

[0336] 1 mL of DPBS (- / -) was diluted with 4.5 μL of iMatrix-511 (Nippi), and the dilution was added to a 12-well plate for cell culture at 500 μL / well, and incubated at 37°C for 1 hour or at 4°C overnight. Then, the dilution was removed before use.

[0337] (2) Culture of epithelial cells

[0338] After pulling out 5 hairs from the subjects (humans), they were immediately immersed in Antibiotic Mix for a while. Then, 1 to 2 mm of the hair including the outer root sheath was cut in Antibiotic Mix, and one sample was seeded per well in 250 μL of mixed culture medium (EpiLife+MEF-Conditioned culture medium+Antibiotic Mix) in a 12-well plate coated with a collagen solution (collagen acidic solution I-PC, final concentration 0.5 mg / mL (atelocollagen)).

[0339] 250 μL of the mixed culture medium was added 1 day or 2 days after sowing, and 500 μL of the mixed culture medium was added 3 days after sowing. Then, the total liquid volume of the culture medium was confirmed to be about 0.75 mL at the time point 3 days after sowing, and static culture was carried out until 6 days after sowing.

[0340] At 6 days after seeding, if the proliferating cells have not extended more than 1 to 2 mm in the radial direction around the hair shaft, remove 250 μL of the mixed medium and replace with 250 μL of MEF-Conditioned medium. This MEF-Conditioned medium replacement is continued until the cells extend more than 1 to 2 mm in the radial direction.

[0341] Then, after confirming that the proliferating cells have extended more than 1 to 2 mm in the radial direction with the hair shaft as the center, the medium is replaced. When replacing the medium, the above mixed medium is completely removed and 500 μL of EpiLife medium is added. It should be noted that at the time point of 6 days after seeding, if the cells have extended more than 1 to 2 mm in the radial direction with the hair shaft as the center, the medium is replaced with EpiLife medium without replacing the above MEF-Conditioned medium.

[0342] Two days after the medium was replaced, 500 μL of EpiLife medium was added again, and two days after the medium was added, the medium was replaced with 500 μL of EpiLife medium. Then, the medium was replenished and replaced in the same manner every two days.

[0343] In the development area of ​​cells proliferating from the outer root sheath, before the cells become too dense, specifically, when the confluence of the cells reaches 50-80%, the EpiLife medium is removed and washed with DPBS. Then, 0.5 mL of TrypLESelect (Gibco) is added, and after standing in a 37°C incubator, the cells are peeled off from the plate with a pipette and recovered in a 15 mL test tube. After centrifugation, the recovered cells are removed from the supernatant and suspended in 1 mL of MEF-Conditioned medium.

[0344] Then, the cells were seeded at 50,000 cells / well on a 6-well plate coated with a collagen solution (collagen acid solution I-PC, final concentration 0.5 mg / mL (atelocollagen)) and cultured in a 37°C incubator. The medium was replaced several times with 500 μL of EpiLife medium until the confluence reached 50-80%. At the stage of 50-80% confluence, iPS cell induction was performed.

[0345] (3) Induction of iPS cells

[0346] First, the EpiLife medium was replaced with the morphogenetic solution, and after standing in a 37°C incubator for 4 hours, the medium was replaced with 2 mL of EpiLife medium containing 10 μM Y27632 (EpiLife medium containing Y27632) for culturing.

[0347] On the second day, after removing the culture medium and washing with DPBS (- / -), 1 mL of TrypLESelect was added and the plate was left to stand in a 37°C incubator. The cells were detached from the plate with a pipette. After adding 2 mL of trypsin neutralization solution (Kurabo), 3 mL of EpiLife medium containing Y27632 was added and the cells were recovered in a 15 mL test tube. After centrifugation of the recovered cells, the supernatant was removed and the cells were suspended in 1 mL of EpiLife medium containing Y27632.

[0348] Then, the cells were seeded at 10,000 cells / well on a 12-well plate coated with iMatrix-511 (Nippi) and cultured in a 37°C incubator (subculture day 0). After subculture for 1 to 3 days, 10 μM Essential 8 containing Y27632 was added every day. TM Flex medium (Essential 8 containing Y27632). After 4 days of subculturing, the medium was completely replaced with Essential 8 containing Y27632, and the medium was changed every day thereafter.

[0349] (4) Immunohistochemical staining

[0350] One day after subculturing, cell immunostaining for iPS cell marker genes (OCT4, SOX2, NANOG, and TRA1-81) was performed according to the following steps.

[0351] First, the cells were washed with PBS and then fixed with 4% paraformaldehyde, permeabilized by adding PBS containing 0.5% TritonX-100 and leaving for 10 minutes, and then blocked with PBS containing 10% goat serum for 30 minutes.

[0352] Next, after washing the cells with PBS, the primary antibody was added and left to stand overnight at 4°C. After washing again with PBS, the fluorescently labeled secondary antibody (Alexa647 or Alexa488) and Dapi were added and left to stand for 30 minutes. After washing with PBS, the fluorescence images were acquired by fluorescence microscopy.

[0353] (5) Results

[0354] Figure 2 Figure 2 shows the state of epithelial cells extending from the outer root sheath 12 days after seeding on a 12-well plate in the culture of epithelial cells (2). Figure 2 As shown, it was confirmed that epithelial cells began to proliferate in large numbers starting from the periphery of the outer root sheath.

[0355] in addition, Figure 3 The state of cells 20 days after passage in (3) iPS cell induction is shown.

[0356] like Figure 3 As shown, by carrying out the production method of Example 1, a large number of iPS cell colonies were confirmed.

[0357] also, Figure 4 It was shown that the proliferated cells expressed iPS cell markers and that iPS cells that had acquired pluripotency had been produced.

[0358] <Comparative Example 1>

[0359] Induction of iPS cells was carried out in the same manner as in Example 1, except that the extracellular matrix used in the induction of iPS cells (3) in Example 1 was changed to vitronectin.

[0360] As a result, unlike Example 1 using a plate coated with iMatrix-511, apoptosis was induced in Comparative Example 1 using a plate coated with vitronectin, and almost all cells died after one week of subculture.

[0361] <Comparative Example 2>

[0362] In the induction of iPS cells in (3) Example 1, the episomal vector used was changed to Episomal iPSC Reprogramming Vectors (manufactured by Invitrogen) that do not have p53 inhibitory activity. At this time, except for the vector used, the induction of iPS cells was carried out according to the same steps as in Example 1, and the number of iPS cell colonies formed was confirmed.

[0363] The results show that the colony formation rate of iPS cells in Example 1 is significantly better than that in Comparative Example 2.

[0364] <Inspection>

[0365] It is clear from the results of Example 1 and Comparative Examples 1 and 2 that iPS cell colonies can be efficiently obtained by using Example 1, which is the production method of the present invention. Specifically, it is clear from the results of Comparative Example 1 that when performing adhesion culture of iPS cells, culturing on an adhesion surface coated with a laminin fragment such as iMatrix can inhibit cell apoptosis and promote the proliferation of iPS cells.

[0366] In addition, the results of Comparative Example 2 show that the formation of iPS cell colonies can be promoted by inhibiting the function of p53 when introducing reprogramming factors. In other words, by inhibiting the function of p53 while expressing reprogramming factors, the formation of iPS cell colonies can be promoted and the efficiency of iPS cell production can be improved.

[0367] <Example 2>

[0368] (1) Reagents used

[0369] Reagents that are repeated in Example 1 will be omitted as appropriate.

[0370] (a) Mixed culture medium A (primary culture medium)

[0371] Contains 90% of Essential 8 TM Penicillin-streptomycin mixed solution (added to a final concentration of 100 U / ml penicillin and 100 μg / ml streptomycin, nacalai tesque, inc.), Supplement S7 (Gibco, final concentration was a 100-fold dilution of the stock solution), 20 ng / ml EGF (epithelial cell growth factor (EGF), human, Fujifilm Wako Pure Chemical Industries, Ltd.), 20 ng / ml IGF-I (insulin-like growth factor 1), 200 ng / ml hydrocortisone and 10 μg / ml transferrin were added to Flex medium (Gibco, Supplement already added), 10% CTSKnockout SR XenoFree (Gibco) medium.

[0372] (b) Mixed culture medium B (secondary culture medium)

[0373] Mix the Essential 8 TM Flex medium replaced with EpiLife TM Mixed medium B was prepared by adding 1% paraformaldehyde and 1% paraformaldehyde to the culture medium, with 60 μM calcium (Gibco) and other conditions being the same as those of mixed medium A.

[0374] (2) Culture of epithelial cells

[0375] Hair was plucked from a subject (human) and sterilized with AntibioticMix according to the same procedure as in Example 1. Then, one sample was seeded per well in 250 μL of mixed culture medium A in a 12-well plate coated with collagen.

[0376] 250 μL of mixed culture medium A was added 1 day or 2 days after sowing, and 250 μL of mixed culture medium A was added 3 days after sowing. Then, the total liquid volume of the culture medium was confirmed to be about 0.75 mL at the time point 3 days after sowing, and static culture was carried out until 6 days after sowing.

[0377] Six days after seeding, remove 250 μL of the culture medium and replace with 250 μL of mixed culture medium A. Replace the culture medium with mixed culture medium A in the same manner every other day until the cells extend more than 1 to 2 mm in the radial direction around the hair stem. Cultivate with mixed culture medium A for 2 to 3 weeks.

[0378] After confirming that the proliferating cells have extended more than 1 to 2 mm in the radial direction around the hair shaft, the medium is replaced with mixed medium B. In replacing mixed medium B, all mixed medium A is removed and 500 μL of mixed medium B is added.

[0379] Two days after the medium was replaced, the entire medium was replaced with 500 μL of mixed medium B. Thereafter, the entire medium was replaced in the same manner every two days.

[0380] After culturing in mixed medium B for 1 to 2 weeks, in the development area of ​​cells proliferating from the outer root sheath, before the cells become too dense, specifically, when the confluence reaches 50 to 80%, the medium is removed and washed with DPBS. Then, 0.5 mL of TrypLESelect (Gibco) is added, and after standing in a 37°C incubator, the cells are peeled off from the plate with a pipette and recovered in a 15 mL test tube. The recovered cells are centrifuged, the supernatant is removed, and suspended in 1 mL of mixed medium B.

[0381] Then, 50,000 cells / well were seeded on a 6-well plate coated with collagen solution and cultured in a 37°C incubator. The medium was replaced several times with 500 μL of mixed medium B until the confluence reached 50-80%. At the stage of 50-80% confluence, iPS cell induction was performed.

[0382] (3) Induction of iPS cells

[0383] The induction of iPS cells was carried out in the same manner as in Example 1, except that the EpiLife medium containing Y27632 used in the culture after the morphological transformation was made into a mixed medium B containing 10 μM Y27632. The obtained cells were subjected to immunohistostaining as in Example 1, and it was confirmed that they expressed iPS cell markers. In addition, the three germ layer differentiation ability of the obtained cells was also confirmed.

[0384] The results of Example 2 show that epithelial cells can be obtained from tissues obtained by hair plucking under serum-free and xeno-free conditions (see Figure 5 ). Then, the epithelial cells can be used to induce iPS cells under serum-free and xeno-free conditions. That is, according to the present invention, iPS cells can be produced using tissue obtained by plucking hair under serum-free and xeno-free conditions.

[0385] in addition, Figure 6 The iPS cells obtained in Example 2 are shown. Figure 7 Shows Figure 6 The results of cell immunostaining of iPS cell marker genes (OCT4, SOX2, NANOG, and TRA1-81) in iPS cells were obtained. This confirmed that the proliferated iPS cells expressed multiple iPS cell markers and were pluripotent.

[0386] [Example 3] Expression of epithelial cell markers

[0387] The expression of epithelial cell markers was confirmed for the epithelial cells obtained in the culture of epithelial cells (2) in Example 1. Using cells seeded on a 12-well plate for 7 days, epithelial cells were labeled using keratin-14 (KRT14) and CD200 as primary antibodies and Alexa647 or Alexa488 as secondary antibodies in the same manner as in the above (4) immunohistostaining.

[0388] As a result, it was confirmed that the obtained cells were epithelial cells expressing both KRT14 and CD200 ( Figure 8 ).

[0389] Industrial Applicability

[0390] The present invention can be applied to improving the efficiency of methods for inducing iPS cells, developing pharmaceutical preparations using iPS cells, and the like.

[0391] Explanation of symbols

[0392] S1 Cultivation Process

[0393] S11 First step

[0394] S12 Second step

[0395] S13 Step 3

[0396] S2 initialization process

[0397] S3 adhesion culture step.

Claims

1. A method for producing artificial pluripotent stem cells using epithelial cells, the method comprising: The initialization step includes introducing a reprogramming gene or its translation product for performing nuclear initialization into epithelial cells; The adhesion culture step comprises the step of subjecting the cells subjected to the initialization step to adhesion culture on a culture surface coated with laminin or a fragment thereof.

2. The manufacturing method according to claim 1, wherein: The epithelial lineage cells are derived from the outer hair root sheath.

3. The manufacturing method according to claim 1, wherein: The epithelial lineage cells are derived from tissue obtained by plucking hair.

4. The manufacturing method according to claim 1, wherein: The manufacturing method further includes: a culturing step of culturing the epithelial cells used in the initialization step, and The culturing step includes a step of culturing tissue containing the outer hair root sheath and proliferating epithelial cells.

5. The manufacturing method according to claim 4, wherein: The tissue containing the outer root sheath is cultured in a state of being attached to the hair.

6. The manufacturing method according to claim 4, wherein: Tissue containing the outer root sheath is obtained from hair obtained by plucking.

7. The manufacturing method according to claim 1, wherein: The initialization process includes inhibiting the function of p53.

8. The manufacturing method according to claim 7, wherein: The functional inhibition of p53 is induced by one or more substances selected from the following (a) to (c): (a) Chemical inhibitors of p53; (b) dominant negative mutants of p53 and nucleic acids encoding them; and (c) siRNA, shRNA and their encoding DNA targeting p53.

9. The manufacturing method according to claim 1, wherein: The production method comprises: culturing the cells into which the reprogramming gene or its translation product has been introduced in the initialization step in a culture medium containing a ROCK inhibitor.

10. The manufacturing method according to claim 4, wherein: The culturing step includes a step of contacting the obtained tissue containing the outer root sheath with a disinfectant for less than 1 minute before culturing.

11. The manufacturing method according to claim 4, wherein: The culturing process comprises: A step of seeding the obtained tissue containing the outer hair root sheath on a plate coated with an extracellular matrix; and The process of culturing the tissue containing the outer root sheath seeded on the plate for 1 to 3 days while supplementing with fresh medium without replacing the entire medium, and then statically culturing the tissue containing the outer root sheath for 2 to 7 days without replacing or supplementing the medium to allow epithelial cells to proliferate.

12. The manufacturing method according to claim 1, wherein: The laminin is laminin 511.

13. The manufacturing method according to claim 1, wherein: The reprogramming genes include Oct3 / 4 gene, Klf4 gene and Sox2 gene.

14. The manufacturing method according to claim 13, wherein: The reprogramming gene includes c-Myc gene or L-Myc gene.

15. The manufacturing method according to any one of claims 1 to 14, wherein: The epithelial cells are keratinocytes.

16. The manufacturing method according to claim 4, wherein: The culture medium used in the culturing step is a serum-free and / or xeno-free culture medium.

17. The manufacturing method according to claim 16, wherein: The culturing step includes: a step of culturing in a stem cell culture medium.

18. The manufacturing method according to claim 17, wherein: The culturing step includes: culturing in the stem cell culture medium and then culturing in an epithelial cell culture medium.

19. The manufacturing method according to claim 16, wherein: The culturing process comprises: A step of culturing in a high calcium medium having a calcium concentration of 0.2 to 4.0 mM; and After culturing in the high calcium medium, the cells are then cultured in a low calcium medium having a calcium concentration of 40 to 80 μM.

20. The manufacturing method according to any one of claims 17 to 19, wherein: The stem cell culture medium and / or the high calcium culture medium contains epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone, transferrin, basic fibroblast growth factor (bFGF), TGFβ1 (transforming growth factor-β1), L-ascorbic acid, selenium and insulin.

21. The manufacturing method according to claim 18 or 19, wherein: The epithelial cell culture medium and / or the low calcium culture medium contains epithelial cell growth factor (EGF), insulin-like growth factor-I (IGF-I), hydrocortisone and transferrin.

22. The manufacturing method according to any one of claims 4 to 14 and 16 to 19, wherein: The manufacturing method does not include a step of degrading components of an extracellular matrix in the tissue containing the outer root sheath before seeding the tissue containing the outer root sheath.

23. The manufacturing method according to claim 22, wherein: The degradation step is a step of treating the sample with one or more selected from trypsin, collagenase, and dispase.

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