Cells having ability to induce skin appendages, and production method thereof

Abstract

[Problem] To directly convert somatic cells into cells that have an ability to form a stratified epithelial tissue capable of acting as the body outer skin and also have an ability to induce skin appendages. [Solution] A production method of cells that have an ability to induce skin appendages, said method comprising a step for introducing into somatic cells at least one gene that is relatively strongly expressed in cells having the ability to induce skin appendages.

Description

Cells capable of inducing skin appendages and method for producing the same

[0001] The present invention relates to cells capable of skin appendage induction induced from somatic cells that do not have the ability to induce skin appendages, and a method for producing the same. The present invention also relates to a cell preparation for skin and skin appendage regeneration, a method for treating skin ulcers, a method for treating alopecia, xeroderma, and asteatosis, and a platform for developing a skin and skin appendage regeneration method, all of which utilize the cells capable of skin appendage induction. Furthermore, the present invention relates to a composition for preparing cells for inducing somatic cells that do not have the ability to induce skin appendages into cells capable of induction of skin appendages.

[0002] Skin appendages consist of hair follicles, sebaceous glands, sweat glands, etc., and play roles in protecting against mechanical damage, maintaining heat, moisturizing, and regulating body temperature. Skin appendages are formed by interactions between epithelial and mesenchymal tissues during fetal organ development (see Non-Patent Document 1).

[0003] Typical pathologies caused by the loss, damage, or dysfunction of skin appendages include alopecia and asteatosis, which can lead to thermoregulation disorders, dry skin that causes itching, and cosmetic problems.

[0004] Clinically, it is difficult to regenerate lost skin appendages, and it is usually necessary to transplant existing appendages from other parts of the body using skin grafting or skin flap surgery.

[0005] Therefore, methods have been developed to regenerate and generate skin appendages in adults, such as transplanting cells derived from the skin of fetal or newborn animals to reconstruct skin appendages (see Non-Patent Documents 2 and 3), and obtaining appendage-like structures through organoid formation from pluripotent stem cells (see Non-Patent Documents 4 and 5). However, it has been difficult to regenerate and generate sufficient amounts of skin appendages in a short period of time.

[0006] The interaction between epithelial cells present on the surface of the body and mesenchymal cells present in the dermis or subcutaneous tissue, as well as environmental factors, play an important role in the development and regeneration of skin appendages (see Non-Patent Documents 6 and 7). In particular, recent studies using genetically modified animals have suggested that the expression of genes involved in skin development, such as the LEF1 gene and the SHH gene, is an important environmental factor in the regeneration of skin appendages, particularly hair follicles (Non-Patent Documents 8-10).

[0007] During development, once a somatic cell has committed to a cell lineage, it continues to proliferate and differentiate within that same cell lineage. In contrast, cell lineage conversion, which is not physiologically recognized, is called direct conversion, and techniques for direct conversion of skin fibroblasts into muscle cells (Non-Patent Document 11), nerve cells (Non-Patent Document 12), cardiomyocytes (Non-Patent Document 13), hepatocytes (Non-Patent Document 14), and cells capable of forming stratified squamous epithelium (Patent Document 1, Non-Patent Document 15) have been reported.

[0008] Against the background of such conventional technology, there is a strong desire to develop cells with skin appendage inducibility that can be directly induced from somatic cells that do not have this ability, such as skin fibroblasts and adipose-derived mesenchymal cells, and that can regenerate and neogenesis skin appendages, thereby providing a cell source for the treatment of skin appendage defects, disorders, and dysfunction.However, there have yet to be any reports of cells with skin appendage inducibility being directly converted from somatic cells that do not have this ability.

[0009] International Publication No. 2016 / 072105

[0010] Lee J , Boscke R , Tang PC , Hartman BH , HellerS , Koehler KR . “Cell Research”,2018, 22(1), p242-254Thank you SH, Morgan DL, Walker RJ, Bates RR. “J Invest Dermatol”, 1970, 55(6), p379-89.Worst PK, Mackenzie IC, Fusenig NE. “Cell Tissue Res”, 1982, 225(1), p65-77.Lee J, Boscke R, Tang PC, Hartman BH, HellerS, Koehler KR. “Cell Rep”,2018, 22(1), p242-254.Lee J, Rabbani CC, Gao H, Steinhart MR,Woodruff BM, Pflum ZE, Kim A, Heller S, Liu Y, Shipchandler TZ, Koehler KR. ”Nature”, 2020, 582(7812), p399-404.Sennett R, Wang Z, Rezza A, Grisanti L,Roitershtein N, Sicchio C, Mok KW, Heitman NJ, Clavel C, Ma'ayan A, Rendl M. “Dev Cell”, 2015, 34(5), p577-591.Yang H, Adam RC, Ge Y, Hua ZL, Fuchs E. “Cell”, 2017, 169(3), p483-496.Lim CH, Sun Q, Ratti K, Lee SH, Zheng Y,Takeo M, Lee W, Rabbani P, Plikus MV, Cain JE, Wang DH, Watkins DN, Millar S,Taketo MM, Myung P, Cotsarelis G, Ito M. “Nat Commun”, 2018, 9(1), 4903.Phan QM, Fine GM, Salz L, Herrera GG, WildmanB, Driskell IM, Driskell RR.“Elife”, 2020, 9, e60066. Sun X, Are A, Annusver K, Sivan U, Jacob T, Dalessandri T, Joost S, Fullgrabe A, Gerling M, Kasper M. “Elife”, 2020, 9, e46756. Davis RL, Weintraub H, Lassar AB, “Cell”, 1987 Dec 24, 51(6), p987-1000Vierbuchen T1, Ostermeier A, Pang ZP, KokubuY, Sudhof TC, Wernig M, “Nature”, 2010,463(7284), p1035-1041Ieda M, Fu JD, Delgado-Olguin P, VedanthamV, Hayashi Y, Bruneau BG, Srivastava D, “Cell”, 2010, 142(3), p375-386Sekiya S, Suzuki A, “Nature”, 2011, 475(7356), p390-393Kurita M, Araoka T, Hishida T, O'Keefe DD, Takahashi Y, Sakamoto A, Sakurai M, Suzuki K, Wu J, Yamamoto M, Hernandez-Benitez R, Ocampo A, Reddy P, Shokhirev MN, Magistretti P, Nunez Delicado E, Eto H, Harii K, Izpisua Belmonte JC, “Nature”, 2018, 561(7722), p243-247.

[0011] Therefore, in order to solve the problems of the prior art, one of the objects of the present invention is to directly convert somatic cells that do not have skin appendage inducibility into cells capable of inducing skin appendages such as hair, hair follicles, and sebaceous glands. More specifically, an object of the present invention is to establish a technology for providing a source of cells capable of forming skin appendages such as hair, hair follicles, and sebaceous glands. Another object of the present invention is to provide various uses for cells capable of inducing skin appendages induced from such somatic cells.

[0012] In order to solve the above-mentioned problems, the present inventors have adopted adipose tissue-derived mesenchymal cells as representative somatic cells that do not have the ability to induce skin appendages, and have found that by introducing genes that are likely to characterize fetal skin epithelial cells, fetal skin mesenchymal cells, and mesenchymal cells that constitute hair follicles, which have a strong ability to reconstitute skin appendages, it is possible to produce a group of cells with the ability to induce skin appendages that can regenerate and generate new hair, hair follicles, and sebaceous glands by transplanting the mixture into a skin chamber attached to the back of an immunodeficient animal.

[0013] That is, the present invention provides the following aspects: 1. A method for producing cells capable of skin appendage induction, comprising the step of introducing a transgene containing at least one gene that is relatively highly expressed in cells capable of skin appendage induction into somatic cells that do not have this ability.

[0014] 2. A method for producing cells capable of inducing skin appendages, comprising the step of introducing a transgene containing (1) the LEF1 gene, and (2) one, two, or three genes selected from the DNP63A gene, the GRHL2 gene, and the TFAP2A gene, into somatic cells that do not have the ability to induce skin appendages.

[0015] 3. A method for producing cells capable of inducing skin appendages, comprising the step of introducing a transgene containing four genes, namely, the LEF1 gene, the DNP63A gene, the GRHL2 gene, and the TFAP2A gene, into somatic cells that do not have the ability to induce skin appendages.

[0016] 4. A method for producing cells capable of inducing skin appendages, comprising the step of introducing a transgene containing: (1) the LEF1 gene; and (2) at least one, two, three, or four genes selected from the group consisting of the DNP63A gene, the GRHL2 gene, the TFAP2A gene, and at least one of the MYC family genes, into somatic cells that do not have the ability to inducibly form skin appendages.

[0017] 5. A method for producing cells capable of inducing skin appendages, comprising the step of introducing an introduced gene containing at least five genes, namely the LEF1 gene, the DNP63A gene, the GRHL2 gene, the TFAP2A gene, and at least one of the MYC family genes, into somatic cells that do not have the ability to induce skin appendages.

[0018] 6. The method for producing cells capable of inducing skin appendages according to 4 or 5 above, wherein the transgene contains a cMYC gene as the MYC family gene.

[0019] 7. A method for producing cells capable of inducing skin appendages, comprising the step of introducing an introduced gene containing one or more genes selected from the group consisting of the SHH gene, the ETV1 gene, the FOXD1 gene, the PRDM1 gene, and the LEF1 gene into somatic cells that do not have the ability to induce skin appendages.

[0020] 8. A method for producing cells capable of inducing skin appendages, comprising the step of introducing a transgene containing the SHH gene into somatic cells that do not have the ability to inducibly form skin appendages.

[0021] 9. A method for producing cells capable of inducing skin appendages, comprising the step of introducing a transgene containing the SHH gene and the LEF1 gene into somatic cells that do not have the ability to inducibly form skin appendages.

[0022] 10. A method for producing cells with skin appendage induction ability, comprising the step of introducing an introduced gene containing one, two, or three genes selected from the ETV1 gene, the PRDM1 gene, and the FOXD1 gene into somatic cells that do not have the ability to induce skin appendages.

[0023] 11. A method for producing cells with skin appendage induction ability, comprising the step of introducing an introduced gene containing (1) the SHH gene, and (2) one or more genes selected from the ETV1 gene, PRDM1 gene, FOXD1 gene, and LEF1 gene into somatic cells that do not have the ability to induce skin appendages.

[0024] 12. A method for producing cells capable of inducing skin appendages, comprising the step of introducing transgenes comprising: (1) the ETV1 gene and the PRDM1 gene; (2) the FOXD1 gene and the PRDM1 gene; or (3) the ETV1 gene, the PRDM1 gene, and the FOXD1 gene; into somatic cells that do not have the ability to induce skin appendages.

[0025] 13. The method for producing cells capable of inducing skin appendages according to any one of items 7 to 12 above, wherein when multiple types of genes are introduced, multiple cells with different introduced genes are prepared.

[0026] 14. The method for producing cells capable of inducing skin appendages according to any one of 1 to 13 above, wherein the somatic cells are derived from a human.

[0027] 15. A method for producing cells capable of inducing skin appendages according to any one of items 1 to 14 above, wherein the somatic cells are somatic cells differentiated from skin fibroblasts, subcutaneous adipose tissue-derived stromal cells (subcutaneous adipocytes), embryonic fibroblasts, adipocytes, muscle cells, osteoblasts, chondrocytes, circulating mononuclear cells, ES cells, mesenchymal stem cells, or the like.

[0028] 16. Cells capable of inducing skin appendages, produced by the production method described in any one of 1 to 15 above.

[0029] 17. A cell preparation comprising the cells capable of inducing skin appendages described in 16 above.

[0030] 18. A cell preparation comprising: (1) a first cell having the ability to induce skin appendages, produced by a process of introducing an introduced gene containing the SHH gene, or the SHH gene and the LEF1 gene, into somatic cells that do not have the ability to induce skin appendages; and (2) a second cell having the ability to induce skin appendages, produced by a process of introducing an introduced gene containing one or more genes selected from the ETV1 gene, the PRDM1 gene, and the FOXD1 gene into somatic cells that do not have the ability to induce skin appendages.

[0031] 19. The cell preparation according to claim 18, wherein the transgenes of the second cells include: (1) the ETV1 gene and the PRDM1 gene; (2) the FOXD1 gene and the PRDM1 gene; or (3) the ETV1 gene, the PRDM1 gene, and the FOXD1 gene.

[0032] 20. A cell preparation according to any one of items 17 to 19 above, which contains a scaffold material.

[0033] 21. The cell preparation according to claim 20, wherein the scaffold material is collagen.

[0034] 22. The cell preparation described in any one of items 17 to 21 above, wherein the cell preparation is for skin tissue regeneration or hair regeneration.

[0035] 23. The cell preparation according to any one of the above 17 to 22, wherein the cell preparation is a cell aggregate having a sheet-like structure or a three-dimensional structure.

[0036] 24. A non-human mammal in which skin appendages have been formed, produced by administering to a non-human mammal the cells having the ability to induce skin appendages described in 16 above or the cell preparation described in any one of 17 to 23 above, and causing the cells having the ability to induce skin appendages to form skin appendages within the body of the mammal.

[0037] 25. A method for determining the efficacy of a test substance, comprising the steps of administering the test substance to the non-human mammal described in 24 above, and determining the efficacy of the test substance on the skin and / or skin appendage tissues.

[0038] 26. A method for assessing the influence of external factors, comprising the steps of applying stress such as an anticancer drug or radiation to the non-human mammal described in 24 above, and assessing the stress on the skin and / or skin appendage tissues.

[0039] 27. A composition for adjusting cells capable of inducing skin appendages, comprising a transgene used in the manufacturing method described in any one of 1 to 12 above.

[0040] 28. The cell-regulating composition according to claim 27, wherein the transgene is contained in a form that can be introduced into somatic cells.

[0041] 29. Skin and skin appendage-like tissues produced by culturing the cells having the ability to induce skin appendages as described in 16 above.

[0042] 30. A method for analyzing the efficacy of a test substance, comprising the steps of administering a test substance to the skin and skin appendage-like tissue described in 29 above, and analyzing the efficacy of the test substance on the skin and / or skin appendage-like tissue.

[0043] 31. A method for analyzing the effects of external factors, comprising the steps of applying stress such as an anticancer drug or radiation to the skin and skin appendage-like tissue described in 29 above, and analyzing the stress on the skin and / or skin appendage-like tissue.

[0044] 32. A kit of vectors specialized for introducing the gene according to any one of 1 to 12 above into somatic cells.

[0045] 33. A method for evaluating the skin appendage induction ability of cells, using the cells having skin appendage induction ability described in 16 above.

[0046] 34. A vector for producing cells capable of inducing skin appendages, which expresses the LEF1 gene, the DNP63A gene, the GRHL2 gene, the TFAP2A gene, and the cMYC gene.

[0047] 35. A vector for producing cells capable of inducing skin appendages, which expresses the SHH gene, ETV1 gene, PRDM1 gene, FOXD1 gene, and LEF1 gene.

[0048] 36 A vector kit comprising: a first vector intended for producing cells with skin appendage inducibility, the first vector expressing the SHH gene, or the SHH gene and the LEF1 gene; and a second vector intended for producing cells with skin appendage inducibility, the second vector expressing one or more genes selected from the ETV1 gene, the PRDM1 gene, and the FOXD1 gene.

[0049] 37. The vector kit according to claim 36, wherein the second vector expresses: (1) the ETV1 gene and the PRDM1 gene; (2) the FOXD1 gene and the PRDM1 gene; or (3) the ETV1 gene, the PRDM1 gene, and the FOXD1 gene.

[0050] 38. A kit of the vector according to claim 34 or 35 or the vector according to claim 32, 36 or 37, wherein the vector is a viral vector.

[0051] 39. A cell preparation comprising cells capable of inducing skin appendages, produced by the method described in any one of 2 to 6 above, and mesenchymal cells capable of inducing skin appendages.

[0052] 40. The cell preparation described in 39 above, wherein the mesenchymal cells capable of inducing skin appendages are cells capable of inducing skin appendages produced by the production method described in any one of 7 to 12 above.

[0053] 41. A cell preparation comprising cells capable of forming skin appendages, produced by the method described in any one of items 7 to 12 above, and epithelial cells capable of forming skin appendages.

[0054] 42. The cell preparation according to claim 41, wherein the epithelial cells capable of inducing skin appendages are cells capable of inducing skin appendages produced by the production method according to any one of claims 2 to 6.

[0055] The present invention provides cells capable of forming tissues with properties equivalent to those of skin, including skin appendages. Such cells capable of reconstituting skin appendages can provide effective medical treatments or cell preparations for treating conditions caused by the loss or dysfunction of skin appendages, such as alopecia, xerosis, and asteatosis, as well as skin ulcers, such as burns, trauma, pressure ulcers, diabetic ulcers, and skin ulcers caused by peripheral circulatory insufficiency. Furthermore, the present invention can generate cells capable of inducing skin appendages from a patient's somatic cells (not limited to skin-derived cells, such as blood or adipose tissue stromal cells), and perform various analyses of the generated cells, thereby contributing to the elucidation of the pathology or treatment of diseases. In particular, cells capable of inducing skin appendages generated from human somatic cells are suitable as materials for drug discovery and development, and are also suitable for confirming drug efficacy. Furthermore, the use of cells capable of inducing skin appendages, which possess the opposing properties of self-renewal and differentiation, can contribute to the elucidation of pathologies originating from stem cells, such as cancer and sarcoma. Furthermore, by transplanting the cells produced by the present invention or applying the present invention to somatic cells in vivo, the cells can be used as a therapeutic means and method to induce regeneration and neogenesis of skin appendages and to satisfy skin appendage function in patients with pathological conditions in which skin and skin appendages are missing, such as skin ulcers, or conditions in which skin appendages and their function have deteriorated due to age-related changes, or after healing of skin ulcers, resulting in a lack of skin appendages.

[0056]

[0033] Figure 1 is a schematic diagram of a transplantation test of adult mouse skin-derived cells, showing the isolation and culture of epithelial cells and mesenchymal cells from adult mice and transplantation into immunodeficient animals. Figure 2 is a photograph showing the results of a transplantation test of adult mouse skin-derived cells (Reference Example 1), showing that transplantation of epithelial cells and mesenchymal cells derived from adult mouse skin did not result in regeneration of skin appendages. Figure 3 is a schematic diagram of a transplantation test of neonatal mouse skin-derived cells, showing the isolation and culture of epithelial cells and mesenchymal cells from neonatal mouse skin and transplantation into immunodeficient animals. Figure 4 is a photograph showing the results of a transplantation test of neonatal mouse skin-derived cells (Reference Example 2), showing that transplantation of epithelial cells and mesenchymal cells derived from neonatal mouse skin resulted in regeneration of skin appendages. Figure 5 is a schematic diagram of a transplantation test of induced epithelial cells prepared by gene transfer into mesenchymal cells derived from adult mouse adipose tissue cells, and mesenchymal cells derived from neonatal mouse skin. Photographs showing the results of a transplantation test (Comparative Example 1) of induced epithelial cells prepared by gene transfer of DNP63A, GRHL2, TFAP2A, and cMYC into mesenchymal cells derived from adult mouse adipose tissue cells, and mesenchymal cells derived from neonatal mouse skin, demonstrating that transplantation of induced epithelial cells prepared by gene transfer of DNP63A, GRHL2, TFAP2A, and cMYC into mesenchymal cells derived from adult mouse adipose tissue cells, and mesenchymal cells derived from neonatal mouse skin, did not result in regeneration of skin appendages. Photographs showing the results of a gene transfer test (Comparative Example 2) of DNP63A, GRHL2, TFAP2A, c-MYC, and SHH into mesenchymal cells derived from adult mouse adipose tissue cells, demonstrating that epithelial cells could not be induced even when DNP63A, GRHL2, TFAP2A, c-MYC, and SHH were gene transferred into mesenchymal cells derived from adult mouse adipose tissue cells. 1 is a photograph showing the results of a gene transfer test (Comparative Example 3) of DNP63A, GRHL2, TFAP2A, c-MYC, and FOXD1 into mouse adult adipose-derived mesenchymal cells, demonstrating that epithelial cells can be induced by gene transfer of DNP63A, GRHL2, TFAP2A, c-MYC, and FOXD1 into mouse adult adipose-derived mesenchymal cells.Photographs showing the results of a transplantation test (Comparative Example 3) of induced epithelial cells prepared by gene transfer of DNP63A, GRHL2, TFAP2A, cMYC, and FOXD1 into mesenchymal cells derived from adult mouse adipose tissue cells, and mesenchymal cells derived from neonatal mouse skin, demonstrating that transplantation of induced epithelial cells prepared by gene transfer of DNP63A, GRHL2, TFAP2A, cMYC, and FOXD1 into mesenchymal cells derived from adult mouse adipose tissue cells, and mesenchymal cells derived from neonatal mouse skin, did not result in regeneration of skin appendages. Photographs showing the results of a gene transfer test (Example 1) of DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into mesenchymal cells derived from adult mouse adipose tissue cells, demonstrating that epithelial cells can be induced by gene transfer of DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into mesenchymal cells derived from adult mouse adipose tissue cells. This photograph shows the results of a transplantation test (Example 1) using induced epithelial cells prepared by transfecting adult mouse adipose tissue cell-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, cMYC, and LEF1, and mesenchymal cells derived from neonatal mouse skin. It demonstrates that transplantation of induced epithelial cells prepared by transfecting adult mouse adipose tissue cell-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, cMYC, and LEF1, and mesenchymal cells derived from neonatal mouse skin, results in skin appendage regeneration. The graph shows changes in marker gene expression (PROM1 gene, CRABP1 gene, and VCAN gene) when cultured human fibroblasts were transfected with candidate factors for inducing mesenchymal cells capable of inducing skin appendages. "ALL" indicates changes in marker gene expression when all candidate factors were transfected simultaneously. Each bar represents the mean, and error bars represent the standard deviation. Statistically significant changes compared to NC (control without gene transfer) are indicated with an * (* p<0.05, ** p<0.01, ***p<0.001). Cultured human fibroblasts were transfected with candidate factors for inducing mesenchymal cells with skin appendage inducibility, and then alkaline phosphatase staining was performed. The figure shows the number of positive cells when all candidate factors were transfected simultaneously.Each bar graph shows the average value, and the error bars show the standard deviation. Changes that were statistically significant compared to NC (control without gene transfection) are indicated with an * (* p<0.05, ** p<0.01, *** p<0.001). The figures show the number of alkaline phosphatase-positive cells when cultured human fibroblasts were transfected with a single candidate factor for inducing mesenchymal cells with skin appendage-inducing ability, and when multiple candidate factors deemed highly effective based on the results of marker gene expression and alkaline phosphatase expression were transfected in combination. A + indicates the addition of the factor, and a - indicates its absence. Each bar graph shows the average value, and the error bars show the standard deviation. Changes that were statistically significant compared to NC (control without gene transfection: result on the far right) are indicated with an * (* p<0.05, ** p<0.01, ***p<0.001). 1 shows a schematic diagram of a transplantation test of induced epithelial cells prepared by genetically introducing DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, and induced mesenchymal cells prepared by genetically introducing candidate factors for inducing mesenchymal cells with skin appendage inducibility into adult mouse adipose-derived mesenchymal cells. 1 shows photographs showing the results of a transplantation test (Example 2-1) of induced epithelial cells prepared by genetically introducing DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, induced mesenchymal cells prepared by genetically introducing ETV1, FOXD1, and PRDM1 into adult mouse adipose-derived mesenchymal cells, and induced mesenchymal cells prepared by genetically introducing SHH and LEF1 into adult mouse adipose-derived mesenchymal cells, demonstrating that skin appendage regeneration can be achieved by transplantation of these induced cells.FIG. 1 is a cross-sectional photograph showing the results of a transplantation test (Example 2-1) of induced epithelial cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1, induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with ETV1, FOXD1, and PRDM1, and induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with SHH and LEF1. Histologically, mature hair shafts (arrow 1), hair matrix-like tissue (arrow 2), and sebaceous gland-like tissue (arrow 3) can be seen. FIG. 1 is a photograph showing the results of a transplantation test (Example 2-2) of induced epithelial cells prepared by introducing DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, induced mesenchymal cells prepared by introducing ETV1, FOXD1, and PRDM1 into adult mouse adipose-derived mesenchymal cells, and induced mesenchymal cells prepared by introducing SHH into adult mouse adipose-derived mesenchymal cells, demonstrating that skin appendage regeneration can be achieved by transplanting these induced cells. FIG. 1 is a photograph showing the results of a transplantation test (Example 2-3) of induced epithelial cells prepared by introducing DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, induced mesenchymal cells prepared by introducing ETV1 and FOXD1 into adult mouse adipose-derived mesenchymal cells, and induced mesenchymal cells prepared by introducing SHH and LEF1 into adult mouse adipose-derived mesenchymal cells, demonstrating that skin appendage regeneration can be achieved by transplanting these induced cells. These photographs show the results of transplantation tests (Example 2-4) of induced epithelial cells prepared by introducing DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, induced mesenchymal cells prepared by introducing FOXD1 and PRDM1 into adult mouse adipose-derived mesenchymal cells, and induced mesenchymal cells prepared by introducing SHH and LEF1 into adult mouse adipose-derived mesenchymal cells, demonstrating that skin appendage regeneration can be achieved by transplanting these induced cells.Photographs showing the results of a transplantation test (Example 2-5) of induced epithelial cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1, induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with ETV1 and PRDM1, and induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with SHH and LEF1, demonstrating that transplantation of these induced cells can achieve regeneration of skin appendages. Photographs showing the results of a transplantation test (Example 2-6) of induced epithelial cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1, and induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with ETV1, FOXD1, PRDM1, and LEF1, demonstrating that transplantation of these induced cells can achieve regeneration of skin appendages.

[0023] Figure 1 shows photographs showing the results of a transplantation test (Example 2-7) of induced epithelial cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1, and induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with ETV1, FOXD1, PRDM1, SHH, and LEF1, demonstrating that transplantation of these induced cells can result in regeneration of skin appendages.

[0024] Figure 1 shows photographs showing the results of a transplantation test (Example 2-8) of induced epithelial cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1, and induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with SHH and LEF1, demonstrating that transplantation of these induced cells can result in regeneration of skin appendages.Photographs showing the results of a transplantation test (Example 2-9) of induced epithelial cells prepared by genetically introducing DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, induced mesenchymal cells prepared by genetically introducing PRDM1 into adult mouse adipose-derived mesenchymal cells, and induced mesenchymal cells prepared by genetically introducing SHH and LEF1 into adult mouse adipose-derived mesenchymal cells, demonstrating that transplantation of these induced cells can achieve regeneration of skin appendages. Photographs showing the results of a transplantation test (Example 2-10) of induced epithelial cells prepared by genetically introducing DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, induced mesenchymal cells prepared by genetically introducing FOXD1 and PRDM1 into adult mouse adipose-derived mesenchymal cells, and induced mesenchymal cells prepared by genetically introducing SHH into adult mouse adipose-derived mesenchymal cells, demonstrating that transplantation of these induced cells can achieve regeneration of skin appendages. (A) is a general image of the transplanted area, and (B) is a magnified image of the hair growth area. These photographs show the results of a transplantation test (Example 2-11) using induced epithelial cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1, induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with PRDM1, and induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with SHH, demonstrating that transplantation of these induced cells can result in regeneration of skin appendages. (A) is a general image of the transplanted area, and (B) is a magnified image of the hair growth area. Photographs showing the results of a transplantation test (Example 2-12) of induced epithelial cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1, induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with FOXD1, and induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with SHH, demonstrating that transplantation of these induced cells can result in regeneration of skin appendages. (A) is a general image of the transplanted area, and (B) is a magnified image of the area where hair growth occurred.

[0023] Figures 1A and 1B are photographs showing the results of a transplantation test (Example 2-13) of induced epithelial cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1, and induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with SHH, demonstrating that transplantation of these induced cells can result in regeneration of skin appendages. (A) is a general image of the transplanted area, and (B) is a magnified image of the hair growth area.

[0024] Figures 1A and 1B are photographs showing the results of a transplantation test (Example 2-14) of induced epithelial cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1, and induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with ETV1, FOXD1, and PRDM1, demonstrating that transplantation of these induced cells can result in the formation of subcutaneous hair follicle-like structures and mature hair shafts. (A) is a photograph of the appearance on day 28 (D28). The dotted line indicates the site where the tissue image was taken. (B) is a cross-sectional photograph of the hematoxylin-eosin (HE) stained tissue image. The dotted square indicates the location of (C). (C) is a magnified image. (D) and (E) are histological images of sections subsequent to (C). The arrow indicates the mature hair shaft, indicating that a continuous shaft was observed within the section. These photographs show the results of a transplantation test (Example 3) using induced epithelial cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, and LEF1; induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with ETV1, FOXD1, and PRDM1; and induced mesenchymal cells prepared by transfecting adult mouse adipose-derived mesenchymal cells with SHH and LEF1. These results demonstrate that transplantation of these induced cells can result in regeneration of skin appendages. (A) is an overall image of the transplanted area, and (B) is an enlarged image of the hair growth area.This figure shows the number of hairs observed at the transplantation site when induced epithelial cells, created by transfecting adult mouse adipose-derived mesenchymal cells with DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1, and one or two types of mesenchymal cells, transfected with a combination of multiple genes relatively highly expressed in mesenchymal cells with skin appendage induction ability, were co-transplanted into a chamber attached to the back of an immunodeficient animal. Results of two experiments were performed for each condition. (A) Silicon chamber mold, (B) prepared silicon chamber, (C) Mouse with silicon chamber attached to its back.

[0057] 1. Method for Producing Cells Capable of Inducing Skin Appendages, and Uses of Cells Capable of Inducing Skin Appendages In the present invention, "cells capable of inducing skin appendages" refer to cells (in other words, epithelial stem cells) that have the ability to function as progenitor cells or stem cells of stratified epithelium, which protects the inside of the body from external factors such as mechanical injury and infection at sites that come into contact with the outside world, such as the epidermis, and that confer reconstitution of skin appendages such as hair follicles and sebaceous glands when transplanted together with appropriate mesenchymal cells, and / or mesenchymal cells that confer reconstitution of skin appendages such as hair follicles and sebaceous glands when transplanted together with appropriate epithelial cells, and also include cells derived from fetal or neonatal skin and cells collected from adult hair follicles and appendages. Furthermore, cells capable of inducing skin appendages also have the ability to form stratified squamous epithelium.

[0058] The method of the present invention for producing cells capable of skin appendage induction is characterized by comprising the step of introducing at least one gene that is expressed relatively more strongly in cells capable of skin appendage induction (e.g., fetal or neonatal skin cells, or cells that constitute skin appendages such as hair follicles and sebaceous glands) into somatic cells that do not have this ability. Hereinafter, the introduced gene will be referred to as a "transgene." The transgene may be multiple genes that are expressed relatively more strongly in cells capable of skin appendage induction, or a gene that is expressed relatively less strongly in cells capable of skin appendage induction may be combined with a gene that is expressed relatively more strongly in cells capable of skin appendage induction.

[0059] Here, "transgene" includes not only genes encoding proteins but also noncoding RNA such as microRNA. Furthermore, "genes that are relatively highly expressed in cells with skin appendage induction ability" refer to genes that have been confirmed to be expressed at higher levels in cells with skin appendage induction ability compared to cells that do not have skin appendage induction ability, such as adult-derived keratinocytes, skin fibroblasts, and adipose-derived mesenchymal cells, by quantitative evaluation of gene expression levels such as real-time PCR, microarrays, and RNA sequencing. Furthermore, "genes that are not relatively highly expressed" refer to genes that are expressed at lower levels in cells with skin appendage induction ability compared to cells that do not have skin appendage induction ability.

[0060] Genes that are relatively highly expressed in epithelial cells with skin appendage inducibility include at least genes encoding the following proteins: DNP63A gene, GRHL2 gene, TFAP2A gene, cMYC gene, and LEF1 gene.

[0061] Genes that are relatively highly expressed in mesenchymal cells with skin appendage inducibility include genes that encode at least the following proteins: SOX2 gene, LEF1 gene, HOXC4 gene, HOXC9 gene, HOXC13 gene, JARID2 gene, HEY1 gene, HEY2 gene, FOXO1 gene, FOXD1 gene, EGR3 gene, MEF2C gene, LHX2 gene, PRRX1 gene, PRRX2 gene, CREB3 gene, ETV1 gene, TFAP2A gene, cMYC gene, TBX6 gene, MSX2 gene, SHH gene, and PRDM1 gene.

[0062] The nucleotide sequences of all genes used in the present invention are known (Table 1). In this specification, NCBI is an abbreviation for the National Center for Biotechnology Information, and the Accession Nos. in Table 1 are also registered in the database provided by NCBI.

[0063]

[0064] Many of these genes are derived from a common source in mammals, including humans, and can be derived from any mammal. However, it is desirable to select an appropriate gene depending on the origin of the somatic cells to be transfected. For example, if the somatic cells to be transfected are derived from humans, the transgene is desirably derived from humans. Furthermore, the transgene may be a mutant gene in which, in addition to the wild-type gene, several amino acids (e.g., 1 to 10, preferably 1 to 6, even more preferably 1 to 4, more preferably 1 to 3, and particularly preferably 1 or 2) have been substituted, deleted, and / or inserted in the amino acid sequence of the gene product, and which encodes a mutant gene product with equivalent functionality to the wild-type gene product. Alternatively, sequences in which codons have been modified and optimized to encode the same amino acids as those encoded by the respective genes may be used.

[0065] In the present invention, the above-mentioned transgene can be prepared according to standard methods based on known sequence information. For example, cDNA of the target gene can be prepared by extracting RNA from mammalian cells and cloning it according to standard methods. Alternatively, the gene can be synthesized as an artificial gene. When synthesizing an artificial gene, codons can be optimized to match the animal from which the somatic cells are to be introduced.

[0066] In the present invention, the "somatic cells" to be induced into cells capable of inducing skin appendages are not particularly limited in type, and cells derived from any tissue or site can be used. Examples of somatic cells used in the present invention include those derived from tissues such as skin, subcutaneous fat, muscle, placenta, bone, cartilage, blood, and corneal stroma. More specifically, dermal fibroblasts, subcutaneous adipose tissue-derived stromal cells (subcutaneous adipocytes), embryonic fibroblasts, adipocytes, muscle cells, osteoblasts, chondrocytes, and circulating mononuclear cells are exemplified. Among these, skin-derived cells, subcutaneous adipose tissue-derived cells, and blood-derived cells are preferred from the viewpoint of minimally invasiveness to the body and more efficiently producing cells capable of inducing skin appendages. Skin fibroblasts, subcutaneous adipose tissue-derived stromal cells, and circulating mononuclear cells are particularly preferred. Thus, the ability to select materials from a variety of cells, particularly easily available cells such as skin-derived cells, subcutaneous adipose tissue-derived cells, and circulating mononuclear cells, offers clinical advantages in terms of reducing the burden on patients and ensuring stable cell availability. Furthermore, commercially available somatic cells may be used as the somatic cells, or somatic cells differentiated from ES cells, mesenchymal stem cells, or the like may also be used.

[0067] Furthermore, the above-mentioned somatic cells are appropriately selected from those derived from mammals such as humans, mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, and monkeys depending on the intended use of the cells capable of inducing skin appendages. However, when the cells are to be used for purposes targeting humans, such as human treatment, elucidation of pathological conditions, or confirmation of drug efficacy, somatic cells derived from humans are preferred. Furthermore, when human-derived somatic cells are used, they may be derived from fetuses, infants, children, or adults. When the cells capable of inducing skin appendages are to be used for purposes such as human treatment, elucidation of pathological conditions, or confirmation of drug efficacy, it is desirable to use somatic cells collected from patients.

[0068] The introduction of the transgene into somatic cells can be carried out using a method commonly used in transfection of animal cells. Specifically, examples of methods for introducing the transgene into somatic cells include methods using vectors; calcium phosphate methods; lipofection methods; electroporation methods; and microinjection methods. Among these, methods using vectors are preferred in terms of introduction efficiency. When using vectors to introduce the transgene into somatic cells, viral vectors, non-viral vectors (including plasmid (DNA) vectors and mRNA vectors), and artificial viruses can be used as vectors. However, viral vectors such as adeno-associated viruses, retroviruses, and lentiviruses are preferably used from the standpoint of safety. When vectors are used and multiple transgenes are used, each transgene may be incorporated into a separate vector, or two or more transgenes may be incorporated into a single vector.

[0069] Thus, the somatic cells into which the transgene has been introduced can be induced to become cells capable of inducing skin appendages.

[0070] It is also possible to induce cells capable of inducing skin appendages from somatic cells in vivo by introducing a transgene into somatic cells present in the body using the above-mentioned gene transfer means or vectors. The transgene may be a gene that is highly expressed relatively in epithelial cells capable of inducing skin appendages, or a gene that is highly expressed relatively in mesenchymal cells capable of inducing skin appendages, but it is preferable to introduce both genes.

[0071] [Induced Epithelial Cells] The method for producing cells capable of skin appendage induction of the present invention, particularly epithelial cells, preferably comprises the step of introducing transgenes containing (1) the LEF1 gene and (2) one, two, or three genes selected from the group consisting of the DNP63A gene, the GRHL2 gene, and the TFAP2A gene into somatic cells that do not have the ability to induce skin appendages. In particular, the method for producing epithelial cells capable of skin appendage induction of the present invention preferably comprises the step of introducing transgenes containing the four genes, the LEF1 gene, the DNP63A gene, the GRHL2 gene, and the TFAP2A gene, into somatic cells that do not have the ability to induce skin appendages.

[0072] Furthermore, the method for producing cells capable of skin appendage induction of the present invention, particularly epithelial cells, preferably comprises the step of introducing a transgene containing (1) the LEF1 gene and (2) at least one, two, three, or four genes selected from the group consisting of the DNP63A gene, the GRHL2 gene, the TFAP2A gene, and at least one of the MYC family genes into somatic cells that do not have the ability to produce skin appendages. In particular, the method for producing epithelial cells capable of skin appendage induction of the present invention preferably comprises the step of introducing a transgene containing at least five genes, the LEF1 gene, the DNP63A gene, the GRHL2 gene, the TFAP2A gene, and at least one of the MYC family genes, into somatic cells that do not have the ability to produce skin appendages.

[0073] Examples of Myc family genes include c-Myc, N-Myc, and L-Myc. These Myc family genes may be used alone or in combination. The c-Myc gene is preferably used as the Myc family gene.

[0074] Furthermore, as seen in the method for inducing stratified squamous epithelium formation ability in Non-Patent Document 15, epithelial cells with skin appendage induction ability can also be produced by adding another gene to the above four or five genes. In other words, the transgene contains at least one of the above four or five genes, and another gene (preferably one that does not inhibit the expression of stratified squamous epithelium formation ability and skin appendage induction ability) may also be added.

[0075] Cells induced into epithelial cells capable of inducing skin appendages can be selected based on their proliferation ability under culture conditions suitable for keratinocyte isolation and expansion, and their epithelial cell characteristics. These epithelial cells exhibit relatively high proliferation potential when cultured on feeder cells suitable for keratinocyte isolation and expansion (e.g., 3T3-J2 feeder cells, 3T3 cells, mouse embryonic fibroblasts, human dermal fibroblasts, etc., inactivated by mitomycin C or radiation treatment) or in serum-free keratinocyte medium, allowing for selection by continuous passaging. Addition of a Rho kinase inhibitor (e.g., Y27632) is also effective in increasing the number of keratinocyte divisions on feeders. Furthermore, the purity of epithelial cells capable of inducing skin appendages can be increased by cell separation using flow cytometry or a magnetic cell sorter, using epithelial cell-specific surface antigens (e.g., CDH1, Epi-CAM). Furthermore, if a reporter gene construct made by linking a drug resistance gene to the promoter of an epithelial cell marker gene (e.g., CDH1, Epi-CAM) is introduced into somatic cells in advance, cells that have acquired the characteristics of epithelial cells will be able to grow in the presence of drugs, and therefore growth in the presence of drugs can be used as an indicator for selection.

[0076] The induced epithelial cells thus obtained, capable of inducing skin appendages, can proliferate when cultured on feeders in liquid medium supplemented with a Rho kinase inhibitor, and can stably proliferate while maintaining their skin appendage inducibility for up to approximately 10 passages, as is typically done. Media typically used for culturing animal cells can be used to culture the induced epithelial cells. An example of a suitable medium for culturing epithelial cells capable of inducing skin appendages is serum-free keratinocyte medium (Keratinocyte-SFM, Life Technologies). It is also useful to add cytokines, such as bFGF, or various pharmacologically active substances that accelerate keratinocyte proliferation under culture conditions.

[0077] The thus obtained induced epithelial cells capable of inducing skin appendages can be mixed and transplanted with neonatal animal skin-derived mesenchymal cells capable of inducing skin appendages into a chamber attached to the back of an immunodeficient animal, resulting in the reconstruction and regeneration of skin appendages. In contrast, when adult-derived skin cells or induced epithelial cells not capable of inducing skin appendages (for example, cells capable of forming stratified squamous epithelium induced by introducing the DNP63A gene, GRHL2 gene, TFAP2A gene, or cMYC gene into somatic cells using the method described in Non-Patent Document 15) are mixed and transplanted with neonatal animal skin-derived mesenchymal cells capable of inducing skin appendages into a chamber attached to the back of an immunodeficient animal, no reconstruction or regeneration of skin appendages can be achieved.

[0078] Similarly, epithelial cells derived from neonatal animals and cells isolated from adult skin appendages can reconstruct and regenerate skin appendages when mixed with mesenchymal cells derived from neonatal animal skin that have the ability to induce skin appendages and transplanted. However, these cells isolated from animals are often contaminated with mesenchymal cells that have the ability to induce skin appendages. In other words, it has been difficult to isolate only epithelial cells with the ability to induce skin appendages from mesenchymal cells with the ability to induce skin appendages. In contrast, the induced epithelial cells thus obtained have the ability to induce skin appendages, but are not contaminated with mesenchymal cells with the ability to induce skin appendages. Taking advantage of this property, mixed transplantation of induced epithelial cells with the ability to induce skin appendages and mesenchymal cells whose ability to induce skin appendages is unknown can be used as an evaluation system to determine whether mesenchymal cells have the ability to induce skin appendages.

[0079] The induced epithelial cells thus obtained, which have the ability to induce skin appendages, can be transplanted into patients with conditions in which there is a quantitative or qualitative deficiency of skin appendages, such as those in the area of ​​skin ulcers, conditions in which skin appendages and their functions have deteriorated due to age-related changes, or after healing of skin ulcers, thereby providing a therapeutic method for regenerating and neogenesis of skin and skin appendages.

[0080] In Non-Patent Document 15, cells capable of forming stratified squamous epithelium are induced from somatic cells in the body that do not have the ability to form stratified squamous epithelium by introducing a gene that induces cells capable of forming stratified squamous epithelium into somatic cells present in the body. Similarly, it is also possible to induce epithelial cells capable of inducing skin appendages from somatic cells in the body by introducing an introduced gene into somatic cells present in the body using the above-mentioned gene introduction means or vector.

[0081] In other words, gene transfer means and vectors that transfer genes that induce epithelial cells with skin appendage induction ability into somatic cells present in the body can be used to transfer genes to patients with pathological conditions in which there is a lack of skin and skin appendages, such as skin ulcers, or conditions in which there is a decline in skin appendages and their function due to age-related changes, or after healing of a skin ulcer, thereby inducing epithelial cells with skin appendage induction ability, thereby providing a treatment method for regenerating and neogenesis of skin and skin appendages.

[0082] [Induced Mesenchymal Cells] The method of the present invention for producing cells capable of inducing skin appendages, particularly mesenchymal cells, preferably comprises the step of introducing at least one gene selected from (1) the PRDM1 gene, (2) the FOXD1 gene, (3) the ETV1 gene, (4) the LEF1 gene, and (5) the SHH gene into somatic cells that do not have the ability to induce skin appendages.

[0083] The method of the present invention for producing cells capable of inducing skin appendages, particularly mesenchymal cells, preferably comprises the step of transferring at least the SHH gene into somatic cells that do not have the ability to inducibly produce skin appendages.

[0084] The method of the present invention for producing cells capable of inducing skin appendages, particularly mesenchymal cells, preferably comprises the step of transferring at least the SHH gene and the LEF1 gene into somatic cells that do not have the ability to inducibly produce skin appendages.

[0085] It is also preferred that the method of the present invention for producing cells capable of inducing skin appendages, particularly mesenchymal cells, includes a step of introducing at least one gene selected from (1) the PRDM1 gene, (2) the FOXD1 gene, and (3) the ETV1 gene into somatic cells that do not have the ability to induce skin appendages.

[0086] It is also preferred that the method of the present invention for producing cells capable of inducing skin appendages, particularly mesenchymal cells, comprises the step of introducing (1) the SHH gene and (2) one or more genes selected from the ETV1 gene, PRDM1 gene, FOXD1 gene, and LEF1 gene into somatic cells that do not have the ability to induce skin appendages.

[0087] It is also preferred that the method of the present invention for producing cells capable of inducing skin appendages, particularly mesenchymal cells, comprises the step of introducing (1) the SHH gene, or the SHH gene and the LEF1 gene, and (2) one or more genes selected from the ETV1 gene, the PRDM1 gene, and the FOXD1 gene, into somatic cells that do not have the ability to induce skin appendages.

[0088] It is also preferred that the method of the present invention for producing cells capable of inducing skin appendages, particularly mesenchymal cells, includes a step of introducing (1) the ETV1 gene and the PRDM1 gene; (2) the FOXD1 gene and the PRDM1 gene; or (3) the ETV1 gene, the PRDM1 gene, and the FOXD1 gene into somatic cells that do not have the ability to induce skin appendages.

[0089] Furthermore, as seen in the method for inducing the ability to form stratified squamous epithelium in Non-Patent Document 15, mesenchymal cells with the ability to induce skin appendages can also be produced by adding other genes to the genes described above.

[0090] The thus obtained induced mesenchymal cells capable of inducing skin appendages can be cultured in, for example, DMEM medium supplemented with 10% FBS (fetal bovine serum) or Advanced-DMEM medium supplemented with N2 supplement. It is also useful to add cytokines such as bFGF, which accelerate the proliferation of mesenchymal cells under culture conditions, or various pharmacologically active substances.

[0091] The thus obtained induced mesenchymal cells capable of inducing skin appendages (for example, induced mesenchymal cells in which the PRDM1 gene and the FOXD1 gene have been introduced into mesenchymal cells that do not have the ability to induce skin appendages, or induced mesenchymal cells in which the LEF1 gene and the SHH gene have been introduced into mesenchymal cells that do not have the ability to induce skin appendages) can be mixed and transplanted with the above-mentioned epithelial cells capable of inducing skin appendages into a chamber attached to the back of an immunodeficient animal, thereby reconstructing and regenerating skin appendages. In contrast, mixed transplantation of adult-derived skin mesenchymal cells with epithelial cells capable of inducing skin appendages into a chamber attached to the back of an immunodeficient animal does not result in the reconstruction or regeneration of skin appendages.

[0092] The thus obtained induced mesenchymal cells capable of skin appendage induction can be mixed and transplanted with epithelial cells derived from the skin of a newborn animal that have the ability to induce skin appendages into a chamber attached to the back of an immunodeficient animal, thereby reconstructing and regenerating skin appendages. Similarly, early mesenchymal cells cultured in primary culture from newborn animals and cells isolated from adult skin appendages can also be mixed and transplanted with epithelial cells capable of skin appendage induction, thereby reconstructing and regenerating skin appendages. However, these cells isolated from animals may be contaminated with epithelial cells capable of skin appendage induction. In contrast, the thus obtained induced mesenchymal cells capable of skin appendage induction have the ability to induce skin appendages, but are not contaminated with epithelial cells with the ability to induce skin appendages. Taking advantage of this property, mixed transplantation of induced mesenchymal cells capable of skin appendage induction with epithelial cells whose ability to induce skin appendages is unknown can be used as an evaluation system for determining whether the epithelial cells have the ability to induce skin appendages.

[0093] The induced mesenchymal cells thus obtained, which have the ability to induce skin appendages, can be transplanted into patients with pathological conditions in which there is a loss of skin and skin appendages, such as on the surface of skin ulcers, or conditions in which there is a quantitative or qualitative deficiency of skin appendages, such as conditions in which skin appendages and their functions have deteriorated due to age-related changes, or after healing of skin ulcers, providing a therapeutic method for regenerating and neogenesis of skin and skin appendages.

[0094] In Non-Patent Document 15, cells capable of forming stratified squamous epithelium are induced from somatic cells in the body that do not have the ability to form stratified squamous epithelium by introducing a gene that induces cells capable of forming stratified squamous epithelium into somatic cells present in the body. Similarly, it is also possible to induce mesenchymal cells capable of inducing skin appendages from somatic cells in the body by introducing an introduced gene into somatic cells present in the body using the above-mentioned gene introduction means or vector.

[0095] That is, gene transfer means and vectors for transferring genes that induce mesenchymal cells with skin appendage inducibility into somatic cells present in the body can be used to transfer genes into patients with pathological conditions in which skin and skin appendages are missing, such as skin ulcers, or conditions in which skin appendages and their functions have decreased due to age-related changes, or after healing of a skin ulcer, thereby inducing mesenchymal cells with skin appendage inducibility, thereby providing a therapeutic method for regenerating and neogenesis of skin and skin appendages. Therefore, gene transfer means and vectors for transferring genes that induce mesenchymal cells with skin appendage inducibility can be used as compositions for preparing such therapeutic preparations.

[0096] As described above, the epithelial cells and mesenchymal cells capable of inducing skin appendages obtained by the present invention have the ability to proliferate and are capable of regenerating skin appendages in vivo, and are therefore effective in treating skin ulcers caused by burns, trauma, iatrogenic injuries (after tumor resection, etc.), pressure ulcers, diabetic ulcers, and skin ulcers due to peripheral circulatory insufficiency, and can be used as a cell preparation (pharmaceutical composition) for regenerating skin and skin appendage tissues.

[0097] When the epithelial cells and mesenchymal cells having skin appendage inducibility are prepared as a cell preparation for regenerating skin and skin appendage tissues, the cell preparation may contain, as needed, a pharmaceutically acceptable diluent carrier in addition to the epithelial cells and mesenchymal cells having skin appendage inducibility. Examples of pharmaceutically acceptable diluent carriers include physiological saline and buffer solutions. Furthermore, the cell preparation may, as needed, contain pharmacologically active ingredients or ingredients that serve as nutrient sources for the epithelial cells and mesenchymal cells having skin appendage inducibility.

[0098] The epithelial cells and mesenchymal cells capable of inducing skin appendages may be cultured to form skin and skin appendage-like tissues, which can be used as tissue-engineered preparations to treat skin diseases. Tissue-engineered preparations include cell aggregates such as sheet-like structures (e.g., epithelial cell sheets) and three-dimensional organ primordia.

[0099] When the above-mentioned epithelial cells and mesenchymal cells having skin appendage induction ability are prepared as tissue engineering preparations for regenerating skin and skin appendage tissues, the epithelial cells and mesenchymal cells having skin appendage induction ability may be used in combination with pharmacologically active ingredients or ingredients that serve as a nutrient source for the epithelial cells and mesenchymal cells having skin appendage induction ability, if necessary.

[0100] The epithelial cells and mesenchymal cells having the ability to induce skin appendages may be used as a scaffolding of an extracellular matrix such as collagen containing mesenchymal cells such as skin fibroblasts and adipose tissue-derived stromal cells to prepare cell aggregates such as sheet-like structures or three-dimensional structures of skin and skin appendage-like tissues or organ primordia, and then applied to the affected skin site.

[0101] When the epithelial cells and mesenchymal cells having the ability to induce skin appendages are prepared as skin and skin appendage-like tissues having a three-dimensional structure for regenerating skin and skin appendages, the epithelial cells and mesenchymal cells having the ability to induce skin appendages may be used in combination with pharmacologically active ingredients or ingredients that serve as a nutrient source for the epithelial cells and mesenchymal cells having the ability to induce skin appendages, as needed. The use of such a scaffold material enables the regeneration of skin and skin appendages at the transplant site more quickly.

[0102] Usable scaffold materials are not particularly limited as long as they are pharmaceutically acceptable and are appropriately selected depending on the site of cartilage tissue to which they are applied, but examples include gel-like, biocompatible materials. Preferred examples of usable scaffold materials include collagen, fibronectin, hyaluronic acid, matrigel, and complexes thereof. These scaffold materials may be used alone or in combination of two or more.

[0103] Furthermore, the shape of the scaffold material is not particularly limited, and may be appropriately designed depending on the shape of the damaged area of ​​the skin and skin appendage tissues to which the cell preparation is to be applied.

[0104] The method of applying the cell preparation to the diseased site of the epithelial tissue is determined appropriately depending on the type of cell preparation, the site of the skin tissue to which it is applied, etc., and examples include a method of directly spraying the cell preparation at the site of the skin ulcer to be treated, a method of directly spraying a tissue-like three-dimensional structure formed under culture conditions, and a method of suturing and fixing a sheet or three-dimensional structure similar to skin grafting.

[0105] Furthermore, the dosage of the cell preparation to be applied to the diseased area of ​​skin tissue can be appropriately determined to be an amount effective for regenerating skin and skin appendage tissue based on the type of cell preparation, the area of ​​epithelial tissue, the severity of symptoms, the age and sex of the patient, etc.

[0106] Furthermore, a non-human mammal having skin and skin appendage tissue formed from the cells having induced skin and skin appendages, after administration of the epithelial cells or mesenchymal cells capable of skin appendage induction, can be used as a tool for evaluating and analyzing the efficacy of a test substance on the skin and / or skin appendage tissue. Specifically, the efficacy of a test substance on the skin and / or skin appendage tissue can be evaluated and analyzed by administering a test substance to a non-human mammal having skin and skin appendage tissue formed from the epithelial cells or mesenchymal cells capable of skin appendage induction and determining and analyzing the efficacy of the test substance on the skin and / or skin appendage tissue. Here, the test substance refers to a substance whose efficacy on the skin and / or skin appendage tissue is to be evaluated and analyzed, and specifically includes candidate substances for therapeutic agents for skin and / or skin appendage diseases. Examples of non-human mammals include mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, etc.

[0107] Furthermore, non-human mammals having skin and skin appendage tissues formed from epithelial cells and mesenchymal cells with the above-mentioned skin appendage induction ability can be used as models for investigating the effects on the skin and / or skin appendage tissues of external factors that damage the skin and / or skin appendage tissues, such as anticancer drugs and radiation.

[0108] Furthermore, three-dimensional structures prepared using scaffolds such as collagen gel containing epithelial cells and mesenchymal cells capable of skin appendage induction can be used as tools for evaluating and analyzing the efficacy of test substances on skin and / or skin appendage tissues. Specifically, the efficacy of a test substance on skin and / or skin appendage tissues can be evaluated and analyzed by administering the test substance to a three-dimensional structure prepared using scaffolds such as collagen gel containing epithelial cells, mesenchymal cells, and skin fibroblasts capable of skin appendage induction and determining and analyzing the efficacy of the test substance on the skin and / or skin appendage tissues. Here, the test substance refers to a substance whose efficacy on skin and skin appendage tissues is to be evaluated and analyzed, and specifically includes candidate substances for therapeutic agents for skin and / or skin appendage diseases. In particular, by using epithelial cells and mesenchymal cells capable of skin appendage induction induced from somatic cells that can be collected with relatively little invasiveness, such as peripheral blood circulating mononuclear cells, it is possible to broadly evaluate and analyze the efficacy of the test substance on a large number of donors with diverse genetic backgrounds.

[0109] Furthermore, the epithelial cells and mesenchymal cells capable of skin appendage induction can be used as tools for elucidating and analyzing various pathologies of skin and skin appendage tissues. Furthermore, epithelial cells and mesenchymal cells capable of skin appendage induction induced from human somatic cells are also useful as tools for drug discovery and development for skin and skin appendage diseases. For example, epithelial cells and mesenchymal cells capable of skin appendage induction can be produced by introducing a transgene into somatic cells extracted from the human body, and these cells can then be cultured to produce skin and skin appendage-like tissues. A test substance can then be administered to the skin and skin appendage-like tissues to evaluate and analyze the efficacy of the test substance on the skin and skin appendage-like tissues, or stress from an anticancer drug, radiation, or the like can be applied to evaluate and analyze the stress on the skin and skin appendage tissues. Evaluation and analysis of the efficacy or stress may be confirmed, for example, by comparing tissues administered or stressed with a test substance with tissues not administered or stressed.

[0110] 2. Experimental Results The present invention will be described in detail below based on examples, but the present invention is not limited to these.

[0111] [Reference Example 1] Transplantation experiment of mouse adult skin-derived epithelial cells and mouse adult skin-derived mesenchymal cells. Skin samples obtained from the backs of adult mice were treated with 0.25% trypsin at 4°C overnight, and the epidermal tissue detached from the dermis and cells collected from the dermal surface were seeded on 3T3-J2 feeder cells in keratinocyte F medium containing the Rho-kinase inhibitor Y27632 (SELLECK) to obtain primary cultured mouse adult skin-derived epithelial cells. Meanwhile, skin samples obtained from the lumbar backs of adult mice were treated with 0.25% trypsin at 4°C overnight, and the dermal tissue detached from the epidermal tissue or subcutaneous adipose tissue was treated with 0.1% collagenase at 37°C for 1 hour, and the collected cells were seeded in DMEM medium supplemented with 10% newborn bovine serum to obtain primary cultured mouse adult skin-derived mesenchymal cells. Primary cultured epithelial cells and primary mesenchymal cells derived from adult mouse skin, cultured for 1-3 days, were transplanted into silicone chambers attached to the backs of immunodeficient animals. Following standard procedures for skin appendage reconstruction assays using silicone chambers, a hole was drilled in the top of the chamber one week after transplantation, and the chamber was removed two weeks after transplantation. Observations were continued for 4-5 weeks after transplantation (Figure 1). The chamber area was epithelialized by the transplanted cells, but no reconstruction or regeneration of skin appendages was observed (Figure 2).

[0112] The 3T3-J2 feeder cells used as feeders are a cell line provided by J-TEC Co., Ltd. They were maintained in a 3T3 cell medium containing 10% newborn bovine serum in DMEM medium according to standard methods, and the day before use as feeder cells, they were treated with 10 μg / ml mitomycin C in medium for 1 hour, and then passaged at a density of 2.0 × 10 cells / well for use as feeders.

[0113] [Reference Example 2] Transplantation experiment of neonatal mouse skin-derived epithelial cells and neonatal mouse skin-derived mesenchymal cells Skin samples obtained from the backs of neonatal mice were treated overnight with 0.25% trypsin at 4°C, and the epidermal tissue detached from the dermis and cells collected from the dermal surface were seeded on 3T3-J2 feeder cells in keratinocyte F medium containing the Rho-kinase inhibitor Y27632 (SELLECK) to obtain primary cultured mouse epithelial cells. Meanwhile, skin samples obtained from the backs of neonatal mice were treated overnight with 0.25% trypsin at 4°C, and the dermal tissue detached from the epidermal tissue and subcutaneous adipose tissue were treated with 0.1% collagenase at 37°C for 1 hour, and the collected cells were seeded in DMEM medium supplemented with 10% neonatal bovine serum to obtain primary cultured mouse mesenchymal cells. Primary epithelial cells derived from neonatal mouse skin and primary mesenchymal cells derived from neonatal mouse skin tissue were cultured for 1-3 days and transplanted into a silicon chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicon chamber, and the silicon chamber was removed two weeks after transplantation. Observation was continued for 4-5 weeks after transplantation (Figure 3). Hair growth and reconstitution and regeneration of skin appendages were observed in the chamber area (Figure 4).

[0114] Comparative Example 1: Transplantation Experiment of Induced Epithelial Cells Capable of Forming Stratified Squamous Epithelium and Neonatal Mouse Skin-Derived Mesenchymal Cells. Primary cultured mesenchymal cells derived from adult mouse adipose tissue were obtained by treating adult mouse subcutaneous adipose tissue with 0.1% collagenase for 1 hour at 37°C and seeding the harvested cells in DMEM medium supplemented with 10% neonatal bovine serum. After two passages, induced epithelial cells capable of forming stratified squamous epithelium were generated by gene transfer of the DNP63A gene, GRHL2 gene, TFAP2A gene, and c-MYC gene using an adeno-associated virus vector (AAV). The induced epithelial cells and primary cultured mesenchymal cells derived from neonatal mouse skin tissue were transplanted into a silicone chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber. Two weeks after transplantation, the silicone chamber was removed, and observations were continued for 4–5 weeks after transplantation (Figure 5). Although the chamber site was epithelialized by the transplanted cells, no reconstruction or regeneration of the skin appendages was achieved (FIG. 6).

[0115] [Comparative Example 2] In order to develop a method for inducing epithelial cells capable of inducing skin appendages from primary cultured mesenchymal cells derived from mouse adult adipose tissue by gene transfer, primary cultured mesenchymal cells derived from mouse adult adipose tissue were passaged twice, and then the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and SHH gene were transferred using AAV, but no induced epithelial cells were obtained (Figure 7).

[0116] [Comparative Example 3] In order to develop a method for inducing epithelial cells capable of inducing skin appendages from mouse adult adipose-derived mesenchymal primary culture cells by gene transfer, mouse adult adipose-derived mesenchymal primary culture cells were passaged twice, and then the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and FOXD1 gene were transferred using AAV, and induced epithelial cells were obtained (Figure 8).

[0117] The induced epithelial cells thus obtained and primary cultured mesenchymal cells derived from neonatal mouse skin tissue were transplanted into a silicon chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed. Observation was continued for 4-5 weeks after transplantation. The chamber site was epithelialized by the transplanted cells, but reconstruction or regeneration of skin appendages was not observed (Figure 9).

[0118] Example 1 Transplantation experiment of induced epithelial cells with skin appendage induction ability and mesenchymal cells derived from neonatal mouse skin To develop a method for inducing epithelial cells with skin appendage induction ability from primary cultured mesenchymal cells derived from mouse adult adipose tissue by gene transfer, primary cultured mesenchymal cells derived from mouse adult adipose tissue were passaged twice, and then the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene were transferred using AAV, resulting in induced epithelial cells (Figure 10).

[0119] The induced epithelial cells thus obtained and primary cultured mesenchymal cells derived from mouse neonatal skin tissue were transplanted into a silicon chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed. Observation was continued for 4 to 5 weeks after transplantation. Hair growth and reconstitution and regeneration of skin appendages were observed at the chamber site (Figure 11). The induced epithelial cells thus obtained fulfilled the properties of induced epithelial cells with the ability to induce skin appendages. Details are described in Example 1 below.

[0120] Comparative Example 4 Transplantation experiment of epithelial cells derived from neonatal mouse skin and induced mesenchymal cells Induced cells are obtained by gene transfer into primary cultured mesenchymal cells derived from adult mouse adipose tissue. When co-transplanted with epithelial cells capable of inducing skin appendages, these induced cells induce mesenchymal cells that reconstruct and regenerate skin appendages. As candidate genes for inducing mesenchymal cells with skin appendage inducibility, genes that are relatively highly expressed in mesenchymal cells with skin appendage inducibility (SOX2 gene, LEF1 gene, HOXA9 gene, HOXC4 gene, HOXC9 gene, HOXC13 gene, JARID2 gene, HEY1 gene, HEY2 gene, FOXO1 gene, FOXD1 gene, EGR3 gene, MEF2C gene, LHX2 gene, PRRX1 gene, CREB3 gene, ETV1 gene, TFAP2A gene, cMYC gene, TBX6 gene, MSX2 gene, SHH gene, PRDM1 gene) were selected.

[0121] We considered primary cultured epithelial cells derived from neonatal mouse skin to be epithelial cells capable of skin appendage induction. Epithelial cells were cultured for 2-3 passages to remove contaminating mesenchymal cells derived from neonatal mouse skin during primary culture. Primary mesenchymal cells derived from adult mouse adipose tissue were passaged twice and then transplanted into a silicone chamber attached to the back of immunodeficient animals together with cells transfected with a retroviral vector containing a combination of genes relatively highly expressed in mesenchymal cells capable of skin appendage induction. While some mice showed a small amount of hair growth and regeneration of skin appendages at the chamber site, no consistent trend was observed depending on the transgene. Therefore, it was highly likely that the primary cultured epithelial cells derived from neonatal mouse skin, cultured for 2-3 passages to remove mesenchymal cells derived from neonatal mouse skin, were actually contaminated with mesenchymal cells derived from neonatal mouse skin. It was difficult to obtain epithelial cells free of mesenchymal contamination while maintaining the skin appendage induction ability of primary cultured epithelial cells derived from neonatal mouse skin.

[0122] In this regard, induced epithelial cells obtained by transfecting mouse adult adipose-derived primary mesenchymal cells with the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene (Examples 1 and 2) or the DNP63A gene, GRHL2 gene, TFAP2A gene, and LEF1 gene (Example 3) have the ability to induce skin appendages, but are not contaminated with mesenchymal cells with skin appendage induction ability. Utilizing this property, as described in Examples 2 and 3, by co-transplanting the cells with induced epithelial cells with skin appendage induction ability, they could be used to evaluate whether certain mesenchymal cells have the ability to induce skin appendages.

[0123] Example 2 Transplantation Experiment of Induced Epithelial Cells Capable of Inducing Skin Appendages and Induced Mesenchymal Cells Capable of Inducing Skin Appendages First, the SHH gene and LEF1 gene were selected as candidates, as studies using genetically modified animals have suggested that they are important environmental factors for hair follicle regeneration. Mouse adult adipose-derived primary mesenchymal cells were passaged twice, and then transfected with the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene using AAV to create induced epithelial cells capable of inducing skin appendages (Example 1). Mouse adult adipose-derived primary mesenchymal cells were passaged twice, and then transfected with mesenchymal cells transfected with the SHH gene alone using a retroviral vector (Example 2-13) or mesenchymal cells transfected with a combination of the SHH gene and the LEF1 gene (Example 2-8) were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed. Observation was continued for 4 to 5 weeks after transplantation. A small amount of hair growth was observed in the chamber area (Figures 24 and 29).

[0124] Next, to further identify factors that induce mesenchymal cells with skin appendage-inducing ability, which, when co-transplanted with induced epithelial cells with skin appendage-inducing ability, would result in skin appendage reconstruction and regeneration, we examined the changes in expression of marker genes (PROM1, CRABP1, and VCAN) for mesenchymal cells with skin appendage-inducing ability (Figure 12) and alkaline phosphatase expression (Figure 13) upon gene transfer of each candidate gene. Since single gene transfer enhanced marker gene and alkaline phosphatase expression, FOXD1, HEY1, HEY2, ETV1, and TFAP2A genes were selected as likely candidates. Furthermore, we examined the changes in alkaline phosphatase expression upon gene transfer of multiple candidate genes in combination (Figure 14) to guide the selection of candidate genes. Through this series of analyses, we ultimately concluded that ETV1 and FOXD1 were particularly effective factors for inducing induced mesenchymal cells with skin appendage-inducing ability, and their effectiveness was confirmed in subsequent in vivo transplantation experiments.

[0125] To select factors that induce mesenchymal cells with skin appendage induction capacity through a mechanism different from that of the SHH gene, LEF1 gene, and environmental factors, the ETV1 gene, FOXD1 gene, and PRDM1 gene were selected as candidates. Primary mesenchymal cells derived from mouse adult adipose tissue were passaged twice, and then transfected with the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene using AAV to create induced epithelial cells with skin appendage induction capacity (Example 1). Primary mesenchymal cells derived from mouse adult adipose tissue were passaged twice, and then transfected with a combination of the ETV1 gene, FOXD1 gene, and PRDM1 gene using a retroviral vector (Example 2-14). These cells were then co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber. Two weeks after transplantation, the silicone chamber was removed. The animals were then observed for 4 to 5 weeks after transplantation. Histological examination revealed hair follicle-like structures and mature hair shafts in the subcutaneous tissue at the center of the cell transplantation area, in an area where hair is not normally found (Figure 30).

[0126] It was thought that by introducing a combination of the ETV1 gene, FOXD1 gene, and PRDM1 gene, mesenchymal cells with the ability to induce skin appendages could be induced through a mechanism different from that of the SHH gene, LEF1 gene, or environmental factors.This suggests that transplanting multiple types of cells introduced with these genes may be able to more efficiently induce hair growth and reconstruct and regenerate skin appendages.

[0127] To select factors for inducing mesenchymal cells with skin appendage-inducing ability that, when co-transplanted with induced epithelial cells with skin appendage-inducing ability, would reconstruct and regenerate skin appendages, induced epithelial cells with skin appendage-inducing ability (Example 1) were prepared by transfecting primary mesenchymal cells derived from adult mouse adipose tissue with the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene using AAV. This was followed by co-transplantation of one or two types of mesenchymal cells transfected with a combination of multiple genes relatively highly expressed in mesenchymal cells with skin appendage-inducing ability using a retroviral vector into a chamber attached to the back of an immunodeficient animal (Figure 15). One week after transplantation, a hole was drilled in the top of the silicone chamber. Two weeks after transplantation, the silicone chamber was removed, and observations were continued for 4–5 weeks after transplantation. The genes transfected into the first and second induced mesenchymal cells are listed in Table 2. In animals co-transplanted with one or two types of induced mesenchymal cells transfected with the 13 combinations described in Examples 2-1 to 2-13, hair growth and reconstruction and regeneration of skin appendages were observed at the chamber site.

[0128]

[0129] Two experiments were performed for each transgene combination, and the number of hairs observed to grow under a stereomicroscope was counted. The results showed that combining transgenes and cells resulted in greater hair growth and the reconstruction and regeneration of skin appendages. Figure 32 shows the results of the number of hairs grown in each of the two experiments in Examples 2-1 to 2-13. It was also found that even when using the same combination of multiple transgenes, better results could be obtained by preparing and transplanting multiple induced mesenchymal cells. For example, compared to the first induced mesenchymal cells in Example 2-6, which were transfected with the ETV1 gene, FOXD1 gene, PRDM1 gene, and SHH gene, the transplantation of a single cell was superior to the transplantation of a first induced mesenchymal cell transfected with the SHH gene and a second induced mesenchymal cell transfected with the ETV1 gene, FOXD1 gene, and PRDM1 gene.

[0130] It was confirmed that the induced mesenchymal cells thus obtained, when co-transplanted with induced epithelial cells capable of inducing skin appendages, fulfill the properties of mesenchymal cells capable of inducing skin appendages, thereby reconstructing and regenerating skin appendages.

[0131] Example 3: Furthermore, induced epithelial cells were generated by transfecting mouse adult adipose-derived primary mesenchymal cells with the DNP63A gene, GRHL2 gene, TFAP2A gene, and LEF1 gene using AAV after two passages, and mesenchymal cells transfected with the ETV1 gene, FOXD1 gene, and PRDM1 gene using retroviral vectors, and mesenchymal cells transfected with the LEF1 gene and SHH gene using retroviral vectors after two passages, and these cells were co-transfected into a chamber attached to the back of an immunodeficient animal (Figure 15). One week after transplantation, a hole was drilled in the top of the silicone chamber, and the silicone chamber was removed two weeks after transplantation. Observation was continued for 4 to 5 weeks after transplantation. Hair growth and reconstitution and regeneration of skin appendages were observed at the chamber site, confirming that the induced epithelial cells generated by transfecting the DNP63A gene, GRHL2 gene, TFAP2A gene, and LEF1 gene using AAV satisfy the properties of epithelial cells capable of inducing skin appendages. In the examples, a viral vector was used to introduce the gene, but the present invention is not limited to this, and any vector that allows the expression of the introduced gene, such as a plasmid vector or an mRNA vector, can be used for administration.

[0132] 1-1 Induction of primary cultured mesenchymal cells derived from mouse adult adipose tissue into induced epithelial cells capable of inducing skin appendages. AAVs expressing the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene were created for gene transfer into mouse adult adipose tissue-derived mesenchymal cells under culture conditions.

[0133] AAV was produced using human Gateway entry clones purchased from NBRC (National Institute of Technology and Evaluation, Biotechnology Division), plasmids purchased from manufacturers (System Biosciences, Gene Copoeia, and Origene), and cDNA prepared from mRNA extracted from BJ fibroblast (ATCC) as templates. The coding sequence was PCR-amplified using the In-Fusion® HD Cloning Kit (Clonetech) and cloned into the pAAV-CAG-GFP (Addgene Plasmid #37825) backbone. The pAAV plasmid was then cloned into AAV-DJ Rep-Cap Plasmid (Cell Biolabs) according to the method described in Reference 2 (Grieger, JC, Choi, VW & Samulski, RJ. Production and characterization of adeno-associated viral vectors. "Nat Protocol," 2006, 1(3), pp. 1412-1428). AAV was titered using standard qPCR.

[0134] Subcutaneous fat samples obtained from the lumbar region of adult mice (C57BL / 6) were minced and treated with 0.1% collagenase at 37°C for 1 hour. The collected cells were seeded in DMEM medium supplemented with 10% newborn bovine serum to obtain primary mesenchymal cells derived from mouse adult adipose tissue. After two subcultures, the cells were cryopreserved in DMEM medium supplemented with 15% newborn bovine serum containing 10% DMSO. The frozen cells were thawed and, after one subculture, seeded at 50,000-100,000 cells per well in DMEM medium supplemented with 10% newborn bovine serum. The next day, 10 cells were cultured in DMEM medium supplemented with 10% newborn bovine serum. 10 Gene Copy (GC) DNP63A gene expression AAVDJ, 10 9 GRHL2 gene expression AAVDJ in GC, 5 x 10 9 TFAP2A gene expression in GC AAVDJ, 5 x 10 9 GC cMYC gene expression AAVDJ, 2 x 10 9GCs were infected with AAVDJ expressing the LEF1 gene. The medium was replaced with fresh medium on days 2, 3, and 5. From day 6, the medium was replaced with Keratinocyte F medium containing the Rho-kinase inhibitor Y27632 (Wako), and the medium was replaced every day thereafter.

[0135] Keratinocyte F medium was prepared according to standard procedures. In this study, 225 ml of F12 medium (WAKO) and 225 ml of DME / F12 medium (WAKO) were mixed, to which 25 ml of newborn bovine serum was added. The medium was then adjusted to contain 24 μg / ml of adenine (Sigma), 8.4 ng / ml of cholera toxin (WAKO), 5 μg / ml of insulin (WAKO), 0.4 μg / ml of hydrocortisone (Sigma), 100 U / ml of penicillin (WAKO), 100 μg / ml of streptomycin (WAKO), and 10 ng / ml of EGF (WAKO). Furthermore, the Keratinocyte F medium was adjusted to 10 μM of Y-27632 (Selleck).

[0136] To observe the appearance of epithelial-like colonies as shown in Figure 10 between days 10 and 15, the cells were passaged on day 15 onto 3T3-J2 feeder cells prepared in a mitomycin-treated 6-well plate. After passage, the medium was changed every two days, and when the cells became subconfluent (approximately one week later), they were subcultured at a ratio of 1:3 to 1:8 to increase the cell number. The cells were then cryopreserved in Y27632-added keratinocyte F medium containing 10% DMSO, thawed, expanded, and used for transplantation experiments as needed.

[0137] 1-2 Regeneration of Skin Appendages Using Induced Epithelial Cells with Skin Appendage Induction Ability Induced from Primary Mesenchymal Cells Derived from Adult Mouse Adipose Tissue and Mesenchymal Cells Derived from Neonatal Mouse Skin The induced epithelial cells obtained in Example 1-1 and primary mesenchymal cells derived from neonatal mouse skin tissue were transplanted into a silicone chamber attached to the back of an immunodeficient animal using the following procedure. Induced epithelial cells were cultured to subconfluence in Keratinocyte F medium on 3T3-J2 feeder cells prepared in two 10 cm cell culture dishes (Biolamo). The cells were treated with 0.25% trypsin at 37°C for 2 minutes, and the feeder cells were removed by aspirating the trypsin solution. The cells were then further treated with 0.25% trypsin at 37°C for 5-10 minutes and collected in 10 ml of Keratinocyte F medium to prepare a suspension containing induced epithelial cells. Meanwhile, skin samples obtained from the backs of newborn mice were treated overnight with 0.25% trypsin at 4°C, and then the epidermal tissue was peeled off and the dermal tissue and subcutaneous fat tissue were treated with 0.1% collagenase at 37°C for 1 hour. The collected cells were seeded in one well of a 6-well plate (Biolamo) in DMEM medium supplemented with 10% newborn bovine serum. Primary mesenchymal cells derived from mouse newborn skin were cultured for 3 days, then treated with 0.25% trypsin at 37°C for 5-10 minutes, and recovered in DMEM medium supplemented with 10% newborn bovine serum to prepare a suspension of primary mesenchymal cells derived from newborn skin tissue. Both suspensions were filtered through a 100 μm cell strainer, mixed, and centrifuged at 300 g for 5 minutes. The cell pellet was suspended in 100 μl of a 1:1 mixture of Keratinocyte F medium and DMEM medium supplemented with 10% neonatal bovine serum. The resulting solution was transplanted into a dome-shaped silicone chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber. Two weeks after transplantation, the chamber was removed. Observation continued for 4–5 weeks after transplantation. Hair growth and regeneration of skin appendages were observed at the chamber site (Figure 11). The resulting induced epithelial cells fulfilled the properties of induced epithelial cells with skin appendage induction ability.

[0138] BALB / cAJcl-nu / nu mice (derived from the Central Institute for Experimental Animals) were used as immunodeficient animals. Silicon chambers were prepared using a method similar to that described in reference 3 (Lichti U, Anders J, Yuspa SH. Isolation and short-term culture of primary keratinocytes, hair follicle populations, and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. "NatProtocol", 2008, 3(5), pp. 799-810). A mold with an inner diameter of 10 mm, a flange width of 3 mm, and a thickness of 0.5 mm was created using a 3D printer, and mold-making silicone (HVT-4000, Engraving Japan) was poured into the mold. Figure 33(A) shows the mold for the silicon chamber, and Figure 33(B) shows the resulting silicon chamber. A circular incision was made in the skin on the back of the mouse, and a silicone chamber was inserted and attached with nylon thread. Figure 33(C) shows a mouse with a silicone chamber sewn onto its back.

[0139] 2-1 Induction of primary cultured mesenchymal cells derived from mouse adult adipose tissue into induced mesenchymal cells capable of inducing skin appendages We created retroviruses expressing the ETV1 gene, FOXD1 gene, PRDM1 gene, SHH gene, and LEF1 gene for gene transfer into mouse adult adipose tissue-derived mesenchymal cells under culture conditions.

[0140] Retroviral plasmids were prepared using cDNA prepared from mRNA extracted from human Gateway entry clones purchased from NBRC (National Institute of Technology and Evaluation, Biotechnology Division), BJ fibroblast (ATCC), and human fibroblasts (Takara Bio) as templates. The coding sequences were PCR-amplified using the In-Fusion® HD Cloning Kit (Clonetech) and subcloned into PMXs retrovirus. 293FT cells (Thermo Fisher Scientific) were transfected with packaging plasmids (pCMV-gagpol-PA, pCMV-VSVg) using Lipofectamine 2000 (Thermo Fisher Scientific). The supernatant was used as the retroviral solution after a medium change.

[0141] Subcutaneous fat samples from the lumbar region of adult C57BL / 6 mice were minced and treated with 0.1% collagenase for 1 hour at 37°C. The collected cells were seeded in DMEM supplemented with 10% neonatal bovine serum (10% neonatal bovine serum) to obtain primary mesenchymal cells derived from mouse adult adipose tissue. After two passages, the cells were cultured in DMEM supplemented with 10% neonatal bovine serum (4 μg / ml polybrene) containing 10% SHH gene-expressing retrovirus solution or LEF1 gene-expressing retrovirus solution to generate the first induced mesenchymal cells expressing the SHH gene or LEF1 gene. Similarly, the second induced mesenchymal cells were cultured in DMEM supplemented with 10% neonatal bovine serum (4 μg / ml polybrene) containing 10% ETV1 gene-expressing retrovirus solution, FOXD1 gene-expressing retrovirus solution, or PRDM1 gene-expressing retrovirus solution to generate the second induced mesenchymal cells expressing the ETV1 gene, FOXD1 gene, or PRDM1 gene.

[0142] The induced epithelial cells obtained in Example 1-1 (generated by AAV expressing the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene) and two types of induced mesenchymal cells obtained in Example 2-1 (first induced mesenchymal cells expressed by a retrovirus expressing the SHH gene and LEF1 gene, and second induced mesenchymal cells expressed by a retrovirus expressing the ETV1 gene, FOXD1 gene, and PRDM1 gene) were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber. Two weeks after transplantation, the silicone chamber was removed, and observations were continued up to four weeks after transplantation. Hair growth, reconstitution, and regeneration of skin appendages were observed at the chamber site (Figure 16). Histological examination confirmed the regeneration of mature hair shafts, hair matrix tissue, and sebaceous gland tissue at the same site (Figure 17).

[0143] 2-2 In an experimental procedure similar to Example 1-1, induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal. A hole was drilled in the top of the silicone chamber one week after transplantation. The silicone chamber was removed two weeks after transplantation, and observations were continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 18).

[0144] 2-3 In an experimental procedure similar to Example 1-1, induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal. First, induced mesenchymal cells 1 induced by the SHH gene and LEF1 gene, and second, induced mesenchymal cells 2 induced by the ETV1 gene and FOXD1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber. Two weeks after transplantation, the silicone chamber was removed, and observations were continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 19).

[0145] 2-4 In an experimental procedure similar to Example 1-1, induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal. First, induced mesenchymal cells induced by the SHH gene and LEF1 gene, and second, induced mesenchymal cells induced by the FOXD1 gene and PRDM1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber. Two weeks after transplantation, the silicone chamber was removed, and observations were continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 20).

[0146] 2-5 In an experimental procedure similar to Example 1-1, induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal. First, induced mesenchymal cells induced by the SHH gene and LEF1 gene, and second, induced mesenchymal cells 2 induced by the ETV1 gene and PRDM1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber. Two weeks after transplantation, the silicone chamber was removed, and observations were continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 21).

[0147] 2-6 Induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene using an experimental method similar to Example 1-1, and induced mesenchymal cells induced by the ETV1 gene, FOXD1 gene, PRDM1 gene, and SHH gene using an experimental method similar to Example 2-1, were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber, and the silicone chamber was removed two weeks after transplantation. Observation was continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 22).

[0148] 2-7 Induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene using an experimental method similar to Example 1-1, and induced mesenchymal cells induced by the ETV1 gene, FOXD1 gene, PRDM1 gene, SHH gene, and LEF1 gene using an experimental method similar to Example 2-1, were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber, and the silicone chamber was removed two weeks after transplantation. Observation was continued for up to four weeks after transplantation. Regeneration of skin appendages was observed at the chamber site (Figure 23).

[0149] 2-8 Induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene using an experimental method similar to Example 1-1, and induced mesenchymal cells induced by the SHH gene and LEF1 gene using an experimental method similar to Example 2-1, were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber, and the silicone chamber was removed two weeks after transplantation. Observation was continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 24).

[0150] 2-9 In an experimental procedure similar to Example 1-1, induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal with first induced mesenchymal cells induced by the SHH gene and LEF1 gene and second induced mesenchymal cells induced by the PRDM1 gene using an experimental procedure similar to Example 2-1. One week after transplantation, a hole was drilled in the top of the silicone chamber, and the silicone chamber was removed two weeks after transplantation. Observation was continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 25).

[0151] 2-10 In an experimental procedure similar to Example 1-1, induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal with first induced mesenchymal cells induced by the SHH gene and second induced mesenchymal cells induced by the FOXD1 gene and PRDM1 gene, using an experimental procedure similar to Example 2-1. One week after transplantation, a hole was drilled in the top of the silicone chamber, and the silicone chamber was removed two weeks after transplantation. Observation was continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 26).

[0152] 2-11 In an experimental procedure similar to Example 1-1, induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal with first induced mesenchymal cells induced by the SHH gene and second induced mesenchymal cells induced by the PRDM1 gene using an experimental procedure similar to Example 2-1. One week after transplantation, a hole was drilled in the top of the silicone chamber, and the silicone chamber was removed two weeks after transplantation. Observation was continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 27).

[0153] 2-12 In an experimental procedure similar to Example 1-1, induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene were co-transplanted into a chamber attached to the back of an immunodeficient animal with first induced mesenchymal cells induced by the SHH gene and second induced mesenchymal cells induced by the FOXD1 gene using an experimental procedure similar to Example 2-1. One week after transplantation, a hole was drilled in the top of the silicone chamber, and the silicone chamber was removed two weeks after transplantation. Observation was continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 28).

[0154] 2-13 Induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene using an experimental method similar to Example 1-1, and induced mesenchymal cells induced by the SHH gene using an experimental method similar to Example 2-1, were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber, and the silicone chamber was removed two weeks after transplantation. Observation was continued for up to four weeks after transplantation. Regeneration of skin appendages was observed at the chamber site (Figure 29).

[0155] 2-14 Induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, or LEF1 gene using an experimental method similar to that of Example 1-1, and induced mesenchymal cells induced by the ETV1 gene, FOXD1 gene, or PRDM1 gene using an experimental method similar to that of Example 2-1 were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber, and the silicone chamber was removed two weeks after transplantation. Observation was continued for 4 to 5 weeks after transplantation. Figure 30 (A) is a photograph of the external appearance on day 28 (D28), and (B) is a cross-sectional photograph of the tissue image stained with hematoxylin and eosin (HE) (the top photograph is a full-length photograph, and the bottom three photographs are enlarged portions of the photograph). Although hair growth was not observed in the external photograph, histological examination revealed hair follicle-like structures and mature hair shafts in the subcutaneous tissue at the center of the cell transplantation site, an area where hair is not normally found. The induced mesenchymal cells induced by the ETV1 gene, FOXD1 gene, and PRDM1 gene produced in this example were transplanted together with the first induced mesenchymal cells as the second induced mesenchymal cells in Examples 2-1 and 2-2, but were also confirmed to have the ability to induce hair follicle-like structures and mature hair shafts even when used alone. Such induced mesenchymal cells with inducibility can be used to regenerate skin appendages by transplanting them, for example, into areas where SHH or LEF1 is physiologically highly expressed. Furthermore, the results of this example demonstrate that skin and skin appendage-like tissues can be generated in vitro using transgenes containing the ETV1 gene, FOXD1 gene, and PRDM1 gene, and can be used for applications such as transplantation. Furthermore, by co-transplanting cells that physiologically overexpress SHH, they can also be used as part of a cell population with the ability to induce skin appendages.

[0156] Skin appendage regeneration by induced mesenchymal cells and other induced epithelial cells with skin appendage induction capacity induced from primary cultured mesenchymal cells derived from mouse adult adipose tissue. In this example, four genes were used as transgenes for induced epithelial cells: DNP63A, GRHL2, TFAP2A, and LEF1. Using an experimental method similar to Example 1-1, induced epithelial cells induced by the DNP63A, GRHL2, TFAP2A, and LEF1 genes were co-transplanted into a chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was drilled in the top of the silicone chamber. The chamber was removed two weeks after transplantation, and observations were continued for up to four weeks after transplantation. Skin appendage regeneration was observed at the chamber site (Figure 31).

[0157] As can be seen from Examples 1 and 3, induced epithelial cells capable of inducing skin appendages could be produced by introducing five genes (DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene) or four genes (DNP63A gene, GRHL2 gene, TFAP2A gene, and LEF1 gene) into somatic cells lacking the ability to induce skin appendages. Since induced epithelial cells could not be obtained in Comparative Examples 1 to 3, it was found that the introduction of the LEF1 gene is necessary to produce epithelial cells capable of inducing skin appendages. It is preferable to introduce one or more genes selected from the DNP63A gene, GRHL2 gene, TFAP2A gene, and c-MYC gene as the gene to be introduced together with the LEF1 gene.

[0158] Furthermore, Example 2-13 demonstrated that induced mesenchymal cells capable of skin appendage induction could be produced by genetically transferring the SHH gene into somatic cells lacking skin appendage induction capacity. Example 2-8 demonstrated that induced mesenchymal cells capable of skin appendage induction could be produced by genetically transferring the SHH gene and LEF1 gene into somatic cells lacking skin appendage induction capacity. Furthermore, Example 2-14 demonstrated that induced mesenchymal cells capable of skin appendage induction could be produced by genetically transferring the ETV1 gene, PRDM1 gene, and FOXD1 gene into somatic cells lacking skin appendage induction capacity. Figure 32 suggests that simultaneous transplantation of these cells can result in higher skin appendage induction capacity. In particular, it suggests that simultaneous transplantation of cells transfected with the ETV1 gene, PRDM1 gene, and part of the FOXD1 gene and cells transfected with the SHH gene alone or the SHH gene and LEF1 gene results in induced mesenchymal cells with higher skin appendage induction capacity.

Claims

[Claim 1] A method for producing cells having skin appendage inducibility, comprising the step of introducing an introduced gene containing at least one type of gene that is relatively highly expressed in cells having skin appendage inducibility into somatic cells that do not have skin appendage inducibility.