WO2022244502A1 - 皮膚付属器誘導能を有する細胞、及びその製造方法 - Google Patents

皮膚付属器誘導能を有する細胞、及びその製造方法 Download PDF

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WO2022244502A1
WO2022244502A1 PCT/JP2022/015867 JP2022015867W WO2022244502A1 WO 2022244502 A1 WO2022244502 A1 WO 2022244502A1 JP 2022015867 W JP2022015867 W JP 2022015867W WO 2022244502 A1 WO2022244502 A1 WO 2022244502A1
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gene
cells
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skin
skin appendages
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昌和 栗田
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to cells with the ability to induce skin appendages that are induced from somatic cells that do not have the ability to induce skin appendages, and methods for producing the same.
  • the present invention also provides cell preparations for regeneration of skin and skin appendages, methods for treating skin ulcers, methods for treating baldness, xerosis, and sebum deficiency using the cells having the ability to induce skin appendages. It relates to a skin and skin appendage regeneration method development platform.
  • the present invention relates to a cell preparation composition for inducing somatic cells that do not have the ability to induce skin appendages into cells that have the ability to induce skin appendages.
  • Skin appendages consist of hair follicles, sebaceous glands, sweat glands, etc., and play roles such as protection from mechanical damage, heat retention, moisture retention, and body temperature regulation. Skin appendages are formed during the fetal period and in the process of organogenesis through interactions between epithelial tissue and mesenchymal tissue (see Non-Patent Document 1).
  • Alopecia and sebum deficiency are typical pathological conditions caused by defects, disorders, and dysfunctions of skin appendages. In addition to causing pruritus due to thermoregulatory disturbance and dry skin, it causes cosmetic disorders.
  • Non-Patent Documents 2 and 3 a method of transplanting cells derived from fetal or neonatal animal skin to obtain reconstitution of skin appendages.
  • a method for obtaining an appendage-like structure through formation of an organ-like culture (organoid) from potent stem cells has been developed (see Non-Patent Documents 4 and 5). Getting a new birth was difficult.
  • Non-Patent Documents 6 and 7 The interaction between epithelial cells present on the surface of the body and mesenchymal cells present in the dermis or subcutaneous layer and environmental factors play important roles in the regeneration and regeneration of skin appendages (see Non-Patent Documents 6 and 7). .
  • genes involved in skin development such as the LEF1 gene and SHH gene, is important as an environmental factor for the regeneration of skin appendages, especially hair follicles ( Non-Patent Documents 8-10).
  • Non-Patent Document 11 skin fibroblasts to muscle cells
  • Non-Patent Document 12 nerve cells
  • Non-Patent Document 13 cardiomyocytes
  • Non-Patent Document 14 hepatocytes
  • Patent Document 1 and Non-Patent Document 15 cells having the ability to form stratified squamous epithelium
  • the present invention provides cells that are capable of inducing skin appendages such as hair, hair follicles, sebaceous glands, etc.
  • One of the purposes is to convert directly from somatic cells. More specifically, the present invention aims to establish a technique for providing a source of cells capable of forming skin appendages such as hair, follicles and sebaceous glands.
  • Another object of the present invention is to provide various uses of cells having the ability to induce skin appendages derived from such somatic cells.
  • adipose tissue-derived mesenchymal cells as representative somatic cells that do not have the ability to induce skin appendages, and developed fetal cells that have a strong ability to reconstitute skin appendages.
  • genes that are highly likely to characterize skin epithelial cells, fetal skin mesenchymal cells, and mesenchymal cells that make up hair follicles by mixed transplantation into skin chambers attached to the back of immunodeficient animals They have found that it is possible to produce a group of cells having the ability to induce skin appendages that can induce regeneration and neogenesis of hair, hair follicles and sebaceous glands.
  • the present invention provides inventions in the following aspects.
  • Inducing skin appendage inducibility including the step of introducing a transgene containing at least one gene that is relatively strongly expressed in cells with skin appendage inducibility into somatic cells that do not have skin appendage inducibility.
  • LEF1 gene (2) one, two, or three genes selected from DNP63A gene, GRHL2 gene, and TFAP2A gene;
  • a method for producing cells capable of inducing skin appendages comprising the step of transfecting a transgene containing A into a somatic cell having no ability to induce skin appendages.
  • LEF1 gene (2) at least one, two, three, or four genes selected from at least one of DNP63A gene, GRHL2 gene, TFAP2A gene, and MYC family gene;
  • a method for producing cells capable of inducing skin appendages comprising the step of transfecting a transgene containing A into a somatic cell having no ability to induce skin appendages.
  • Skin appendages including the step of introducing a transgene containing at least five of at least one of the LEF1 gene, DNP63A gene, GRHL2 gene, TFAP2A gene, and MYC family gene into somatic cells that do not have the ability to induce skin appendages
  • a transgene containing at least five of at least one of the LEF1 gene, DNP63A gene, GRHL2 gene, TFAP2A gene, and MYC family gene into somatic cells that do not have the ability to induce skin appendages
  • a method for producing cells having the ability to induce organogenesis including the step of introducing a transgene containing at least five of at least one of the LEF1 gene, DNP63A gene, GRHL2 gene, TFAP2A gene, and MYC family gene into somatic cells that do not have the ability to induce skin appendages
  • a method for producing cells capable of inducing skin appendages comprising the step of transfecting a transgene containing the SHH gene into somatic cells that do not have the ability to induce skin appendages.
  • 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 induce skin appendages.
  • a method for producing cells capable of inducing skin appendages comprising the step of transfecting a transgene containing A into a somatic cell having no ability to induce skin appendages.
  • ETV1 gene and PRDM1 gene (1) ETV1 gene and PRDM1 gene; (2) FOXD1 gene and PRDM1 gene; or (3) ETV1 gene, PRDM1 gene, and FOXD1 gene;
  • a method for producing cells capable of inducing skin appendages comprising the step of transfecting a transgene containing A into a somatic cell having no ability to induce skin appendages.
  • the somatic cells are skin fibroblasts, subcutaneous adipose tissue-derived stromal cells (subcutaneous adipocytes), embryonic fibroblasts, adipocytes, muscle cells, osteoblasts, chondrocytes, circulating mononuclear cells 15.
  • the method for producing cells having the ability to induce skin appendages according to any one of 1 to 14 above, which are somatic cells differentiated from, ES cells, or mesenchymal stem cells.
  • a first cell capable of inducing skin appendages produced through the step of introducing an SHH gene, or a transgene containing an SHH gene and a LEF1 gene, into a somatic cell having no ability to induce skin appendages.
  • Skin appendages produced through the step of transfecting a transgene containing one or more genes selected from the ETV1 gene, PRDM1 gene, and FOXD1 gene into somatic cells that are incapable of inducing skin appendages.
  • a second cell having inducibility A cell preparation containing
  • the transgene of said second cell comprises (1) ETV1 gene and PRDM1 gene; (2) FOXD1 gene and PRDM1 gene; or (3) ETV1 gene, PRDM1 gene, and FOXD1 gene; 19.
  • a method of determining the efficacy of a test substance comprising the step 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 tissue.
  • a method for determining the effects of external factors including the step of applying stress such as anticancer drugs or radiation to the non-human mammal described in 24 above, and determining the stress on the skin and/or skin appendage tissue. .
  • a method of analyzing the efficacy of a test substance comprising 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.
  • a vector kit specialized for introducing the gene according to any one of 1 to 12 above into somatic cells.
  • a method for evaluating the ability of cells to induce skin appendages using the cells having the ability to induce skin appendages according to 16 above.
  • a vector intended to produce cells having the ability to induce skin appendages and expressing the LEF1 gene, DNP63A gene, GRHL2 gene, TFAP2A gene and cMYC gene.
  • a vector intended to produce cells having the ability to induce skin appendages and expressing the SHH gene, ETV1 gene, PRDM1 gene, FOXD1 gene, and LEF1 gene.
  • first vector intended to produce cells having the ability to induce skin appendages, the first vector expressing an SHH gene or an SHH gene and a LEF1 gene; a second vector intended to produce cells having the ability to induce skin appendages, the second vector expressing one or more genes selected from the ETV1 gene, the PRDM1 gene, and the FOXD1 gene; , a vector kit containing.
  • a cell preparation comprising cells capable of inducing skin appendages produced by the production method according to any one of 2 to 6 above and mesenchymal cells capable of inducing skin appendages.
  • a cell preparation comprising cells having the ability to induce skin appendages produced by the production method according to any one of 7 to 12 above and epithelial cells having the ability to induce skin appendages.
  • cells that have the ability to form tissue with properties equivalent to those of the skin, including skin appendages.
  • Cells that reconstitute such skin appendages are used in pathological conditions caused by deficiencies and dysfunctions of skin appendages such as baldness, xerosis, and sebum deficiency, as well as burns, trauma, bedsores, diabetic ulcers, and peripheral circulatory insufficiency.
  • cells having the ability to induce skin appendages are prepared from a patient's somatic cells (not limited to skin-derived cells, e.g., blood, adipose tissue stromal cells, etc.), and the prepared cells are Performing various analyzes can contribute to elucidation of pathological conditions or treatment of diseases.
  • somatic cells not limited to skin-derived cells, e.g., blood, adipose tissue stromal cells, etc.
  • cells with the ability to induce skin appendages prepared from human somatic cells are suitable as materials for drug discovery and drug development in terms of drug efficacy confirmation.
  • transplantation of the cells prepared by the present invention or the application of the present invention to somatic cells in vivo may result in the loss of skin and skin appendages such as skin ulcers, and skin appendages due to age-related changes.
  • therapeutic means and treatments for inducing regeneration and neogenesis of skin appendages and satisfying the function of skin appendages in patients with a condition in which skin appendages are deficient, such as in a state of reduced function or after healing of a skin ulcer. can be used as a method.
  • FIG. 10 is a photograph showing the results of an adult mouse skin-derived cell transplantation test (Reference Example 1), showing that skin appendages were not regenerated even when adult mouse skin-derived epithelial cells and mesenchymal cells were transplanted. It is This is a schematic of transplantation test of neonatal mouse skin-derived cells, and represents isolation and culture of epithelial and mesenchymal cells from neonatal mouse skin and transplantation into immunodeficient animals.
  • Transplantation test of induced epithelial cells prepared by transfecting DNP63A, GRHL2, TFAP2A, and cMYC into mesenchymal cells derived from adult mouse adipose tissue cells and mesenchymal cells derived from neonatal mouse skin (Comparative Example 1) It is a photograph showing , induced epithelial cells created by transfecting DNP63A, GRHL2, TFAP2A, and cMYC into mesenchymal cells derived from adult mouse adipose tissue cells, and transplanted mesenchymal cells derived from neonatal mouse skin. It has also been shown that skin appendages were not regenerated.
  • transfection of GRHL2, TFAP2A, c-MYC, and FOXD1 can induce epithelial cells.
  • Transplantation test of induced epithelial cells prepared by transfecting DNP63A, GRHL2, TFAP2A, cMYC, and FOXD1 into mesenchymal cells derived from adult mouse adipose tissue cells and mesenchymal cells derived from neonatal mouse skin (Comparative Example 3) Induced epithelial cells prepared by transfecting DNP63A, GRHL2, TFAP2A, cMYC, and FOXD1 into mesenchymal cells derived from adult mouse adipose tissue cells, and mesenchymal cells derived from neonatal mouse skin.
  • Transplantation test of induced epithelial cells prepared by transfecting DNP63A, GRHL2, TFAP2A, cMYC, and LEF1 into mesenchymal cells derived from adult mouse adipose tissue cells and mesenchymal cells derived from neonatal mouse skin (Example 1) Induced epithelial cells prepared by transfecting DNP63A, GRHL2, TFAP2A, cMYC, and LEF1 into mesenchymal cells derived from adult mouse adipose tissue cells, and mesenchymal cells derived from neonatal mouse skin. It has been shown that regeneration of skin appendages can be obtained by transplanting .
  • marker genes PROM1 gene, CRABP1 gene, VCAN gene
  • ALL is a change in marker gene expression when all candidate factors are transfected simultaneously.
  • Each bar graph represents the mean and error bars represent the standard deviation.
  • Statistically significant changes compared to NC (control without gene transfer) are indicated by * (* p ⁇ 0.05, ** p ⁇ 0.01, ***p ⁇ 0.001). It shows the number of positive cells when alkaline phosphatase staining was performed after gene introduction of a candidate factor for inducing mesenchymal cells with the ability to induce skin appendages into cultured human fibroblasts.
  • ALL is the number of positive cells when all candidate factors are transfected simultaneously. Each bar graph represents the mean and error bars represent the standard deviation. Statistically significant changes compared to NC (control without gene transfer) are indicated by * (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001). From the results of marker gene expression and alkaline phosphatase expression, it was determined that the effectiveness of single gene transfer among the candidate factors for inducing mesenchymal cells with the ability to induce skin appendages into cultured human fibroblasts is high. The figure shows the number of alkaline phosphatase-positive cells when a combination of multiple candidate factors was transfected. + indicates that the factor is added, and - indicates that it is not added.
  • Transplantation test of induced mesenchymal cells created by transfecting SHH and LEF1 into adult mouse adipose-derived mesenchymal cells (Example 2-1) A photograph showing the results, showing that regeneration of cutaneous appendages can be obtained by transplantation of these induced cells.
  • Induced mesenchymal cells prepared by transfecting SHH into adult mouse adipose-derived mesenchymal cells Transplantation test (Example 2-2) results of induced mesenchymal cells prepared by transfecting SHH , showing that regeneration of cutaneous appendages can be obtained by transplantation of these induced cells.
  • Induced epithelial cells created by transfecting DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, and ETV1 and FOXD1 genes into adult mouse adipose-derived mesenchymal cells The results of a transplantation test (Example 2-3) of induced mesenchymal cells prepared by introduction and induced mesenchymal cells prepared by transfecting SHH and LEF1 into adult mouse adipose-derived mesenchymal cells are shown.
  • Fig. 3 is a photograph showing that regeneration of skin appendages can be obtained by transplantation of these induced cells.
  • Induced epithelial cells created by transfecting DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, and FOXD1 and PRDM1 genes into adult mouse adipose-derived mesenchymal cells The results of the transplantation test (Example 2-4) of induced mesenchymal cells prepared by introduction and induced mesenchymal cells prepared by transfecting SHH and LEF1 into adult mouse adipose-derived mesenchymal cells are shown. The photographs shown show that regeneration of cutaneous appendages can be obtained by transplantation of these induced cells.
  • Induced epithelial cells created by transfecting DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, and ETV1 and PRDM1 genes into adult mouse adipose-derived mesenchymal cells The results of the transplantation test (Example 2-5) of induced mesenchymal cells prepared by introduction and induced mesenchymal cells prepared by transfecting SHH and LEF1 into adult mouse adipose-derived mesenchymal cells are shown.
  • Fig. 3 is a photograph showing that regeneration of skin appendages can be obtained by transplantation of these induced cells.
  • Induced epithelial cells generated by transfecting DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, and ETV1, FOXD1, and PRDM1 against adult mouse adipose-derived mesenchymal cells are photographs showing the results of a transplantation test (Example 2-6) of induced mesenchymal cells prepared by transfecting LEF1, showing that regeneration of skin appendages can be obtained by transplantation of these induced cells.
  • FIG. 10 is a photograph showing the results of a transplantation test of induced mesenchymal cells prepared by introduction (Example 2-8). It has been shown that regeneration of cutaneous appendages can be obtained by transplantation of these induced cells.
  • Photographs showing the results of a transplant test (Example 2-9) of induced mesenchymal cells prepared by transfection of induced mesenchymal cells prepared by transfection of SHH and LEF1 into adult mouse adipose-derived mesenchymal cells (Example 2-9) and it has been shown that regeneration of cutaneous appendages can be obtained by transplantation of these induced cells.
  • Photographs showing the results of a transplantation test (Example 2-10) of induced mesenchymal cells prepared by introduction and induced mesenchymal cells prepared by transfecting SHH into adult mouse adipose-derived mesenchymal cells and it has been shown that regeneration of cutaneous appendages can be obtained by transplantation of these induced cells.
  • (A) is a general image of the transplanted part
  • (B) is an enlarged image of the hair growing part.
  • Induced epithelial cells generated by transfection of DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, and PRDM1 into adult mouse adipose-derived mesenchymal cells.
  • FIG. 10 is a photograph showing the results of a transplantation test (Example 2-11) of induced mesenchymal cells prepared by transfection of induced mesenchymal cells and induced mesenchymal cells prepared by introducing SHH into adult mouse adipose-derived mesenchymal cells (Example 2-11). have shown that regeneration of cutaneous appendages can be obtained by transplantation of these induced cells.
  • A is a general image of the transplanted part
  • (B) is an enlarged image of the hair growing part.
  • FIG. 10 is a photograph showing the results of a transplantation test (Example 2-12) of induced mesenchymal cells prepared by transfection of induced mesenchymal cells and induced mesenchymal cells prepared by introducing SHH into adult mouse adipose-derived mesenchymal cells (Example 2-12). have shown that regeneration of cutaneous appendages can be obtained by transplantation of these induced cells.
  • FIG. 2 is a photograph showing the results of a transplantation test (Examples 2-13) of induced mesenchymal cells prepared by the method, showing that regeneration of skin appendages can be obtained by transplantation of these induced cells.
  • (A) is a general image of the transplanted part
  • (B) is an enlarged image of the hair growing part.
  • Induced epithelial cells generated by transfecting DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, and ETV1, FOXD1, PRDM1 against adult mouse adipose-derived mesenchymal cells is a photograph showing the results of a transplantation test (Example 2-14) of induced mesenchymal cells prepared by transfecting the gene, and the transplantation of these induced cells yields a subcutaneous hair follicle-like structure and a mature hair shaft.
  • (A) is a photograph of the appearance on the 28th day (D28), the dotted line is the site where the histological image was collected, (B) is a cross-sectional photograph of the hematoxylin-eosin stained (HE) histological image, and the dotted square is (C) shows the position, (C) is a magnified image, and (D) and (E) are histological images of sections continuous with (C). Arrows indicate mature hair shafts, indicating that a continuous series of shafts was observed in the section.
  • FIG. 10 is a photograph showing the results of a transplantation test (Example 3) of induced mesenchymal cells prepared by transfection of induced mesenchymal cells and induced mesenchymal cells prepared by transfecting SHH and LEF1 into adult mouse adipose-derived mesenchymal cells. have shown that regeneration of cutaneous appendages can be obtained by transplantation of these induced cells.
  • (A) is a general image of the transplanted part
  • (B) is an enlarged image of the hair growing part.
  • Induced epithelial cells created by transfecting DNP63A, GRHL2, TFAP2A, c-MYC, and LEF1 into adult mouse adipose-derived mesenchymal cells, and mesenchymal cells with the ability to induce skin appendages. It is the number of hair growth observed at the transplanted site by co-transplanting one or two types of mesenchymal cells into which multiple genes to be expressed are combined and transfected into a chamber attached to the back of an immunodeficient animal. The results of two experiments are shown for each condition.
  • cells having the ability to induce skin appendages e.g., fetal- or neonatal-derived skin cells, hair follicles, It is characterized by including a step of introducing at least one gene that is relatively strongly expressed in cells that constitute skin appendages such as sebaceous glands.
  • the gene to be introduced is hereinafter referred to as a "transgene".
  • a transgene a plurality of genes that are relatively strongly expressed in cells having the ability to induce skin appendages may be introduced, or in addition to genes that are relatively strongly expressed in cells having the ability to induce skin appendages, Genes that are relatively not strongly expressed may be combined.
  • transgene includes not only genes encoding proteins but also noncoding RNAs such as microRNAs.
  • a gene that is relatively strongly expressed in cells with the ability to induce skin appendages refers to cells that do not have the ability to induce skin appendages, such as adult-derived keratinocytes, skin fibroblasts, and adipose-derived mesenchymal cells. It refers to a gene whose expression level is confirmed by quantitative evaluation methods such as real-time PCR, microarray, RNA sequencing, etc. in cells having the ability to induce skin appendages compared to .
  • the term "relatively less strongly expressed gene” refers to a gene whose expression level is lower in cells that have the ability to induce skin appendages than in cells that do not have the ability to induce skin appendages.
  • Genes that are relatively strongly expressed in epithelial cells that have the ability to induce skin appendages include at least the genes that encode the following proteins. DNP63A gene, GRHL2 gene, TFAP2A gene, cMYC gene, LEF1 gene.
  • Genes that are relatively strongly expressed in mesenchymal cells with the ability to induce skin appendages include at least genes encoding 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, PRDM1 gene.
  • NCBI National Center for Biotechnology Information
  • Accession No. in Table 1 is also registered in the database provided by NCBI.
  • the transgene when using human-derived somatic cells, is preferably human-derived.
  • the transgene has several amino acid sequences (eg, 1 to 10, preferably 1 to 6, more preferably 1 to 4, more preferably 1 to 3) in the amino acid sequence of the gene product. (especially preferably 1 or 2) amino acids are substituted, deleted, and/or inserted, and encodes a mutant gene product having a function equivalent to that of the wild-type gene product. There may be.
  • a sequence in which codons are changed and optimized so as to encode the same amino acid as that encoded by each gene may be used.
  • the transgene can be prepared according to conventional methods based on known sequence information.
  • cDNA of the gene of interest can be prepared by extracting RNA from mammalian-derived cells and cloning it according to a conventional method. It can also be synthesized as an artificial gene. When synthesizing artificial genes, codons can be optimized according to the animal from which the somatic cells to be introduced are derived.
  • the type of "somatic cells” induced into cells having the ability to induce skin appendages is not particularly limited, and those derived from any tissue or site can be used.
  • Somatic cells used in the present invention include, for example, those derived from tissues such as skin, subcutaneous fat, muscle, placenta, bone, cartilage, blood, and corneal stroma, and more specifically, skin fibroblasts. , subcutaneous adipose tissue-derived stromal cells (subcutaneous adipocytes), embryonic fibroblasts, adipocytes, muscle cells, osteoblasts, chondrocytes, and circulating mononuclear cells.
  • skin-derived cells, subcutaneous fat-derived cells, or blood-derived cells are preferable from the viewpoint of minimally invasiveness to the living body and more efficient preparation of cells having the ability to induce skin appendages.
  • Skin fibroblasts, subcutaneous adipose tissue-derived stromal cells, and mononuclear cells in circulating blood are particularly preferred.
  • materials can be selected from various cells, and in particular, easily available cells such as skin-derived cells, subcutaneous fat-derived cells, and circulating mononuclear cells can be used.
  • somatic cells commercial products may be used, and somatic cells differentiated from ES cells, mesenchymal stem cells, or the like can also be used.
  • somatic cells are derived from mammals such as humans, mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, etc. However, when used for the purpose of treating humans, elucidating pathological conditions, confirming drug efficacy, etc., those derived from humans are preferred.
  • human-derived somatic cells when used, they may be derived from fetuses, infants, children, or adults. When cells having the ability to induce skin appendages are used for the purposes of human therapy, elucidation of disease states, confirmation of drug efficacy, etc., it is desirable to use somatic cells collected from patients.
  • transgene into somatic cells can be carried out by a method commonly used for transfection of animal cells.
  • methods for introducing the transgene into somatic cells include methods using vectors; calcium phosphate method; lipofection method; electroporation method; Among these, the method using a vector is preferable from the viewpoint of introduction efficiency.
  • viral vectors, non-viral vectors including plasmid (DNA) vectors and mRNA vectors
  • artificial viruses and the like can be used as vectors.
  • Viral vectors such as adeno-associated virus, retrovirus, and lentivirus are preferably used from the viewpoint of safety.
  • each may be incorporated into a separate vector, or two or more transgenes may be incorporated into one vector. .
  • somatic cells into which the transgene has been introduced can be induced into cells having the ability to induce skin appendages.
  • cells having the ability to induce skin appendages can also be induced from somatic cells in vivo by introducing a transgene into somatic cells existing in vivo using the gene introduction means or vector described above.
  • the transgene may be a gene that is relatively strongly expressed in epithelial cells with the ability to induce skin appendages, or a gene that is relatively strongly expressed in mesenchymal cells with the ability to induce skin appendages. It is possible to have both genes, but it is preferable to introduce both genes.
  • the method for producing cells having the ability to induce skin appendages of the present invention comprises (1) LEF1 gene, and (2) one or two selected from DNP63A gene, GRHL2 gene, and TFAP2A gene. , or 3 genes, into somatic cells that are incapable of inducing cutaneous appendages.
  • a transgene containing four genes, LEF1 gene, DNP63A gene, GRHL2 gene, and TFAP2A gene is introduced into a cell having no skin appendage induction ability. It preferably includes the step of gene introduction into somatic cells.
  • the method for producing cells having the ability to induce skin appendages of the present invention comprises (1) LEF1 gene and (2) at least one of DNP63A gene, GRHL2 gene, TFAP2A gene, and MYC family gene. and at least one, two, three, or four genes selected from , into somatic cells that are incapable of inducing cutaneous appendages.
  • a transgene containing at least five of at least one of LEF1 gene, DNP63A gene, GRHL2 gene, TFAP2A gene, and MYC family gene It preferably includes the step of gene transfer into somatic cells that are incapable of inducing cutaneous appendages.
  • Myc family genes include c-Myc, N-Myc, and L-Myc. These Myc family genes may be used alone, or multiple genes may be used in combination. It is preferable to use the c-Myc gene as the Myc family gene.
  • epithelial cells having the ability to induce skin appendages add other genes to the above 4 genes or 5 genes. It is also possible to manufacture by That is, the transgene contains at least one of the above 4 genes or 5 genes, and further includes other genes (preferably those that do not inhibit the expression of the ability to form stratified squamous epithelium and the ability to induce skin appendages). good too.
  • epithelial cells with the ability to induce skin appendages are suitable feeder cells (3T3-J2 feeder cells, 3T3 cells, mouse embryonic fibroblasts, human skin cells) for isolating and amplifying keratinocytes. Fibroblasts, etc. that have been treated with mitomycin C or radiation to inactivate their proliferation ability) or cultured in keratinocyte serum-free medium exhibit relatively high proliferation ability.
  • Rho kinase inhibitor Y27632 etc.
  • the purity of epithelial cells with the ability to induce skin appendages can be determined by performing cell separation using flow cytometry or a magnetic cell separator using surface antigens specific to epithelial cells (CDH1, Epi-CAM, etc.). can be increased.
  • a reporter gene construct made by binding a drug resistance gene to the promoter of epithelial cell marker genes CDH1, Epi-CAM, etc.
  • the induced epithelial cells thus obtained which have the ability to induce skin appendages, can proliferate when cultured on a feeder in a liquid medium supplemented with a Rho kinase inhibitor. It can proliferate stably while maintaining the organ induction ability.
  • a medium commonly used for culturing animal cells can be used.
  • An example of a suitable medium used 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 and various pharmacologically active substances that accelerate the proliferation of keratinocytes under culture conditions, such as bFGF.
  • the thus-obtained induced epithelial cells having the ability to induce skin appendages are mixed with mesenchymal cells derived from the skin of neonatal animals having the ability to induce skin appendages into a chamber attached to the back of an immunodeficient animal. , giving restructuring and regeneration of skin appendages.
  • adult-derived skin cells induced epithelial cells that do not have the ability to induce skin appendages (for example, DNP63A gene, GRHL2 gene, TFAP2A gene, cMYC gene induced by the method described in Non-Patent Document 15 for somatic cells
  • Cells with stratified squamous epithelium-forming ability induced by the gene transfer of , and mesenchymal cells derived from the skin of neonatal animals with the ability to induce skin appendages can be mixed and transplanted into a chamber attached to the back of an immunodeficient animal. , Reconstruction and regeneration of skin appendages are not obtained.
  • neonatal animal-derived epithelial cells and cells isolated from adult-derived skin appendages are also mixed with neonatal animal skin-derived mesenchymal cells that have the ability to induce skin appendages, thereby producing skin appendages.
  • these cells isolated from animals are often contaminated with mesenchymal cells that have the ability to induce cutaneous appendages.
  • mesenchymal cells capable of inducing skin appendages there been difficult to separate and isolate only epithelial cells capable of inducing skin appendages from mesenchymal cells capable of inducing skin appendages.
  • the induced epithelial cells having the ability to induce skin appendages thus obtained are not contaminated with mesenchymal cells having the ability to induce skin appendages, while having the ability to induce skin appendages.
  • mesenchymal cells have the ability to induce skin appendages by mixed transplantation of induced epithelial cells that have the ability to induce skin appendages and mesenchymal cells that have unknown ability to induce skin appendages. It can be used as an evaluation system for determining whether or not it has
  • the induced epithelial cells having the ability to induce skin appendages obtained in this manner are used in pathological conditions in which skin and skin appendages are deficient such as skin ulcers, and in skin appendages and their functions that are reduced due to age-related changes.
  • transplanting it to a patient with a condition in which skin appendages are insufficient quantitatively or qualitatively, such as after healing of skin ulcers it becomes a therapeutic method for regeneration and regeneration of skin and skin appendages.
  • somatic cells that do not have stratified squamous epithelium-forming ability are introduced by introducing a gene that induces cells having stratified squamous epithelium-forming ability into somatic cells present in vivo.
  • a transgene into somatic cells existing in vivo using the above-mentioned gene introduction means or vector, as if cells having stratified squamous epithelial formation ability are induced from the in vivo body It is also possible to induce epithelial cells having the ability to induce skin appendages from the cells.
  • gene introduction means and vectors for introducing a gene that induces epithelial cells having the ability to induce skin appendages into somatic cells existing in vivo are used for skin and skin appendages such as skin ulcers.
  • Gene transfer is performed in patients with pathological conditions in which skin appendages are deficient, such as conditions in which skin appendages and their functions are degraded due to age-related changes, or after skin ulcers have healed, and skin appendages are induced.
  • By inducing epithelial cells with potential it becomes a therapeutic method for regeneration and regeneration of the skin and skin appendages.
  • the method for producing cells having the ability to induce skin appendages of the present invention comprises (1) PRDM1 gene, (2) FOXD1 gene, (3) ETV1 gene, (4) LEF1 gene, and (5) It is preferable to include the step of transfecting at least one gene selected from SHH genes into somatic cells incapable of inducing skin appendages.
  • the method of the present invention for producing cells having the ability to induce skin appendages, particularly mesenchymal cells preferably includes a step of introducing at least the SHH gene into somatic cells that do not have the ability to induce skin appendages.
  • the method of the present invention for producing cells capable of inducing skin appendages, particularly mesenchymal cells preferably comprises a step of introducing at least the SHH gene and the LEF1 gene into somatic cells not capable of inducing skin appendages. .
  • the method for producing cells having the ability to induce skin appendages of the present invention comprises at least one gene selected from (1) PRDM1 gene, (2) FOXD1 gene, and (3) ETV1 gene. It is also preferred to include the step of gene transfer into somatic cells that are incapable of inducing appendages.
  • the method for producing cells having the ability to induce skin appendages of the present invention comprises (1) SHH gene and (2) one gene selected from ETV1 gene, PRDM1 gene, FOXD1 gene and LEF1 gene. It is also preferable to include the step of introducing the above genes and into somatic cells that are incapable of inducing cutaneous appendages.
  • the method for producing cells having the ability to induce skin appendages of the present invention is selected from (1) SHH gene, or SHH gene and LEF1 gene, and (2) ETV1 gene, PRDM1 gene, and FOXD1 gene. It is also preferable to include the step of transfecting the one or more genes obtained by the method into somatic cells that are incapable of inducing cutaneous appendages.
  • the method for producing cells having the ability to induce skin appendages of the present invention comprises (1) ETV1 gene and PRDM1 gene; (2) FOXD1 gene and PRDM1 gene; or (3) ETV1 gene and PRDM1 gene , and the FOXD1 gene into somatic cells that are incapable of inducing cutaneous appendages.
  • mesenchymal cells having the ability to induce skin appendages add other genes to the genes described above. It is also possible to manufacture by
  • the induced mesenchymal cells having the ability to induce skin appendages thus obtained are cultured, for example, in DMEM medium supplemented with 10% FBS (fetal bovine serum) or Advanced-DMEM medium supplemented with N2-supplement. It is possible. It is also useful to add cytokines and various pharmacologically active substances that accelerate the proliferation of mesenchymal cells under culture conditions, such as bFGF.
  • induced mesenchymal cells having the ability to induce skin appendages for example, induced mesenchymal cells obtained by introducing the PRDM1 gene or FOXD1 gene into mesenchymal cells not having the ability to induce skin appendages
  • LEF1 gene, induced mesenchymal cells transfected with SHH gene are mixed with the epithelial cells having the ability to induce skin appendages into a chamber attached to the back of an immunodeficient animal, thereby cultivating the formation of skin appendages.
  • reconstruction, regeneration for example, induced mesenchymal cells obtained by introducing the PRDM1 gene or FOXD1 gene into mesenchymal cells not having the ability to induce skin appendages
  • LEF1 gene induced mesenchymal cells transfected with SHH gene
  • the induced mesenchymal cells having the ability to induce skin appendages thus obtained are mixed with neonatal animal skin-derived epithelial cells having the ability to induce skin appendages into a chamber attached to the back of an immunodeficient animal. , giving restructuring and regeneration of skin appendages.
  • Early mesenchymal cells obtained by primary culture from neonatal animals and cells isolated from skin appendages derived from adults can also be co-transplanted with epithelial cells that can induce skin appendages. These cells isolated from animals may be contaminated with epithelial cells capable of inducing cutaneous appendages.
  • the induced mesenchymal cells having the ability to induce skin appendages are not contaminated with epithelial cells having the ability to induce skin appendages, while having the ability to induce skin appendages.
  • the epithelial cells have the ability to induce skin appendages. It can be used as an evaluation system for determining whether or not it has.
  • the induced mesenchymal cells thus obtained which have the ability to induce skin appendages, are useful in pathological conditions in which skin and skin appendages are deficient, such as skin ulcers, and in skin appendages and their function deterioration due to age-related changes.
  • transplanting it to a patient with a condition in which skin appendages are lacking quantitatively or qualitatively, such as after skin ulcers have healed it becomes a therapeutic method that regenerates and regenerates the skin and skin appendages.
  • somatic cells that do not have stratified squamous epithelium-forming ability are introduced by introducing a gene that induces cells having stratified squamous epithelium-forming ability into somatic cells present in vivo.
  • a transgene into somatic cells existing in vivo using the above-mentioned gene introduction means or vector, as if cells having stratified squamous epithelial formation ability are induced from the in vivo body It is also possible to induce mesenchymal cells having the ability to induce skin appendages from cells.
  • gene introduction means and vectors for introducing a gene that induces mesenchymal cells having the ability to induce skin appendages into somatic cells existing in vivo are useful for skin and skin appendages such as skin ulcers.
  • Gene transfer is performed in patients with pathological conditions in which skin appendages are deficient, such as conditions in which organs are missing, conditions in which skin appendages and their functions have deteriorated due to age-related changes, or after skin ulcers have healed.
  • gene introduction means and vectors for gene introduction of genes that induce mesenchymal cells having the ability to induce skin appendages are compositions for preparing the therapeutic preparation.
  • the epithelial cells and mesenchymal cells having the ability to induce skin appendages obtained by the present invention have the ability to proliferate and are capable of regenerating skin appendages in vivo. , iatrogenic injury (after tumor resection, etc.), pressure ulcers, diabetic ulcers, and skin ulcers caused by peripheral circulatory insufficiency. composition).
  • the epithelial cells and mesenchymal cells having the ability to induce skin appendages are prepared as cell preparations for regeneration of skin and skin appendage tissue, necessary
  • a pharmaceutically acceptable diluent carrier may be included depending on the application.
  • pharmaceutically acceptable diluent carriers include physiological saline and buffer solutions.
  • the cell preparation may contain a pharmacologically active ingredient, an epithelial cell capable of inducing skin appendages, and a nutrient source for mesenchymal cells, if necessary.
  • tissue engineering preparations that form skin and skin appendage-like tissues under culture conditions.
  • Tissue-engineered preparations can contain cell aggregates such as sheet-like structures (eg, epithelial cell sheets) and organ primordia having a three-dimensional structure.
  • the epithelial cells and mesenchymal cells having the ability to induce skin appendages are produced as tissue-engineered preparations for regeneration of skin and skin appendage tissue, together with the epithelial cells and mesenchymal cells having the ability to induce skin appendages, If necessary, it may be used in combination with a pharmacologically active ingredient or a nutrient source for epithelial cells or mesenchymal cells capable of inducing skin appendages.
  • the epithelial cells and mesenchymal cells having the ability to induce skin appendages are formed into a sheet-like structure using an extracellular matrix such as collagen containing mesenchymal cells such as dermal fibroblasts and adipose tissue-derived stromal cells as a scaffold.
  • cell aggregates such as skin, skin appendage-like tissue, and organ primordium having a three-dimensional structure may be prepared and then applied to the skin disease site.
  • the epithelial cells and mesenchymal cells having the ability to induce skin appendages are produced as skin and skin appendage-like tissue having a three-dimensional structure for regeneration of skin and skin appendages
  • the epithelium having the ability to induce skin appendages Together with cells and mesenchymal cells, if necessary, pharmacologically active ingredients, epithelial cells capable of inducing skin appendages, and ingredients serving as nutritional sources for mesenchymal cells may be used in combination.
  • the use of scaffolding materials in this manner allows for more rapid regeneration of the skin and skin appendages at the graft site.
  • Scaffold materials that can be used are not particularly limited as long as they are pharmaceutically acceptable, and are appropriately selected according to the site of cartilage tissue to be applied. ) materials.
  • Examples of usable scaffold materials include collagen, fibronectin, hyaluronic acid, matrigel, and complexes thereof. These scaffolding materials may be used singly or in combination of two or more.
  • the shape of the scaffolding material is not particularly limited, and may be appropriately designed according to the shape of the damaged site of the skin and skin appendage tissue to which the cell preparation is applied.
  • the method of applying the cell preparation to the diseased site of the epithelial tissue is appropriately set according to the type of the cell preparation, the site of the skin tissue to be applied, etc.
  • Examples include a method of directly spraying a formulation, a method of directly spraying a tissue-like three-dimensional structure constructed under culture conditions, and a method of suturing and fixing a sheet or three-dimensional structure according to skin grafting.
  • the dosage of the cell preparation applied to the diseased part of the skin tissue is determined based on the type of cell preparation, epithelial tissue site, severity of symptoms, age and sex of the patient, etc. can be appropriately set to an effective amount for the regeneration of .
  • a non-human mammal having skin and skin appendage tissue formed from cells having induced skin and skin appendages by administration of the epithelial cells and mesenchymal cells having the ability to induce skin appendages is treated with the skin. and/or as a tool for evaluating and analyzing the efficacy of test substances on skin appendage tissue. That is, a test substance is administered to a non-human mammal having skin and skin appendage tissue formed from epithelial cells and mesenchymal cells having the ability to induce skin appendages, and the skin and/or skin appendage tissue By determining and analyzing the efficacy of the test substance, it is possible to evaluate and analyze the efficacy of the test substance on the skin and/or skin appendage tissue.
  • the test substance is a substance to be evaluated and analyzed for efficacy on the skin and/or skin appendage tissue, and specifically includes a candidate substance for the treatment of skin and/or skin appendage diseases.
  • a candidate substance for the treatment of skin and/or skin appendage diseases include a candidate substance for the treatment of skin and/or skin appendage diseases.
  • non-human mammals mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, etc. are appropriately selected.
  • non-human mammals having skin and skin appendage tissue formed from epithelial cells and mesenchymal cells having the ability to induce skin appendages are treated with anticancer agents, radiation, etc., and skin and / or skin appendages. It can be used as a model to examine the effects of tissue-damaging external factors on the skin and/or skin appendages.
  • the three-dimensional structure prepared by a scaffold such as a collagen gel containing epithelial cells and mesenchymal cells having the ability to induce skin appendages is used to evaluate the efficacy of the test substance on skin and/or skin appendage tissue. It can be used as a tool for analysis. That is, a test substance is administered to a three-dimensional structure made of a scaffold such as a collagen gel containing epithelial cells, mesenchymal cells and dermal fibroblasts having the ability to induce skin appendages, and the skin and/or skin.
  • a scaffold such as a collagen gel containing epithelial cells, mesenchymal cells and dermal fibroblasts having the ability to induce skin appendages, and the skin and/or skin
  • the test substance is a substance to be evaluated and analyzed for efficacy on skin and skin appendage tissue, and specifically includes a candidate substance for a therapeutic drug for skin and/or skin appendage disease.
  • epithelial cells and mesenchymal cells that have the ability to induce skin appendages derived from somatic cells that can be collected with relatively little invasiveness, such as peripheral blood circulating mononuclear cells, many with diverse genetic backgrounds
  • ability to broadly assess and analyze drug efficacy for donors by using epithelial cells and mesenchymal cells that have the ability to induce skin appendages derived from somatic cells that can be collected with relatively little invasiveness, such as peripheral blood circulating mononuclear cells, many with diverse genetic backgrounds.
  • epithelial cells and mesenchymal cells having the ability to induce skin appendages can be used as tools for elucidating and analyzing the pathology of various skin and skin appendage tissues.
  • Epithelial cells and mesenchymal cells with the ability to induce appendages are also useful as tools for drug discovery and drug development for skin and skin appendage diseases.
  • the test substance is administered to the skin and skin appendage-like tissue, and the efficacy of the test substance on the skin and skin appendage-like tissue is evaluated and analyzed. Stress on the organization can be evaluated and analyzed. Evaluation and analysis of drug efficacy or stress may be confirmed by comparing, for example, a tissue administered with a test substance or a tissue subjected to stress and a tissue not administered or subjected to stress.
  • mice skin specimens obtained from the lower back of adult mice were treated with 0.25% trypsin at 4°C overnight, and the dermal tissue or subcutaneous adipose tissue was treated with 0.1% collagenase at 37°C for 1 hour and collected.
  • Mouse adult skin-derived mesenchymal primary culture cells were harvested by seeding the cells in DMEM medium supplemented with 10% newborn bovine serum.
  • Mouse adult skin-derived epithelial primary culture cells and mouse adult skin-derived mesenchymal primary culture cells cultured for 1 to 3 days were transplanted into a silicon chamber attached to the back of an immunodeficient animal.
  • a hole is made in the upper part of the silicon chamber one week after transplantation, and the silicon chamber is removed two weeks after transplantation. Observations were continued for up to a week (Fig. 1). The chamber sites were epithelialized by the transplanted cells, but the reconstitution/regeneration of skin appendages was not obtained (Fig. 2).
  • the 3T3-J2 feeder cells used as feeders are cell lines provided by J-TEC. Using 3T3 cell medium containing 10% neonatal bovine serum added to DMEM medium, cells were maintained according to a conventional method, treated with 10 ⁇ g/ml of mitomycin C for 1 hour on the day before use as feeder cells, and then 2.0 ⁇ 10 Cell/well concentrations were passaged and used as feeders.
  • a skin sample obtained from the back of a neonatal mouse was treated with 0.25% trypsin at 4°C overnight.
  • Mouse neonatal skin-derived mesenchymal primary culture cells were harvested by seeding in DMEM medium supplemented with 10% newborn bovine serum.
  • Mouse neonatal skin-derived epithelial primary cultured cells and mouse neonatal skin tissue-derived mesenchymal primary cultured cells cultured for 1 to 3 days were transplanted into a silicon chamber attached to the back of an immunodeficient animal.
  • a hole was made in the upper part of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed, and observation was continued until 4 to 5 weeks after transplantation (Fig. 3). Hair growth at the chamber site, reconstruction and regeneration of skin appendages were obtained (Fig. 4).
  • Induced epithelial cells were generated.
  • the induced epithelial cells and mouse neonatal skin tissue-derived mesenchymal primary cultured cells were transplanted into a silicon chamber attached to the back of an immunodeficient animal.
  • One week after transplantation a hole was made in the upper part of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed, and observation was continued until 4 to 5 weeks after transplantation (Fig. 5).
  • the chamber sites were epithelialized by the transplanted cells, but the reconstitution/regeneration of the skin appendages was not obtained (Fig. 6).
  • the induced epithelial cells thus obtained and the mesenchymal primary cultured cells derived from mouse neonatal skin tissue were transplanted into a silicon chamber attached to the back of an immunodeficient animal.
  • the chamber sites were epithelialized by the transplanted cells, reconstitution/regeneration of the skin appendages was not obtained (Fig. 9).
  • Example 1 Transplantation experiment of induced epithelial cells having the ability to induce skin appendages and mouse neonatal skin-derived mesenchymal cells Epithelial cells having the ability to induce skin appendages from mouse adult adipose-derived mesenchymal primary cultured cells by gene transfer In order to develop a method to induce As a result, induced epithelial cells were obtained (Fig. 10).
  • the induced epithelial cells and mesenchymal primary cultured cells derived from mouse neonatal skin tissue thus obtained were transplanted into a silicon chamber attached to the back of an immunodeficient animal.
  • One week after transplantation a hole was made in the upper part of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed, and observation was continued until 4 to 5 weeks after transplantation. Hair growth at the chamber site, reconstruction and regeneration of skin appendages were obtained (Fig. 11).
  • the induced epithelial cells thus obtained fulfilled the properties of induced epithelial cells capable of inducing skin appendages. Details will be described in Example 1 below.
  • Epithelial cells derived from mouse neonatal skin are considered to be epithelial cells with the ability to induce skin appendages, and epithelial cells cultured for 2-3 passages to remove mesenchymal cells from mouse neonatal skin that contaminate the primary culture.
  • a retroviral vector was used to combine multiple genes that are relatively strongly expressed in mesenchymal cells that have the ability to induce skin appendages. They were implanted together with the transfected cells into a silicon chamber attached to the back of an immunodeficient animal. In some mice, a small amount of hair growth was observed in the chamber site, and skin appendages were reconstituted and regenerated.
  • DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene (Examples 1 and 2) or DNP63A gene, GRHL2 gene, TFAP2A gene, for mouse adult adipose-derived mesenchymal primary cultured cells, and the induced epithelial cells obtained by transfecting the LEF1 gene (Example 3) have the ability to induce skin appendages, but are not contaminated with mesenchymal cells having the ability to induce skin appendages.
  • Example 2 Transplantation experiment of induced epithelial cells having the ability to induce skin appendages and induced mesenchymal cells having the ability to induce skin appendages
  • SHH gene and LEF1 gene which have been suggested to be important, were selected as candidates.
  • the ability to induce skin appendages created by transfecting the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene using AAV was examined.
  • mesenchymal cells transfected with the SHH gene alone using a retroviral vector (Example 2- 13), or mesenchymal cells transfected with a combination of SHH gene and LEF1 gene (Example 2-8) were co-transplanted into a chamber attached to the back of an immunodeficient animal.
  • a hole was made in the upper part of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed, and observation was continued until 4 to 5 weeks after transplantation. A small number of hairs were observed to grow in the chamber site (Fig. 24, Fig. 29).
  • the ETV1 gene, FOXD1 gene, and PRDM1 gene were candidates. After two passages of mouse adult adipose-derived mesenchymal primary cultured cells, the ability to induce skin appendages created by transfecting the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene using AAV was examined.
  • ETV1 gene, FOXD1 gene, and PRDM1 gene were combined and introduced using a retroviral vector.
  • Leaf cells (Example 2-14) were co-transplanted into a chamber attached to the back of an immunodeficient animal, a hole was made in the upper part of the silicon chamber 1 week after transplantation, and the silicon chamber was opened 2 weeks after transplantation. After removal, observation was continued until 4-5 weeks after transplantation. A histological examination revealed a hair follicle-like structure and a mature hair shaft in the central subcutaneous region of the cell-implanted site, in a site where no hair was originally found (Fig. 30).
  • ETV1 gene, FOXD1 gene, and PRDM1 gene for gene transfer may induce mesenchymal cells with the ability to induce skin appendages through a mechanism different from that of the SHH gene, LEF1 gene, and environmental factors. Therefore, it was suggested that transplantation of multiple types of cells transfected with these genes could more efficiently induce hair growth and reconstitution and regeneration of skin appendages.
  • mice After two passages of adipose-derived mesenchymal primary cultured cells, induction with the ability to induce skin appendages was created by transfecting the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene using AAV.
  • a retroviral vector was used to relatively strongly express mesenchymal cells that have the ability to induce skin appendages.
  • mesenchymal cells transfected with a combination of multiple genes were co-transplanted into a chamber attached to the back of an immunodeficient animal (Fig. 15).
  • Fig. 15 One week after transplantation, a hole was made in the upper part of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed, and observation was continued until 4 to 5 weeks after transplantation.
  • Genes introduced into the first and second induced mesenchymal cells are shown in Table 2.
  • animals co-transplanted with one or two types of induced mesenchymal cells transfected with the 13 types of combinations described in Examples 2-1 to 2-13 hair growth and reconstitution of skin appendages occurred at the chamber site. , got a play.
  • FIG. 32 is a list of the results of the number of hair growths in each of the two experiments of Examples 2-1 to 2-13. It was also found that even when using the same combination of multiple types of transgenes, more excellent results can be obtained by preparing and transplanting multiple induced mesenchymal cells.
  • ETV1 gene, FOXD1 gene, PRDM1 gene, and SHH gene are introduced into one cell and transplanted, as in Example 2-2.
  • the induced mesenchymal cells thus obtained are co-transplanted with induced epithelial cells having the ability to induce skin appendages, thereby providing reconstruction and regeneration of the skin appendages. It was confirmed that the properties as lineage cells were satisfied.
  • Example 3 Furthermore, after 2 passages of mouse adult adipose-derived mesenchymal primary culture cells, induced epithelial cells created by transfecting DNP63A gene, GRHL2 gene, TFAP2A gene, and LEF1 gene using AAV, and mouse adult adipose-derived mesenchymal cells After 2 passages of primary leaf cells, mesenchymal cells transfected with ETV1 gene, FOXD1 gene and PRDM1 gene, and LEF1 gene and SHH gene were transfected using retroviral vectors. They were co-implanted into a chamber attached to the back of an immunodeficient animal (Fig. 15).
  • a viral vector was used for gene introduction, but the invention is not limited to this, and any vector such as a plasmid vector or an mRNA vector can be used for administration that facilitates the expression of the introduced gene.
  • Subcutaneous fat samples obtained from the lumbar region of adult mice (C57BL/6) are minced, treated with 0.1% collagenase at 37°C for 1 hour, and the collected cells are seeded in DMEM medium supplemented with 10% newborn bovine serum.
  • Mouse adult adipose-derived mesenchymal primary cultured cells were collected by the method. After two subcultures, the cells were cryopreserved in DMEM medium supplemented with 15% newborn bovine serum containing 10% DMSO. Frozen cells were thawed, cultured for one subculture, and seeded at 50,000-100,000 cells per well in DMEM medium supplemented with 10 % fetal bovine serum.
  • AAVDJ, 10 9 GC of GRHL2 gene-expressing AAVDJ, 5 ⁇ 10 9 GC of TFAP2A gene-expressing AAVDJ, 5 ⁇ 10 9 GC of cMYC gene-expressing AAVDJ, and 2 ⁇ 10 9 GC of LEF1 gene-expressing AAVDJ were infected.
  • the medium was changed to fresh medium, and from the 6th day, the medium was changed to Keratinocyte F medium containing Y27632 (Wako), which is a Rho-kinase inhibitor.
  • Keratinocyte F medium was prepared according to the standard method. In this test, 225 ml of F12 medium (WAKO) and 225 ml of DME/F12 medium (WAKO) were mixed, 25 ml of newborn bovine serum was added, adenine (Sigma) 24 ⁇ g/ml, cholera toxin (Wako company) 8.4 ng/ml, insulin (WAKO) 5 ⁇ g/ml, hydrocortisone (Sigma) 0.4 ⁇ g/ml, penicillin (WAKO) 100 U/ml, streptomycin (WAKO) 100 ⁇ g/ml, EGF ( WAKO) Adjusted to 10 ng/ml. Furthermore, in this test, the keratinocyte F medium was adjusted to 10 ⁇ M Y-27632 (Selleck) and used.
  • Example 1-1 Regeneration of skin appendages by induced epithelial cells with the ability to induce skin appendages induced from mouse adult adipose-derived mesenchymal primary cultured cells and mouse neonatal skin-derived mesenchymal cells
  • Example 1-1 The obtained induced epithelial cells and mouse neonatal skin tissue-derived mesenchymal primary cultured cells were transplanted into a silicon chamber attached to the back of an immunodeficient animal according to the following procedure. Induced epithelial cells were cultured to subconfluence using keratinocyte F medium on 3T3-J2 feeder cells prepared in two 10 cm cell culture dishes (Biolamo).
  • the cells were collected in DMEM medium supplemented with 10% neonatal bovine serum to obtain a neonatal skin tissue-derived mesenchymal primary cell suspension. Both suspensions were filtered using a 100 ⁇ m cell strainer and mixed, and the cell pellet after centrifugation at 300 G for 5 minutes was mixed 1:1 with keratinocyte F medium and DMEM medium supplemented with 10% newborn bovine serum.
  • the solution suspended in 100 ⁇ l of the culture medium was transplanted into a dome-shaped silicon chamber attached to the back of an immunodeficient animal. One week after transplantation, a hole was made in the upper part of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed, and observation was continued until 4 to 5 weeks after transplantation. Hair growth at the chamber site, reconstruction and regeneration of skin appendages were obtained (Fig. 11).
  • the induced epithelial cells thus obtained fulfilled the properties of induced epithelial cells capable of inducing skin appendages.
  • mice BALB/cAJcl-nu/nu (derived from the Central Institute for Experimental Animals) mice were used as immunodeficient animals. Silicon chambers were used 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 vitroanalysis and for grafting to immunodeficient mice. "NatProtocol", 2008, 3(5), p799-810), which can be attached by a method described in 3D printer, in a mold with an inner diameter of 10mm, a width of the collar of 3mm, and a thickness of 0.5mm.
  • FIG. 33(A) is the mold of the silicon chamber
  • FIG. 33(B) is the created silicon chamber.
  • a circular excision was made on the dorsal skin of the mouse, a silicon chamber was inserted, and it was closed with a nylon thread.
  • FIG. 33(C) is a mouse with a silicon chamber sewn to its back.
  • Human Gateway entry clones purchased from NBRC (National Institute of Technology and Evaluation, Biotechnology field), BJ fibroblast (ATCC), and mRNA collected from human fibroblasts (Takara Bio) were used to create retroviral plasmids.
  • NBRC National Institute of Technology and Evaluation, Biotechnology field
  • ATCC BJ fibroblast
  • Takara Bio mRNA collected from human fibroblasts
  • 293FT cells were transfected with packaging plasmids (pCMV-gagpol-PA, pCMV-VSVg) using Lipofectamine2000 (Thermo Fisher Scientific), and the cell supernatant after medium exchange was used as the retrovirus fluid. board.
  • Subcutaneous fat specimens obtained from the lumbar region of adult mice (C57BL/6) are minced, treated with 0.1% collagenase at 37°C for 1 hour, and the collected cells are seeded in DMEM medium supplemented with 10% newborn bovine serum.
  • Mouse adult adipose-derived mesenchymal primary cultured cells were collected by the method.
  • SHH gene-expressing retrovirus solution and 10% LEF1 gene-expressing retrovirus solution were cultured in DMEM medium supplemented with 10% neonatal bovine serum (polybrene 4ug/ml) to extract SHH gene, First induced mesenchymal cells expressing the LEF1 gene were generated, and similarly, 10% calf newborns containing 10% ETV1 gene-expressing retroviral solution, FOXD1 gene-expressing retroviral solution, and PRDM1 gene-expressing retroviral solution, respectively.
  • a second induced mesenchymal cell expressing ETV1 gene, FOXD1 gene and PRDM1 gene was prepared by culturing in fetal serum-added DMEM medium (polybrene 4 ⁇ g/ml).
  • Example 1-1 Induced epithelial cells obtained in Example 1-1 (DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, LEF1 gene expression AAV) and two types of induced mesenchyme obtained in Example 2-1
  • Lineage cells first induced mesenchymal cells by retrovirus expressing SHH gene and LEF1 gene, and second induced mesenchymal cells by retrovirus expressing ETV1 gene, FOXD1 gene and PRDM1 gene
  • Fig. 16 Histological examination revealed regeneration of mature hair shaft, hair matrix tissue and sebaceous gland tissue at the same site (Fig. 17).
  • Example 2 Induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene by the experimental method according to Example 1-1, and the experimental method according to Example 2-1
  • the first induced mesenchymal cells 1 induced by the SHH gene and the second induced mesenchymal cells induced by the ETV1 gene, FOXD1 gene, and PRDM1 gene were placed on the back of an immunodeficient animal in a chamber. co-implanted.
  • One week after transplantation a hole was made in the upper part of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed, and observation was continued until 4 weeks after transplantation. Regeneration of skin appendages was obtained at the chamber site (Fig. 18).
  • Example 2 Induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene by the experimental method according to Example 1-1, and the experimental method according to Example 2-1
  • First induced mesenchymal cells induced by SHH gene and LEF1 gene, and second induced mesenchymal cells induced by FOXD1 gene and PRDM1 gene were placed together in a chamber attached to the back of an immunodeficient animal. transplanted. One week after transplantation, a hole was made in the upper part of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed, and observation was continued until 4 weeks after transplantation. Regeneration of skin appendages was obtained at the chamber site (Fig. 20).
  • Example 2-5 Induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, and LEF1 gene by the experimental method according to Example 1-1, and the experimental method according to Example 2-1
  • First induced mesenchymal cells induced by SHH gene and LEF1 gene, and second induced mesenchymal cells 2 induced by ETV1 gene and PRDM1 gene were placed on the back of an immunodeficient animal in a chamber. co-implanted.
  • One week after transplantation a hole was made in the upper part of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed, and observation was continued until 4 weeks after transplantation. Regeneration of skin appendages was obtained at the chamber site (Fig. 21).
  • FIG. 30 (A) is a photograph of the appearance on the 28th day (D28), and (B) is a cross-sectional photograph of a hematoxylin-eosin-stained (HE) histological image (upper is a whole photograph, lower three are partially enlarged photographs). ), but hair growth was not observed from the external photograph, but histological examination revealed a hair follicle-like structure and a mature hair shaft in the subcutaneous central part of the cell transplantation site, in a site where hair was not normally observed. rice field.
  • the induced mesenchymal cells induced by the ETV1 gene, FOXD1 gene, and PRDM1 gene produced in this example are the second induced mesenchymal cells in Examples 2-1 and 2-2.
  • inducible mesenchymal cells having such inducibility can be used to transplant skin appendages to sites where SHH or LEF1 is physiologically highly expressed, for example.
  • transgenes including the ETV1 gene, FOXD1 gene, and PRDM1 gene can be used to create skin and skin appendage-like tissue in vitro, and can be used for purposes such as transplantation. can. It can also be used as part of a group of cells that have the ability to induce cutaneous appendages by co-transplanting with cells that physiologically highly express SHH.
  • induced epithelial cells with the ability to induce skin appendages derived from mouse adult adipose-derived mesenchymal primary cultured cells, regeneration of skin appendages by induced mesenchymal cells
  • DNP63A gene a transgene for induced epithelial cells
  • GRHL2 gene a transgene for induced epithelial cells
  • TFAP2A gene a transgene for induced epithelial cells
  • induced epithelial cells induced by the DNP63A gene, GRHL2 gene, TFAP2A gene, and LEF1 gene, SHH gene, and first induced mesenchymal cells induced by the LEF1 gene, Second induced mesenchymal cells induced by the ETV1, FOXD1 and PRDM1 genes were co-implanted into a chamber fitted to the back of immunodeficient animals.
  • One week after transplantation a hole was made in the upper part of the silicon chamber, and two weeks after transplantation, the silicon chamber was removed, and observation was continued until 4 weeks after transplantation. Regeneration of skin appendages was obtained at the chamber site (Fig. 31).
  • DNP63A gene, GRHL2 gene, TFAP2A gene, c-MYC gene, LEF1 gene, or 4 genes of DNP63A gene, GRHL2 gene, TFAP2A gene, LEF1 gene, Induced epithelial cells with the ability to induce skin appendages could be produced by gene transfer into somatic cells that do not have the ability to induce skin appendages. Since no induced epithelial cells could 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.
  • genes to be introduced together with the LEF1 gene one or more genes selected from DNP63A gene, GRHL2 gene, TFAP2A gene, and c-MYC gene are preferably introduced.
  • Examples 2-13 it was possible to produce induced mesenchymal cells with the ability to induce skin appendages by introducing the SHH gene into somatic cells that do not have the ability to induce skin appendages.
  • Examples 2-8 it was possible to produce induced mesenchymal cells having the ability to induce skin appendages by introducing the SHH gene and the LEF1 gene into somatic cells that do not have the ability to induce skin appendages.
  • the ETV1 gene, the PRDM1 gene, and the FOXD1 gene are introduced into somatic cells that do not have the ability to induce skin appendages, thereby producing induced mesenchymal cells that have the ability to induce skin appendages. We were able to.
  • these cells can obtain higher skin appendage induction ability by simultaneous transplantation.
  • cells transfected with part of the ETV1 gene, PRDM1 gene, or FOXD1 gene and cells transfected with the SHH gene alone or the SHH gene and the LEF1 gene it has a higher ability to induce skin appendages. It is suggested that they become induced mesenchymal cells.

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