Skin organoid, graft sheet, full thickness skin tissue, and methods for producing same

Abstract

The present invention provides, as a technique that can be used for obtaining a skin organoid or a skin tissue structure that skillfully mimics the structure and function of the skin of a living body from stem cells, a skin organoid comprising cells that are derived from pluripotent stem cells and have the ability to differentiate into cells constituting the skin, the skin organoid having a lumen, the skin organoid having an epidermal cell layer comprising cells having the ability to differentiate into epidermis on the surface facing the lumen, and the skin organoid having a mesenchymal cell layer, on the outside of the epidermal cell layer, comprising cells having the ability to differentiate into dermis or subcutaneous tissue. The epidermal cell layer is configured by laminating, in this order from the lumen side, a granular layer comprising cells that express loricrin, a spinous layer comprising cells that express KRT10, and a basal layer comprising cells that express KRT5 and p63, and has hair pegs invaginating the mesenchymal cell layer.

Description

Skin organoids, transplant sheets, and full-thickness skin tissues, and methods for producing them

[0001] The present disclosure relates to skin organoids, transplant sheets, and full-thickness skin tissues, as well as methods for producing them. More specifically, the present disclosure relates to skin organoids and transplant sheets induced in vitro from pluripotent stem cells, and full-thickness skin tissues obtained by transplanting and maturing the transplant sheets onto the surface of a living body.

[0002] Technologies have been developed to culture and differentiate stem cells, such as induced stem cells and embryonic stem cells, in three dimensions to obtain tissue structures that mimic the structure and function of parts of living organs. These three-dimensional tissue structures, also known as "organoids" or "mini-organs," are expected to be useful as alternative materials for animal testing and as transplant materials for regenerative medicine.

[0003] Non-Patent Document 1 and Patent Document 1 report that skin organoids were prepared from human pluripotent stem cells, and the structure of the skin organoids was analyzed, resulting in the formation of a multilayer structure comprising an epidermal layer and a dermal layer, as well as hair follicles, sebaceous glands, and neural circuits. Furthermore, it has been reported that when skin organoids were transplanted into the back skin of immunodeficient mice, the skin organoids were incorporated into the mouse epidermis, and human hair grew on the graft.

[0004] Skin is composed of three layers: the epidermis, dermis, and subcutaneous tissue. The epidermis is the outermost layer of the skin and consists of the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. It is primarily composed of keratinocytes. The dermis lies beneath the epidermis and consists of the papillary and reticular layers, composed of capillaries and elastic fibers. The subcutaneous tissue lies beneath the dermis and contains adipocytes, blood vessels, and nerves. The epidermis provides external protection, the dermis provides strength and nutrients, and the subcutaneous tissue provides shock absorption and energy storage. Furthermore, skin contains appendages such as hair, arrector pili muscles, sebaceous glands, and sweat glands, which function in body protection, thermoregulation, and sensory reception. There is a need for technologies to produce skin organoids that closely mimic the structure and function of living skin. For example, skin organoids obtained using the techniques disclosed in the aforementioned Non-Patent Document 1 and Patent Document 1 lack at least sweat glands. Furthermore, Non-Patent Document 1 and Patent Document 1 do not report that host-derived immune cells were able to invade the tissue of skin organoids transplanted into mice.

[0005] Special table 2018-531027 publication

[0006] "Hair-bearing human skin entirely generated from pluripotent stem cells", Jiyoon Lee et. al., Nature, 2020, Vol.582, pages 399-404

[0007] The main purpose of the present disclosure is to provide a technique that can be used to obtain skin organoids or skin tissue structures from stem cells that highly mimic the structure and function of in vivo skin.

[0008] To solve the above problems, the present disclosure provides the following [1] to

[29] . [1] A skin organoid comprising cells induced from pluripotent stem cells and capable of differentiating into cells that constitute skin, wherein the skin organoid has an inner lumen, and an epidermal cell layer on its surface facing the lumen comprising cells capable of differentiating into epidermis, and a mesenchymal cell layer on the outside of the epidermal cell layer comprising cells capable of differentiating into the dermis or subcutaneous tissue, wherein the epidermal cell layer is composed of a granular layer comprising cells that express Loricrin, a spinous layer comprising cells that express KRT10, and a basal layer comprising cells that express KRT5 and p63, stacked in this order from the inner lumen side, and has hair follicles invaginating into the mesenchymal cell layer. [2] The skin organoid according to [1], wherein the mesenchymal cell layer comprises fibroblasts. [3] The skin organoid according to [1] or [2], wherein the pluripotent stem cells are human pluripotent stem cells.

[0009] [4] A transplant sheet comprising cells induced from pluripotent stem cells and capable of differentiating into cells that constitute skin, wherein the transplant sheet has a layered structure in which an epidermal cell layer comprising cells capable of differentiating into epidermis is backed by a mesenchymal cell layer comprising cells capable of differentiating into the dermis or subcutaneous tissue, the epidermal cell layer being composed of a granular layer comprising cells that express loricrin, a spinous layer comprising cells that express KRT10, and a basal layer comprising cells that express KRT5 and p63, laminated in this order, and the transplant sheet has hair piles that invaginate into the mesenchymal cell layer. [5] The transplant sheet according to [4], wherein the mesenchymal cell layer comprises fibroblasts. [6] The transplant sheet according to [4] or [5], wherein the pluripotent stem cells are human pluripotent stem cells.

[0010] [7] A method for producing the skin organoid according to any one of [1] to [3], comprising: (1) culturing an aggregate of pluripotent stem cells in the presence of a substance that activates the Wnt signal pathway; (2) culturing the cell aggregate obtained in step (1) in the presence of a bone morphogenetic protein (BMP), a TGFβ inhibitor, and basic fibroblast growth factor (bFGF); (3) culturing the cell aggregate obtained in step (2) in the presence of an ALK inhibitor and bFGF; (4) culturing the cell aggregate obtained in step (3) in the absence of a substance that activates the Wnt signal pathway, BMP, a TGFβ inhibitor, bFGF, and an ALK inhibitor, to obtain the skin organoid. [8] The manufacturing method according to [7], wherein the substance that activates the Wnt signal pathway is a GSK-3β inhibitor, preferably CHIR99021, and is treated with the cell aggregate at a concentration of 0.1 μM or more and less than 3.0 μM, preferably 0.5 μM or more and 1.5 μM or less. [9] The manufacturing method according to [7] or [8], wherein the TGFβ inhibitor is SB431542.

[10] The manufacturing method according to any one of [7] to [9], wherein the ALK inhibitor is LDN193189.

[11] The manufacturing method according to any one of [7] to

[10] , wherein the pluripotent stem cells are human pluripotent stem cells.

[0011]

[12] A method for producing the transplant sheet described in any one of [4] to [6], comprising the steps of: incising the inner cavity of the skin organoid described in any one of [1] to [3]; and inverting the skin organoid so that the surface facing the inner cavity is exposed on the outer surface of the skin organoid, thereby obtaining the transplant sheet having a layer structure in which the epidermal cell layer is backed by the mesenchymal cell layer.

[0012]

[13] Full-thickness skin tissue induced from pluripotent stem cells, having skin polarity and comprising functional arrector pili muscles, hair follicles and sweat glands.

[14] Full-thickness skin tissue according to

[13] , comprising immune cells.

[15] Full-thickness skin tissue according to

[14] , wherein the immune cells are Langerhans cells or macrophages.

[16] Full-thickness skin tissue according to any one of

[13] to

[15] , wherein the pluripotent stem cells are human pluripotent stem cells.

[0013]

[17] A method for producing a full-thickness skin tissue according to any one of

[13] to

[16] , comprising: (1) incising the inner cavity of the skin organoid according to any one of [1] to [3], and inverting the skin organoid so that the surface facing the inner cavity is exposed on the outer surface of the skin organoid, thereby obtaining a transplant sheet having a layer structure in which the epidermal cell layer is backed by the mesenchymal cell layer; (2) transplanting the transplant sheet to a skin defect in a living body, wherein the transplant sheet is arranged so that the mesenchymal cell layer contacts the biological surface at the transplantation site and the epidermal cell layer is exposed; (3) covering the transplant sheet with a low gas-permeable substrate and maintaining the transplant sheet in a moist environment in the space between the biological surface and the low gas-permeable substrate; (4) removing the low gas-permeable substrate, covering the transplant sheet with a high gas-permeable substrate and maintaining the transplant sheet in a moist environment in the space between the biological surface and the high gas-permeable substrate; (5) A manufacturing method comprising the steps of: (1) removing the highly gas-permeable substrate and maintaining the transplant sheet at the transplant site while aerating it; and (2) re-covering the transplant sheet with the highly gas-permeable substrate and maintaining the transplant sheet in a moist environment in the space between the biological surface and the highly gas-permeable substrate.

[18] The manufacturing method according to

[17] , wherein the transplant sheet is treated with BMP in step (5) or in steps (5) and (6).

[19] The manufacturing method according to

[18] , wherein the BMP treatment inhibits the formation of hair follicles and promotes the formation of sweat glands in the full-thickness skin tissue.

[20] The manufacturing method according to

[18] or

[19] , wherein the BMP is one or more selected from BMP2, BMP4, and BMP5, preferably BMP2 and BMP4.

[0014]

[21] Use of the skin organoid described in any one of [1] to [3], the transplant sheet described in any one of [4] to [6], or the full-thickness skin tissue described in any one of

[13] to

[16] as an alternative to animal experiments.

[22] Use of the skin organoid described in any one of [1] to [3], the transplant sheet described in any one of [4] to [6], or the full-thickness skin tissue described in any one of

[13] to

[16] for regenerative medicine.

[23] A method for producing a chimeric non-human animal for evaluating the response of human skin to a test substance, the method comprising the step of transplanting the transplant sheet described in any one of [4] to [6] into a non-human animal, preferably an immunodeficient non-human animal that lacks adaptive immunity.

[24] A chimeric non-human animal for evaluating the response of human skin to a test substance, the method comprising transplanting the transplant sheet described in any one of [4] to [6] into a non-human animal, preferably an immunodeficient animal that lacks adaptive immunity.

[25] A method for testing the toxicity of a test substance, comprising the steps of administering the test substance to the chimeric non-human animal described in

[24] and evaluating the effect of the test substance on human skin (full-thickness skin tissue).

[26] A method for producing human skin (full-thickness skin tissue) that mimics a disease state, obtained by treating the chimeric non-human animal described in

[24] with a drug, mechanical injury, a pathogenic organism, genome editing, etc.

[27] A chimeric non-human animal described in

[24] that has been treated with a drug, mechanical injury, a pathogenic organism, genome editing, etc., and has human skin (full-thickness skin tissue) that mimics a disease state.

[28] A method for evaluating the effectiveness of the test substance against a disease state, comprising the steps of administering a test substance to the chimeric non-human animal described in

[27] and evaluating the effect of the test substance on human skin (full-thickness skin tissue) that mimics the disease state.

[29] The method of

[28] , wherein the treatment with the drug is topical application of imiquimod to human skin (full thickness skin tissue) to mimic the disease state, and the human skin (full thickness skin tissue) is used as a psoriasis-like model.

[0015] [Definition] In the present disclosure, "skin organoid" refers to a cell aggregate induced to differentiate ex vivo from pluripotent stem cells or embryonic stem cells, comprising a cell population capable of differentiating into the epidermis and a cell population capable of differentiating into the dermis or subcutaneous tissue. "Skin organoid" is preferably a cell aggregate having an inner lumen, with a layer of a cell population capable of differentiating into the epidermis (epidermal cell layer) on the surface (inner surface) facing the lumen, and a layer of a cell population capable of differentiating into the dermis or subcutaneous tissue (mesenchymal cell layer) on the outer surface of the epidermal cell layer. "Skin organoid" more preferably has an epidermal cell layer consisting of the stratum granulosum, stratum spinosum, and stratum basale layer stacked in this order from the inner surface side. A "transplant sheet" is obtained by incising the inner lumen of a skin organoid and inverting the skin organoid so that the epidermal cell layer is exposed on the outer surface of the skin organoid, and has a layered structure in which the epidermal cell layer is backed by a mesenchymal cell layer.

[0016] "Full-thickness skin tissue" refers to a tissue structure induced to differentiate from skin organoids or transplant sheets ex vivo, in which tissues comprising cells constituting the epidermis, cells constituting the dermis, and cells constituting the subcutaneous tissue are arranged in a hierarchical manner. The epidermis of the "full-thickness skin tissue" preferably comprises cells constituting the stratum corneum and / or stratum lucidum, cells constituting the granular layer, cells constituting the stratum spinosum, and cells constituting the basal layer. The dermis of the "full-thickness skin tissue" preferably comprises cells constituting the papillary dermis and cells constituting the reticular dermis. The subcutaneous tissue of the "full-thickness skin tissue" preferably has a fat lobule structure in which accumulations of fat cells containing lipid droplets are separated into lobules by connective tissue fat septa. The "full-thickness skin tissue" additionally comprises skin appendages including hair follicles and sweat glands.

[0017] "Cells that make up the epidermis" primarily refer to keratinocytes, but may also include melanocytes, Langerhans cells, and Merkel cells. Keratinocytes make up the majority of the epidermis and produce keratin, providing the skin's barrier function. Melanocytes reside in the basal layer and produce melanin to protect the skin from ultraviolet rays. Langerhans cells are immune cells found primarily in the spinous layer and function as antigen-presenting cells. Langerhans cells are identified by the expression of Langerin (CD207). Merkel cells reside in the basal layer and function as sensory receptors, sensing touch and pressure.

[0018] A "cell population capable of differentiating into epidermis" refers to cells capable of differentiating into at least one of the above-mentioned cells that constitute the epidermis, and is identified by the expression of at least one marker among the markers of cells that constitute the epidermis. Markers for cells that constitute the basal layer, spinous layer, and granular layer include the following. Cells that constitute the stratum lucidum and stratum corneum can be identified by enucleation.

[0019] Basal layer markers KRT5 and KRT14: Keratins specifically expressed in basal layer cells. p63: A transcription factor of the p53 family that plays an important role in the proliferation and differentiation of keratinocytes. A marker for epidermal stem cells present in the basal layer. p63 is also a marker for surface ectoderm. Spinous layer markers KRT1 and KRT10: Keratins specifically expressed in spinous layer cells. Granular layer marker Loricrin: Synthesized in large quantities in granular layer cells, it plays an important role in the cornification of keratinocytes (the cornification process).

[0020] "Cells that make up the dermis" primarily refer to fibroblasts, and may also include immune cells, vascular endothelial cells, smooth muscle cells, and nerve cells. Immune cells may include macrophages, mast cells, and dendritic cells. "Cells that make up the subcutaneous tissue" primarily refer to adipocytes and fibroblasts. In addition, they may also include immune cells, vascular endothelial cells, smooth muscle cells, and nerve cells, similar to the cells that make up the dermis. The subcutaneous tissue contains large lipid droplets (mainly composed of lipid triglycerides) and exhibits lobulated structures called fat lobules.

[0021] "Cells capable of differentiating into dermis or subcutaneous tissue" refer to cells capable of differentiating into at least one of the cells that make up the dermis or subcutaneous tissue described above, and are identified by the expression of at least one marker among the markers of cells that make up the dermis or subcutaneous tissue. Examples of markers include the following. Adipocytes can be identified by lipid droplet staining.

[0022] Markers for fibroblasts include fibronectin and platelet-derived growth factor receptor (PDGFR), and markers for smooth muscle (arrector pili) cells include α-Smooth Muscle Actin (α-SMA) and integrin alpha-8 (ITAG8).

[0023] Other markers used in the present disclosure are also described below. E-cadherin (epicercadherin) is a protein involved in cell adhesion in epithelial cells, primarily involved in cell-cell adhesion in epithelial tissues. E-cadherin is a marker for epithelial cells in skin and other epithelial tissues. TFAP2 (Transcription Factor AP-2) is a transcription factor involved in the differentiation and proliferation of various cells. TFAP2 is a marker for epidermal ectoderm and epidermal basal cells. CD49f (integrin α6) is involved in cell adhesion. CD49f is a marker for epidermal stem cells present in the basal layer of the epidermis, and is also expressed in hemidesmosomes, which are essential for basal layer keratinocytes to adhere to the basement membrane, making it a marker for the boundary between the epidermis and dermis. AQP5 (aquaporin 5) is a water channel protein and a marker for sweat glands in the skin.

[0024] "Skin polarity" refers to the orientation of cells or cell layers in normal skin, specifically the orientation of cells or cell layers with the epidermis on the apical side and the subcutaneous tissue on the basal side. For example, in the epidermis, the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale are arranged from apical to basal. Skin polarity also refers to the orientation of cells or cell layers in developing skin, and when certain cell types or cell layers are missing due to underdevelopment at certain developmental stages, it refers to the orientation of the other cells or cell layers.

[0025] "Functional arrector pili muscles" refer to muscles that express α-SMA and ITAG8 and are connected to the hair bulb. Hair is composed of the hair shaft, hair root, and hair bulb, and the hair bulb is located at the bottom of the hair root and is the site where hair growth is most active.

[0026] A "hair pile" is a rod-shaped structure formed when the epidermis invaginates into the dermis in the early stages of hair formation. Hair development is induced by the interaction between the epidermis and dermis, with the epidermis first proliferating and thickening locally and then beginning to grow toward the dermis. As the epidermis penetrates deeper into the dermis, a rod-shaped structure called a hair pile is formed. The hair pile is the primordium of the hair follicle and the starting point for the formation of the hair root and hair bulb.

[0027] The present disclosure provides techniques that can be used to obtain skin organoids or skin tissue structures from stem cells that highly mimic the structure and function of in vivo skin.

[0028] This shows an overview of the process for inducing differentiation of skin organoids from human iPS cells (iPSCs). Bright-field microscopy images of iPSC aggregates on Day 0. Bright-field microscopy images of cell aggregates on Day 3. Bright-field microscopy images of cell aggregates on Days 6 and 12. Bright-field microscopy images of cell aggregates (skin organoids) on Day 30. Fluorescent immunostained images of the epidermal and mesenchymal cell layers of cell aggregates (skin organoids) on Day 30. In (A), green fluorescence indicates PDGFRα (a fibroblast marker), and white fluorescence indicates E-cadherin (an epithelial cell marker). In (B), blue fluorescence indicates nuclear staining with Hoechst 33342, green fluorescence indicates KRT5 (a basal layer marker), and white fluorescence indicates E-cadherin. In (C), blue fluorescence indicates nuclear staining with Hoechst 33342, green fluorescence indicates p63 (an epidermal stem cell marker), and white fluorescence indicates E-cadherin. Bright-field microscopy images of skin organoids on day 70. (A) and (B) show immunofluorescent images of the epidermal cell layer and mesenchymal cell layer of skin organoids on day 70, and (C) shows a hematoxylin-eosin stained image. In (A), blue fluorescence indicates nuclear staining with Hoechst 33342, green fluorescence indicates KRT5 (a basal layer marker), and white fluorescence indicates KRT10 (a spinous layer marker). In (B), blue fluorescence indicates nuclear staining with Hoechst 33342, red fluorescence indicates p63 (an epidermal stem cell marker), green fluorescence indicates E-cadherin (an epithelial cell marker), and white fluorescence indicates loricrin (a granular layer marker). (A) A bright-field stereomicroscope image and (B) a fluorescent immunostained image of skin organoids on Day 70. The arrowheads indicate hair follicles. In (B), blue fluorescence indicates nuclear staining with Hoechst 33342, green fluorescence indicates fibronectin (a fibroblast marker), and white fluorescence indicates E-cadherin (an epithelial cell marker). (A) A bright-field microscope image of skin organoids generated on Day -1 with or without CHIR99021 treatment (+), (B) a measurement of the area of ​​the lumen formed by the epidermal cell layer, and (C) a hematoxylin-eosin stained image.Brightfield microscopy images of cell aggregates (human iPS cell line A) on day 3 from skin organoids generated on day -1 without treatment (0 μM) or with treatment at 1 μM, 1.5 μM, or 3 μM CHIR99021 (Scale bar: 200 μm). Brightfield microscopy images of cell aggregates (human iPS cell line BF) on day 3 from skin organoids generated on day -1 with treatment at 1 μM CHIR99021 (Scale bar: 200 μm). Fluorescent immunostaining images of cell aggregates (human iPS cell line A) on day 3 from skin organoids generated on day -1 with treatment at 1 μM CHIR99021 (Scale bar: 200 μm). Green fluorescence indicates E-cadherin (an epithelial cell marker), red fluorescence indicates TFAP2 (a marker for epidermal ectoderm and epidermal basal cells), white fluorescence indicates SOX2, and blue fluorescence indicates nuclear staining with DAPI. This figure shows an overview of the process for inducing differentiation of skin organoids into full-thickness skin tissue. This figure explains the pretreatment process for skin organoids used for cranial window transplantation. The dotted line on the left indicates the border width between the head and tail portions, and this width is indicated by the symbol H. The dotted lines on the right indicate the incision (slit) site for the lumen of the head portion and the partial cut site for the tail portion. This figure shows skin organoids (0 dpt) transplanted into a cranial window. The area surrounded by dotted lines is the epidermal cell layer of the two transplanted skin organoids. This figure shows a photograph of skin organoids (28 dpt). This figure explains the procedure for covering skin organoids with a dressing. This figure explains the scab formed on the surface of the skin organoid (top) and the procedure for re-covering the skin organoid with a dressing (bottom). This figure shows a photograph of full-thickness skin tissue (74 dpt). The left is a top view, and the right is a lateral view. Hematoxylin and eosin stained images of full-thickness skin tissue (74 dpt). HF: hair follicle, SG: sebaceous gland, SwG: sweat gland. Immunostained images of full-thickness skin tissue (74 dpt). In (A), blue fluorescence indicates nuclear staining with Hoechst 33342, and red fluorescence indicates fibronectin (a fibroblast marker).In (B), blue fluorescence indicates nuclear staining with Hoechst 33342, and red fluorescence indicates PDGFRα (a fibroblast marker). In (C), green fluorescence indicates Venus (iPSC-derived), and red fluorescence indicates lipid droplets. This is an immunostained image of a hair follicle from full-thickness skin tissue (74 dpt). Green fluorescence indicates Venus (iPSC-derived), white fluorescence indicates SMA (a smooth muscle cell marker), and red fluorescence indicates ITAG8. This is an immunostained image of a sweat gland (SwG) from full-thickness skin tissue (74 dpt). Green fluorescence indicates Venus (iPSC-derived), blue fluorescence indicates nuclear staining with Hoechst 33342, and red fluorescence indicates AQP5 (a sweat gland marker). This is an immunostained image of full-thickness skin tissue (77 dpt). (B) is an enlarged view of the area enclosed by the dotted line in (A). The solid line indicates the boundary between the epidermis and dermis. Blue fluorescence indicates nuclear staining with Hoechst 33342, and red fluorescence indicates Langerin (a Langerhans cell marker). These are hematoxylin-eosin stained images (top row) and immunostained images (middle and bottom rows) of full-thickness skin tissue (B) with the surface treated with imiquimod (IMQ) and untreated full-thickness skin tissue (A). Blue fluorescence indicates nuclear staining with Hoechst 33342, red fluorescence indicates phosphorylated STAT3 (a cell hyperproliferation marker), and green fluorescence indicates CD68 (a macrophage marker). These are hematoxylin-eosin stained images of the epidermal cell layer of full-thickness skin tissue (70 dpt). These are immunostained images of the epidermal cell layer of full-thickness skin tissue (70 dpt). In (A), green fluorescence indicates Venus (iPSC-derived), red fluorescence indicates Loricrin (a granular layer marker), and white fluorescence indicates KRT10 (a spinous layer marker). In (B), blue fluorescence indicates nuclear staining with Hoechst 33342, green fluorescence indicates Venus, red fluorescence indicates CD49f (a basal layer marker), and white fluorescence indicates KRT10 (a spinous layer marker). In (C), blue fluorescence indicates nuclear staining with Hoechst 33342, green fluorescence indicates Venus, and red fluorescence indicates KRT5 (a basal layer marker). Cell clustering results for human-derived cells are shown in single-cell RNA-seq analysis of full-thickness skin tissue (77 dpt).The results of cell clustering for mouse-derived cells in full-thickness skin tissue (77 dpt) by single-cell RNA-seq analysis are shown. (A) Full-thickness skin tissue continued to be covered with a cover glass, while (B) full-thickness skin tissue was covered with a transparent film dressing. The left side of (A) and (B) are top-view photographs (70 dpt). The center of (A) is a photograph of the cross section of the full-thickness skin tissue cut at the center, and the center of (B) is a lateral view. The right side of (A) and (B) are hematoxylin-eosin stained images (70 dpt). BMP beads (5 μg / ml) and BMP beads (50 μg / ml) were embedded in full-thickness skin tissue (56 dpt). Top-view photographs (A) and (B) show the top view of a section of full-thickness skin tissue at 70 dpt, where BMP beads (5 μg / ml) were implanted (position 1) and BMP beads (50 μg / ml) were implanted (position 2). Fluorescent immunostaining images of full-thickness skin tissue (70 dpt) at positions 1 (A) and 2 (B) are shown. In the left image, the central dotted line indicates the boundary between the untreated area (left) and the BMP beads-implanted area (right). The arrowheads indicate sweat glands. The right image is an enlarged image of the sweat gland shown in the left image. Blue fluorescence indicates nuclear staining with Hoechst 33342; white fluorescence indicates E-cadherin (an epithelial cell marker); red fluorescence indicates KRT5 (a basal layer marker); and red fluorescence indicates AQP5 (a sweat gland marker). (A) Photograph (top view) and hematoxylin-eosin stained image of full-thickness skin tissue (70 dpt) derived from iPSCs from a progeria patient and iPSCs from a healthy individual. (B) Fluorescent immunostained images of full-thickness skin tissue (70 dpt) derived from iPSCs from a progeria patient and iPSCs from a healthy individual. White fluorescence indicates E-cadherin (an epithelial cell marker), red fluorescence indicates type XVII collagen (COL17A1, an epidermal stem cell marker), and blue fluorescence indicates nuclear staining with DAPI. Results of a wound healing experiment using full-thickness skin tissue derived from iPSCs from a progeria patient and iPSCs from a healthy individual are shown. (A) Left: Photograph (top view) of full-thickness skin tissue (70 dpt). The area enclosed by the dotted line is the site of a skin punch biopsy. Right: Enlarged view of the biopsy site, showing the wound.(B) is a photograph of full-thickness skin tissue 10 days after wound creation. The left is the biopsy site, and the right is a magnified view. The arrow indicates the remaining wound. Immunostaining of full-thickness skin tissue 10 days after wound creation is shown (Scale bar: 500 μm). Green fluorescence indicates E-cadherin (an epithelial cell marker), red fluorescence indicates keratin 17 (KRT17), and blue fluorescence indicates nuclear staining with DAPI.

[0029] Preferred embodiments for carrying out the present disclosure will be described below with reference to the drawings. Note that the embodiments described below are examples of typical embodiments of the present disclosure, and should not be construed as narrowing the scope of the present disclosure.

[0030] 1. Skin Organoids and Their Manufacturing Method (1) Histological Structure of Skin Organoids The skin organoids disclosed herein are induced in vitro from human pluripotent stem cells (iPSCs) and consist of two regions that are observed at different brightness levels in bright-field observation. Specifically, one portion of the skin organoid contains a fluid-filled space (lumen) and is observed as a bright region in bright-field observation due to the high optical transparency of the lumen (see Figure 6-1). Hereinafter, this portion will be referred to as the "Head portion." The other portion of the skin organoid does not contain a lumen and is observed as a dark region in bright-field observation due to reduced optical transparency caused by the presence of solid cells (see Figure 6-1). Hereinafter, this portion will be referred to as the "Tail portion." The referenced figures are of cell aggregates, skin organoids, or full-thickness skin tissues created under specific conditions in the examples, and the reference to these figures is intended to facilitate understanding of the skin organoids, etc., according to the present disclosure, and is not intended to allow a limited interpretation of the skin organoids, etc., according to the present disclosure.

[0031] In the skin organoid, the head portion and the tail portion are continuous. In one embodiment, the skin organoid can have a gourd-like three-dimensional shape consisting of a head portion and a tail portion having a larger volume than the head portion.

[0032] The tail portion is composed of fibroblasts, which will differentiate into cells that will form the dermis or subcutaneous tissue in the future.

[0033] The lumen of the head portion is formed by a membrane-like (balloon-like) arrangement of epidermal cells containing cells capable of differentiating into epidermis. In other words, the surface facing the lumen (inner surface) is composed of a layer of epidermal cells (epidermal cell layer) containing cells capable of differentiating into epidermis. The epidermal cell layer has a structure characteristic of the developing epidermis, exhibiting a multilayer structure including at least the granular layer (containing loricrin-positive cells), the spinous layer (containing KRT10-positive cells), and the basal layer (containing KRT5 / p63-positive cells) (see Figure 6-2). The granular layer, spinous layer, and basal layer are layered in this order from the lumen side. Hereinafter, the lumen side will be referred to as the "inside" and the opposite side as the "outside."

[0034] The head portion has a layer containing fibroblasts (fibroblast layer) outside the epidermal cell layer (see Figure 6-2). The fibroblast layer of the head portion is continuous with the tail portion, which also contains fibroblasts. Both the fibroblast layer of the head portion and the tail portion will eventually become the dermis or subcutaneous tissue. Hereinafter, the fibroblast layer of the head portion and the tail portion will be collectively referred to as the "mesenchymal cell layer." The mesenchymal cell layer contains cells that have the ability to differentiate into the dermis or subcutaneous tissue.

[0035] Skin organoids contain hair piles, which are precursor tissues of hair follicles. Hair piles are formed when cells from the epidermal cell layer of the head portion invaginate into the mesenchymal cell layer (including the fibroblast layer of the head portion) (see Figures 6-2 and 7).

[0036] Below, the development of the histological structure of skin organoids, along with their production method, is described in more detail.

[0037] (2) Differentiation induction of skin organoids The method for producing skin organoids according to the present disclosure comprises the following steps. An overview of the steps is shown in Figure 1, particularly based on Test Example 1. - Pretreatment step (activation of the Wnt signaling pathway) - Step of inducing differentiation of epidermal ectoderm - Step of promoting differentiation of neural crest cells - Maturation culture

[0038] (2-1) Pretreatment step (activation of the Wnt signaling pathway): In this step, iPSC cell aggregates are cultured in the presence of a substance that activates the Wnt signaling pathway. Activation of the Wnt signaling pathway in iPSCs promotes the development of epidermal cell layers during the differentiation induction step of the next epidermal ectoderm.

[0039] iPSCs refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells," including iPSCs established by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPSCs established by selecting using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPSCs created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.), iPSCs established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28 (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), iPSCs created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and iPSCs created by Sakurada et al. (JP Patent Publication No. 2008-307007).In addition, many other papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7, iPSCs are disclosed in various publications, including the Japanese Patent Application Laid-Open Nos. 2008-307007, 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, and WO2009-007852. In the present disclosure, any of these known iPSCs can be used. Various iPSC lines established by the NIH, RIKEN, Kyoto University, and other institutions can be used. Examples of iPSCs include RIKEN's 1383D6 (HPS1006), WTC-11 (AICS, UCSFi001-A), HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 strains, and Kyoto University's 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, and 648A1 strains. Commercially available iPSC strains are also available, such as the 771-3G strain (Reprocell, RCRP003N). Human iPSCs also include iPSC strains established from cells of patients with various genetic disorders.

[0040] The iPSC aggregates used in this step can be prepared according to conventional techniques. However, it is preferable to form cell aggregates by seeding dispersed iPSCs into 96-well V-bottom plates and culturing them, preferably at a seeding density of 3,500-4,000 cells / well. In V-bottom plates, seeded cells concentrate at a single point at the deepest point of the well bottom, promoting cell aggregate formation and enabling the efficient production of cell aggregates that firmly maintain a spherical shape compared to U-bottom or flat-bottom plates. Furthermore, the inventors' studies have demonstrated that a seeding density of 3,500-4,000 cells / well promotes the differentiation and formation of epidermal ectoderm in the subsequent step.

[0041] The substance that activates the Wnt signaling pathway may be a known substance that belongs to the Wnt ligand or GSK3β inhibitor group. Examples of Wnt ligands include Wnt3 and Wnt1. GSK3β inhibitors include CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile), CHIR98014 (2-[[2-[(5-nitro-6-aminopyridin-2-yl)amino]ethyl]amino]-4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)pyrimidine), TDZD-8 (4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), and TWS-119 (3-[6-(3-aminophenyl)- Examples include 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), SB415286 (3-[(3-chloro-4-hydroxyphenyl)amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), AR-AO144-18, CT99021, CT20026, BIO, BIO-acetoxime, pyridocarbazole-cyclopentadienyl ruthenium complex, OTDZT, alpha-4-dibromoacetophenone, and lithium. The GSK3β inhibitor is not limited to these, and antisense oligonucleotides and siRNAs against GSK3β mRNA, antibodies that bind to GSK3β, dominant-negative GSK3β mutants, etc. may also be used as GSK3β inhibitors. Two or more GSK3β inhibitors may be used in combination.

[0042] The concentration of the substance that activates the Wnt signaling pathway is not particularly limited as long as the desired effect of promoting the development of the epidermal cell layer is obtained, and is adjusted appropriately depending on the type of Wnt signaling pathway activator added. For example, when CHIR99021 is used, the concentration is 0.1 to less than 3.0 μM, preferably 0.1 to 2.0 μM, more preferably 0.1 to 1.5 μM, particularly preferably 0.5 to less than 3.0 μM, even more particularly preferably 0.5 to 2.0 μM, and particularly preferably 1.0 to 1.5 μM.

[0043] The treatment time with the substance that activates the Wnt signaling pathway is not particularly limited as long as the desired effect of promoting the development of the epidermal cell layer is achieved, but is, for example, 6 to 48 hours, preferably 12 to 36 hours, and particularly 24 hours. The treatment with the substance that activates the Wnt signaling pathway is preferably carried out immediately before the step of inducing differentiation of the epidermal ectoderm. In other words, this step and the next step are consecutive steps, and it is preferable that there is no culture in the absence of the substance that activates the Wnt signaling pathway between the two steps.

[0044] The cell aggregates obtained by this process are typically spherical in shape with a diameter of approximately 100-1000 μm (see FIG. 2).

[0045] (2-2) Step of inducing differentiation of epidermal ectoderm In this step, the cell aggregates obtained in the pretreatment step are cultured in the presence of bone morphogenetic protein (BMP), a TGFβ inhibitor, and basic fibroblast growth factor (bFGF) (the day the culture was initiated is referred to as "Day 0"). Treatment with BMP, a TGFβ inhibitor, and bFGF induces and develops epidermal ectoderm on the outermost surface of the cell aggregates (see Figure 3). Cell masses containing neural crest cells (NCCs) are formed within the cell aggregates. The epidermal ectoderm forms a membrane-like (balloon-like) shape that surrounds the neural crest cell mass.

[0046] The inventors' studies have revealed that some iPSC cell lines exhibit insufficient epidermal cell layer formation. By performing the above-described pretreatment step (activation of the Wnt signaling pathway) prior to this step, even cell lines that normally exhibit insufficient epidermal cell layer formation can be enhanced to obtain cell aggregates containing sufficient cells capable of differentiating into epidermis. In particular, by using a Wnt signaling pathway activator at a concentration of 0.5 μM to 2.0 μM, preferably 1.0 μM to 1.5 μM, and particularly 1.0 μM, good epidermal ectoderm formation can be achieved without any differences between cell lines (see Test Example 2).

[0047] The BMP may be BMP4 or a compound with a similar agonistic effect, preferably BMP4. Two or more BMPs may be used in combination. Examples of compounds include 2-[[(4-Bromophenyl)methyl]thio]benzoxazole (SB4), a BMP4 agonist. The concentration of BMP is not particularly limited as long as it allows differentiation and formation of the desired epidermal ectoderm and neural crest cells. It is adjusted appropriately depending on the type of BMP added. For example, when BMP4 is used, the concentration is 0.1-50 ng / ml, preferably 1-10 ng / ml, and particularly 5 ng / ml.

[0048] TGFβ inhibitors can be substances that inhibit the binding of TGFβ to its receptor or substances that inhibit downstream signals after TGFβ binds to the receptor, such as phosphorylation of TGFβ type I receptor by TGFβ type II receptor and phosphorylation of Smad by phosphorylated TGFβ type I receptor. As a TGFβ inhibitor, SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzami de), A83-01 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), LDN1931 89 (4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), GW788388 (4-[4-[3 -(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), SM16 (4 -[4-(1,3-Benzodioxol-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-2-yl]-bicyclo[2.2.2]octane- 1-carboxamide), IN-1130 (3-[[5-(6-Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]met hyl]-benzamide), GW6604 (2-Phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine) and SB505124 (2-[ 4-(1,3-Benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine). Two or more TGFβ inhibitors may be used in combination.

[0049] The concentration of the TGFβ inhibitor is not particularly limited as long as it allows for the differentiation and formation of the desired epidermal ectoderm and neural crest cells, and is adjusted appropriately depending on the type of TGFβ inhibitor added. For example, when SB431542 is used, the concentration is 1-50 μM, preferably 5-20 μM, and particularly 10 μM.

[0050] The concentration of bFGF is not particularly limited as long as it allows differentiation and formation of the desired epidermal ectoderm and neural crest cells, but is, for example, 0.1-50 ng / ml, preferably 1-10 ng / ml, and particularly 4 ng / ml.

[0051] The treatment time with BMP, TGFβ inhibitor, and bFGF is not particularly limited as long as it allows differentiation and formation of the desired epidermal ectoderm and neural crest cells, but is, for example, 1-5 days, preferably 2-4 days, and particularly 3 days (up to Day 3).

[0052] The cell aggregates obtained by this process are typically spherical in shape with a diameter of approximately 200-1500 μm (see FIG. 3).

[0053] (2-3) Step of Promoting Neural Crest Cell Differentiation In this step, the cell aggregates obtained in step (2-2) are cultured in the presence of an ALK inhibitor and bFGF. Treatment with the ALK inhibitor and bFGF promotes neural crest cell differentiation. In this step, neural crest cells present inside the cell aggregates migrate further outward from the epidermal ectoderm (see the left diagram in Figure 4). The cell aggregates change from a state in which a neural crest cell mass is present inside and a membrane (balloon) of epidermal ectoderm is present on the outside (see the left diagram in Figure 4) to a state in which a membrane of epidermal ectoderm is present inside and a layer of neural crest cells is present on the outside (see the right diagram in Figure 4).

[0054] ALK inhibitors include SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide), A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), an ALK4,5,7 inhibitor, and LDN193189 (4-[6-(4-piperazin-1-yl-phenyl)-pyrazolo[1,5-α]pyrimidin-3-yl]-quinoline), an ALK2,3,6 inhibitor. hydrochloride), 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine, ALK5 inhibitor Wnt3a / BIO, BMP4, GW788388 (4-[4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl]-N-(tetrahydro-2H-pyran-4-yl)benzamide), SM16, IN-1130 (3-((5-(6- Examples of ALK inhibitors that can be used include (2-phenyl-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide), GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine), and SB505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride). ALK inhibitors are not limited to these, and antisense oligonucleotides and siRNAs against ALK mRNA, antibodies that bind to ALK, and dominant-negative ALK mutants can also be used. Two or more ALK inhibitors may also be used in combination.

[0055] The concentration of the ALK inhibitor is not particularly limited as long as it promotes neural crest cell differentiation and is adjusted appropriately depending on the type of ALK inhibitor added. For example, when LDN193189 is used, the concentration is 0.005-1 μM, more preferably 0.01-0.5 μM, and particularly 0.03-0.2 μM. The concentration of the ALK inhibitor may be constant or variable within these concentration ranges, preferably decreasing stepwise. In a preferred embodiment of the stepwise concentration change, the starting concentration on Days 1-4 (Days 3-6) is (e.g., 0.2 μM), and the concentration is approximately 1 / 1.6 of the starting concentration (e.g., 0.125 μM) on Days 4-6 (Days 6-8), approximately half of that concentration (e.g., 0.062 μM) on Days 6-8 (Days 8-10), and approximately half of that concentration (e.g., 0.031 μM) on Days 8-10 (Days 10-12).

[0056] The concentration of bFGF is not particularly limited as long as it achieves the desired differentiation and migration of neural crest cells, but is, for example, 2-250 ng / ml, more preferably 4-100 ng / ml, and particularly 8-50 ng / ml. The bFGF concentration may be constant or variable within these ranges, preferably decreasing stepwise. In a preferred embodiment of this stepwise change in concentration, the starting concentration on Days 1-4 (Days 3-6) is (e.g., 53.2 ng / ml), followed by approximately 1 / 1.6 of the starting concentration (e.g., 33.25 ng / ml) on Days 4-6 (Days 6-8), approximately half of that concentration (e.g., 16.6 ng / ml) on Days 6-8 (Days 8-10), and approximately half of that concentration (e.g., 8.3 ng / ml) on Days 8-10 (Days 10-12).

[0057] In addition to the ALK inhibitor and bFGF, the culture in this step may also contain a BMP and a TGFβ inhibitor. The BMP and TGFβ inhibitor may be contained in the culture medium used in step (2-2) and used for the culture in this step by bringing the culture medium into this step together with the cell aggregates, or may be newly added to the culture medium in this step.

[0058] The treatment time with the ALK inhibitor and bFGF is not particularly limited as long as the desired differentiation and migration of neural crest cells is achieved, but is, for example, 6-12 days, preferably 8-10 days, and particularly 9 days (Days 3-12).

[0059] (2-4) Maturation Culture: In this step, the cell aggregates obtained in step (2-3) are cultured for maturation in the absence of substances that activate the Wnt signaling pathway, BMP, TGFβ inhibitors, bFGF, and ALK2 / 3 inhibitors to obtain skin organoids. During maturation culture, the cell aggregates develop into skin organoids, including tissue regions that differentiate into the epidermis, dermis, and subcutaneous tissue. Specifically, the epidermal cell layer first gradually thickens, and the lumen formed by the epidermal cell layer expands, forming a head portion (see Figure 5-1). The epidermal cell layer exhibits a layered structure characteristic of the epidermis during embryonic development, consisting of a basal layer (KRT5, p63 positive) and a periderm (E-cadherin positive) (see Figure 5-2). Furthermore, the epidermal cell layer exhibits a layered structure characteristic of the epidermis of a more developed embryo, consisting of a basal layer (KRT5 positive), a spinous layer (KRT10 positive), and a granular layer (Loricrin positive) (see Figure 6-2). On the opposite side of the head portion, a tail portion is formed, which contains neural crest cell-derived dermal fibroblasts (see Figure 5-1). The tail portion is continuous with the fibroblast layer adjacent to the epidermal cell layer of the head portion (see Figure 6-2). Together, the fibroblast layers of the tail and head portions constitute a mesenchymal cell layer that will eventually form the dermis and subcutaneous tissue. Furthermore, hair pegs, which are the initial hair follicle tissue, are formed in the epidermal cell layer. The hair pegs are formed by invaginating from the epidermal cell layer into the mesenchymal cell layer (fibroblast layer) (see Figure 6-2 and arrowheads in Figure 7).

[0060] Maturation culture can be performed using a conventional, general-purpose medium, such as a 1:1 mixture of Advanced-DMEM / F12 (Gibco) and Neurobasal medium (Gibco), supplemented with B-27 minus vitamin A supplement (Gibco, final concentration 0.5x), N-2 supplement (Gibco, final concentration 0.5x), GlutaMax (Gibco, final concentration 1x), 2-Mercaptoethanol (Wako, final concentration 0.1 mM), and Normocin (InvivoGen, final concentration 100 μg / ml).

[0061] The period of maturation culture is not particularly limited as long as the desired skin organoids are formed, but is, for example, about 40-80 days, preferably about 50-70 days, and particularly about 58 days (Day 12-70).

[0062] At the start of this process, during this process, or at the end of this process, the cell aggregates (or skin organoids) to be cultured may be selected based on a certain indicator. Examples of indicators include the formation of a head portion or a hair pile. For example, by selecting cell aggregates whose head portion accounts for a certain percentage (e.g., 40% or more) of the total at the start of this process and subjecting them to maturation culture, skin organoids with good histological characteristics as described above can be obtained. Furthermore, for example, by selecting skin organoids with hair piles formed in the epidermal cell layer at the end of this process and with a certain boundary width between the head portion and the tail portion (symbol H in the left diagram of Figure 10) (e.g., 2 mm or more) and subjecting them to the next process, full-thickness skin tissue with good histological characteristics as described below can be obtained.

[0063] 2. Full-thickness skin tissue and its manufacturing method (1) Histological structure of full-thickness skin tissue The full-thickness skin tissue according to the present disclosure is derived from human pluripotent stem cells and can be obtained by transplanting the above-described skin organoids into a skin defect in a living organism (host) and growing them in the transplanted area. The full-thickness skin tissue is composed of the epidermis, dermis, and subcutaneous tissue, and has skin polarity (see Figure 16-1). The full-thickness skin tissue also includes functional arrector pili muscles (see Figure 17-1), hair follicles, sweat glands, and sebaceous glands (see Figure 16-1, symbol SwG: sweat gland).

[0064] The epidermis of full-thickness skin tissue has a four-layer structure consisting of the basal layer, spinous layer, and granular layer, which are differentiated from the epidermal cell layer of the skin organoid, and a layer composed of enucleated cells (corresponding to the stratum corneum or stratum lucidum), and also contains a dermis, which is differentiated from the mesenchymal cell layer of the skin organoid (Figure 18-1).

[0065] Skin organoids have spaced hair follicles that grow hair all the way to the surface (see Figure 15). The hair follicles invaginate from the epidermis and reside in the dermis and subcutaneous tissue (see Figure 16-1, symbol HF: hair follicle). "Spaced hair follicles" refer to hair follicles that grow hair at regular intervals, as observed in living skin, rather than hair follicles that form densely packed or ingrown hairs. The hair bulb of the hair follicle is connected to a functional arrector pili muscle that expresses SMA (smooth muscle actin) and ITAG8 (see Figure 17-1).

[0066] Sweat glands (SwG) invaginate from the epidermis and are present in the dermis and subcutaneous tissue, forming a tubular structure (see Figures 16-1 and 17-2). Sebaceous glands (SG) also invaginate from the epidermis and are present in the dermis and subcutaneous tissue, forming a tubular structure.

[0067] The full-thickness skin tissue may also contain immune cells (see Figure 17-3). The immune cells are presumed to be host-derived and infiltrate from the host tissue into the epidermis of the full-thickness skin tissue during the "maintenance process by covering with a highly gas-permeable substrate" described below. The immune cells may include Langerhans cells, macrophages, dendritic cells, mast cells, neutrophils, lymphocytes, and the like. Because these immune cells are involved in the inflammatory response in full-thickness skin organoids (see Figure 17-4), full-thickness skin organoids may serve as model skin tissue equipped with the immune system of normal skin or an immune system that closely mimics it.

[0068] (2) Differentiation induction of full-thickness skin tissue The development of the histological structure of full-thickness skin tissue will be described in more detail below, along with its manufacturing method. The manufacturing method of full-thickness skin tissue according to the present disclosure comprises the following steps. An overview of the steps is shown in Figure 9, particularly based on Test Example 3. - Pretreatment step (preparation of transplant sheet) - Transplantation step - Maintenance step by covering with low gas-permeable substrate - Maintenance step by covering with high gas-permeable substrate - Aeration step - Maintenance step by re-covering with high gas-permeable substrate - Recovery of full-thickness skin tissue

[0069] (2-1) Pretreatment step (preparation of transplant sheet) In this step, the lumen of the above-mentioned skin organoid is cut open, and the skin organoid is inverted so that the epidermal cell layer is exposed on the outer surface of the skin organoid, thereby obtaining a transplant sheet having a structure in which the epidermal cell layer is backed by a mesenchymal cell layer.

[0070] The head portion of the skin organoid exhibits a layered structure with an epidermal cell layer (apical side) on the lumen side and a mesenchymal cell layer (basal side) on the outer side, exhibiting a layered structure with the polarity of normal skin reversed. Slits are made on all four sides of the head portion to open up the lumen of the skin organoid (see Figure 10), and the polarity of the skin organoid tissue is reversed by cutting further until the epidermal cell layer is autonomously exposed to the outside. This results in a sheet-shaped skin organoid (transplant sheet) with a layered structure in which the epidermal cell layer is backed by a mesenchymal cell layer. The tail portion may be cut in a straight line parallel to the interface with the head portion, and all or part of it may be excised (see Figure 10).

[0071] (2-2) Transplantation Step In this step, the transplant sheet is transplanted into the skin defect area of ​​a living body (host). More specifically, the transplant sheet is placed at the transplantation site so that the mesenchymal cell layer of the skin organoid contacts the surface of the living body and the epidermal cell layer is exposed and visible from the outside (see Figure 11).

[0072] The skin defect to be transplanted must be configured to supply the nutrients and oxygen necessary for the growth of skin organoids. The skin defect is preferably a site where at least the epidermis of the skin is deleted to expose the dermis, or a site where the epidermis and dermis are deleted to expose the subcutaneous tissue. Furthermore, a cranial window (a site where the skin of the head is removed together with the skull to expose the brain surface) can also be used as the skin defect.

[0073] The host is not particularly limited, but is preferably an immunodeficient animal in order to ensure good survival of the transplant sheet.

[0074] (2-3) Maintenance step by covering with a low-gas-permeable substrate: In this step, the transplant sheet is covered with a low-gas-permeable substrate, and the transplant sheet is maintained in a moist environment in the space between the biological surface and the low-gas-permeable substrate, allowing the dermal organoids to grow. Covering with a low-gas-permeable substrate stably holds the dermal organoids at the transplant site and contributes to maintaining the polarity of the dermal organoids.

[0075] The low-gas-permeable substrate is preferably made of a material that does not allow water vapor to pass through in order to maintain the skin organoids in a humid environment, or that has lower water vapor permeability than the highly gas-permeable substrate described below. Furthermore, it is preferable that the substrate be made of a transparent material so that the transplant site can be viewed from the outside. Low-gas-permeable substrates include glass, polydimethylsiloxane (PDMS), etc., and general-purpose cover glass is particularly suitable. In this process, even if a highly gas-permeable substrate described below is used instead of the low-gas-permeable substrate, it is possible to obtain the same effect as when a low-gas-permeable substrate is used. In other words, even if a highly gas-permeable substrate is used, it is possible to maintain a humid environment in the space between the biological surface and the substrate, allowing skin organoids to grow, and achieving the effect of maintaining the polarity of the skin in the skin organoids.

[0076] If necessary, an aqueous solvent such as saline is injected into the space between the biological surface and the low-gas-permeable substrate, and the space is sealed with an adhesive to create a moist environment. The aqueous solvent is not limited to saline, and various buffer solutions and hydrogels may also be used, as long as it can maintain the skin organoids in a moist environment and does not adversely affect cell viability. The saline solution may be absorbed into absorbent cotton and placed in the space.

[0077] This step is carried out for, for example, 24-32 days, preferably 26-30 days, and particularly 28 days (up to 28 dpt), with the day of transplantation being 0 day post-transplantation (dpt).

[0078] In this process, pigmentation of hair follicles is observed in the epidermal cell layer of the skin organoids (see Figure 12), hair follicles elongate toward the surface of the epidermal cell layer, and the invasion of biological blood vessels into the skin organoids is observed.

[0079] (2-4) Maintenance step by covering with a highly gas-permeable substrate: In this step, the low-gas-permeable substrate is removed, and the transplant sheet is covered with a highly gas-permeable substrate. The transplant sheet is maintained in a humid environment in the space between the biological surface and the highly gas-permeable substrate, and dermal organoids are grown (see FIG. 13). Switching from the low-gas-permeable substrate to the highly gas-permeable substrate allows the ambient air that has permeated the substrate to come into contact with the epidermal cell layer, thereby helping to maintain the polarity of the skin of the dermal organoids and promoting the growth of hair follicles. By the completion of this step, the dermal organoids are expected to have grown to possess all of the histological characteristics of the full-thickness skin tissue disclosed herein.

[0080] The highly gas-permeable substrate is made of a material that is more air permeable than the low-gas-permeable substrate described above and that can maintain skin organoids in a moist environment. A wound dressing used to cover and protect wounds can be used as the highly gas-permeable substrate. The dressing is preferably made of a transparent material so that the transplant site can be viewed from the outside. Dressings include traditional gauze and, more recently, hydrocolloids, hydrogels, foam dressings, and film dressings. However, transparent, thin film dressings are preferred from the perspective of high gas permeability. Commercially available transparent film dressings can be used, such as Tegaderm (3M).

[0081] If necessary, an aqueous solvent such as saline is injected into the space between the biological surface and the highly gas-permeable substrate, and the space is sealed using an adhesive. The aqueous solvent is not limited to saline, and various buffer solutions and hydrogels may be used as long as it can maintain the skin organoids in a moist environment and does not adversely affect cell viability. The saline solution may be absorbed into absorbent cotton and placed in the space.

[0082] This step may last, for example, 15-27 days, preferably 18-24 days, and particularly 21 days (up to 49 dpt).

[0083] During this process, the epidermal cell layer becomes pigmented and reveals the hair bulb and root within the hair follicle.

[0084] (2-5) Aeration Step In this step, the highly gas-permeable substrate is removed, and the transplant sheet (full-thickness skin tissue) is further maintained at the transplant site in a state where it is in direct contact with the outside air.

[0085] When the highly gas-permeable substrate is removed, the full-thickness skin tissue dries and a scab forms (see the upper part of Figure 14), so during this step, it is preferable to excise the scab that forms using a microciser. The excision of the scab may be performed multiple times as necessary.

[0086] This step may last, for example, 10-18 days, preferably 12-16 days, and particularly 14 days (up to 63 dpt).

[0087] In this step, the full-thickness skin tissue may be treated with BMP. BMP treatment makes it possible to induce sweat glands more preferentially than hair follicles. It is expected that the higher the treatment concentration of BMP, the more hair follicle formation is suppressed and the more sweat gland formation is promoted. By adjusting the treatment concentration of BMP, it is possible to obtain full-thickness skin tissue with a desired ratio of hair follicles to sweat glands. When the full-thickness skin tissue obtained by the present disclosure is used as a substitute for skin in a living body for transplantation or testing, setting the ratio of hair follicles to sweat glands in the full-thickness skin tissue to a value close to that of the skin being replaced can be useful for improving the effectiveness of transplantation or testing.

[0088] BMP treatment can be performed by applying or dropping a BMP solution onto the surface of full-thickness skin tissue, or by injecting a BMP solution into the surface of full-thickness skin tissue. Alternatively, BMP treatment can be performed by incising the surface of full-thickness skin tissue and embedding a carrier (e.g., microbeads) impregnated with BMP so that the BMP is continuously released from the carrier into the full-thickness skin tissue.

[0089] The BMP may be one or more selected from BMP2, BMP4, and BMP5, but preferably BMP2 and BMP4 are used in combination.

[0090] The duration of the BMP treatment may be part of or the entire duration of the aeration step. Furthermore, when the BMP is administered by a method in which the BMP is continuously released into the full-thickness skin tissue by embedding microbeads or the like, the end of the BMP treatment period may be any timing before the full-thickness skin tissue is harvested.

[0091] (2-6) Maintenance step by re-covering with highly gas-permeable substrate In this step, the transplant sheet is covered with a highly gas-permeable substrate, and the transplant sheet is maintained in a moist environment in the space between the body surface and the highly gas-permeable substrate, allowing further growth of full-thickness skin tissue.

[0092] This process may last for, for example, 10-18 days, preferably 12-16 days, and particularly 14 days (up to 77 dpt). After completion of this process, the full-thickness skin tissue is excised from the graft site and collected.

[0093] 3. Use of Skin Organoids, Transplant Sheets, and Full-Thickness Skin Tissue The skin organoids, transplant sheets, and full-thickness skin tissues disclosed herein have the histological structure characteristic of human skin described above, and can be suitably used as an alternative to animal testing and in applications such as regenerative medicine.

[0094] To evaluate the effects of test substances such as chemicals, pharmaceuticals, and cosmetics on human skin, various tests such as skin toxicity tests, skin allergy tests, skin absorption tests, and wound healing tests are conducted using laboratory animals such as mice, rats, guinea pigs, rabbits, and minipigs. The skin organoids, transplant sheets, and full-thickness skin tissues according to the present disclosure can be used in place of animal skin in these tests, and when skin organoids and transplant sheets are used, conventional in vivo tests can also be replaced with in vitro tests.

[0095] Furthermore, by using a chimeric non-human animal in which a transplant sheet according to the present disclosure has been transplanted into an experimental animal to form human full-thickness skin tissue, the effects of a test substance on human skin can be evaluated more accurately than in conventional tests using experimental animals. In this case, it is preferable to use an immunodeficient non-human animal that lacks adaptive immunity as the experimental animal. A disease model animal that simulates a human disease may also be used as the experimental animal. Various disease model animals are available, and disease model animals for diseases related to the skin, such as allergic dermatitis, pruritus, liver disease, vascular disease, and diabetes, can be used. By using a chimeric non-human animal in which a transplant sheet according to the present disclosure has been transplanted into these disease model animals to form human full-thickness skin tissue, the effects of a disease on the host side on the grafted human full-thickness skin tissue can be evaluated, and further, the effects of a test substance on human full-thickness skin tissue under the pathological condition of the disease can be evaluated.

[0096] Furthermore, by using chimeric non-human animals grafted with transplant sheets of iPSCs derived from cells of patients with genetic diseases, it is possible to evaluate the effects of test substances on the skin of humans with the genetic disease. Genetic diseases that manifest as skin symptoms include, for example, progeria, epidermolysis bullosa, ichthyosis, incontinentia pigmenti, xeroderma pigmentosum, and tuberous sclerosis.

[0097] In tests using chimeric non-human animals with human full-thickness skin tissue, the test substance may be applied or dropped onto the skin surface of the human full-thickness skin tissue, or administered intradermally or subcutaneously. The test substance may also be administered orally or intravascularly to the chimeric non-human animal. By comparing the condition of the human full-thickness skin tissue between chimeric non-human animals treated with the test substance and untreated chimeric non-human animals, the effects of the test substance on human skin, such as toxicity, can be evaluated and determined.

[0098] Additionally, experimental animals have traditionally been treated with drugs, mechanical injury, pathogenic organisms, genome editing, etc. to create model animals that mimic various human disease states. A chimeric non-human animal, in which a transplant sheet according to the present disclosure has been transplanted to form human full-thickness skin tissue, can be treated with drugs, mechanical injury, pathogenic organisms, genome editing, etc. in the same manner as in the past to create human skin (full-thickness skin tissue) that mimics a disease state. This disease-mimicking human skin can be used to evaluate the effects of a test substance on human skin exhibiting symptoms of the disease. For example, applying the drug imiquimod (IMQ) to the surface of the human full-thickness skin tissue of a chimeric non-human animal can induce skin inflammation and create a psoriasis model. A candidate therapeutic substance can be applied to the skin of the human full-thickness skin tissue or administered internally to this psoriasis model, and the state of the human full-thickness skin tissue can be compared with a control to evaluate and determine the therapeutic effect of the candidate therapeutic substance on psoriasis.

[0099] Autologous cultured epidermis and cell sheets made by culturing mesenchymal stem cells or iPSC-derived epidermal cells in sheet form are currently used as materials for skin regenerative medicine. The skin organoids, transplant sheets, and full-thickness skin tissues disclosed herein are expected to be useful in skin regenerative medicine as an alternative to these regenerative materials.

[0100] [Test Example 1: Differentiation Induction of Skin Organoids] 1. iPS Cell (iPSC) Maintenance Culture iPSCs were cultured in 6-well plates. iPSC lines 1383D6 (RIKEN, HPS1006), 771-3G (Reprocell, RCRP003N), or WTC-11 (AICS, UCSFi001-A) were used. Stem Fit AK02N (Ajinomoto, hereafter referred to as "AK02N") medium was used. Medium changes were performed every other day, and cells were passaged every week. The cell density was adjusted to 80% confluence before passage.

[0101] 2. Preparation of cell suspension for differentiation induction. After confirming that iPSCs in maintenance culture reached 80% confluence, the medium was removed and the cells were washed once with 2 ml of D-PBS (Nacalai). Cells were detached from the plate by adding 1 ml of Accutase (Nacalai) and treating in a 37°C CO2 incubator for 5 minutes. The detached cells were suspended in 1 ml of RPMI1640 and transferred to a 15 ml tube containing 4 ml of RPMI1640, followed by centrifugation at 300 xg for 5 minutes. The supernatant was removed, and the remaining cell pellet was resuspended in 1 ml of AK02N containing 10 μM Y-27632. The cell number was then counted using a hemocytometer. The cells were resuspended in a 15 ml tube, and additional Y-27632 was added to a final concentration of 20 μM to prepare a cell suspension for differentiation induction.

[0102] 3. Induction of iPSC aggregates into skin organoids (1) Formation of iPSC aggregates (Day -2) The differentiation-inducing cell suspension was used to initiate the process for inducing differentiation into skin organoids. An overview of the process is shown in Figure 1.

[0103] Two days before differentiation induction (Day -2), the cell suspension for differentiation induction was dispensed into a 96-well V-bottom plate at 4,000 cells / 100 μl of AK02N medium / well, and the plate was centrifuged at 100 g for 6 minutes to pellet the cells. The plate was then incubated at 37°C in a CO2 incubator for 24 hours (Day -1).

[0104] (2) Activation of the Wnt Signaling Pathway (Day -1) On Day -1, 100 μl of AK02N containing CHIR99021 (Tocris, final concentration 1.5 μM) was added to each well and incubated at 37°C in a CO2 incubator for 24 hours to allow iPSC aggregate formation (Day 0). Bright-field microscopy images of iPSC aggregates on Day 0 are shown in Figure 2.

[0105] (3) Induction of epidermal ectoderm differentiation (Day 0) On Day 0, iPSC aggregates were removed and washed in a 60 mm Petri dish (Nunc) containing 6 ml of D-PBS. After transfer to a 60 mm Petri dish containing 6 ml of Essential 6 medium (Gibco, hereafter referred to as "E6") and acclimated to the medium, a single iPSC aggregate was collected together with 50 μl of E6 and transferred to a 96-well U-bottom plate (Greiner). E6 was prepared as a differentiation-inducing medium by adding BMP4 (R&D, final concentration 5 ng / ml), SB431542 (Selleck, final concentration 10 μM), bFGF (Nacalai, final concentration 4 ng / ml), and Cultrex (R&D, final concentration 2%). 50 μl of this medium was added to each well of a 96-well U-bottom plate containing iPSC aggregates and cultured in a CO2 incubator at 37°C for 3 days (Day 3).

[0106] Figure 3 shows bright-field microscopy images of cell aggregates on day 3. Surface ectoderm formed in the outermost layer of the cell aggregates. Cell clusters containing neural crest cells (NCCs) formed within the cell aggregates. The epidermal ectoderm formed a membrane-like (balloon-like) structure surrounding the neural crest cell clusters. In bright-field microscopy, neural crest cell clusters were observed as dark regions within the cell aggregates, while the epidermal ectoderm was observed as a bright membrane surrounding the neural crest cell clusters. The epidermal ectoderm expressed TFAP2 and p63, and TFAP2 and p63 expression was confirmed in the epidermal ectoderm and its derived tissues up to day 30.

[0107] (4) Promotion of neural crest cell differentiation (Day 3) After the formation of epidermal ectoderm was confirmed, 25 μl / well of E6 containing LDN193189 (Selleck, final concentration 0.2 μM) and basic FGF (final concentration 50 ng / ml) was added and the cells were cultured in a CO2 incubator at 37°C for 3 days (Day 6).

[0108] On Day 6, 75 μl of E6 was added, resulting in a total of 200 μl of medium being added to each well of the 96-well U-bottom plate. Two days later, half of the differentiation medium (100 μl) was removed and 100 μl of fresh E6 was added (Day 8). Two days later, half of the differentiation medium (100 μl) was removed and 100 μl of fresh E6 was added (Day 10). The cells were cultured for another two days (Day 12). The medium composition for Days 6-8, 8-10, and 10-12 is shown below.

[0109] Day 3-6 BMP4: 4 ng / ml SB431542: 8 μM bFGF: 53.2 ng / ml LDN193189: 0.2 μM Day 6-8 BMP4: 2.5 ng / ml SB431542: 5 μM bFGF: 32.25 ng / ml LDN193189: 0.125 μM Day 8-10 BMP4: 1.25 ng / ml SB431542: 2.5 μM bFGF: 16.6 ng / ml LDN193189: 0.062 μM Day 10-12 BMP4: 0.63 ng / ml SB431542: 1.25 μM bFGF: 8.3 ng / ml LDN193189: 0.031 μM

[0110] Bright-field microscopy images of cell aggregates on Day 6 and Day 12 are shown in Figure 4. On Day 6, neural crest cells migrated from the interior of the cell aggregate to the outermost layer of the epidermal ectoderm (the outermost surface of the cell aggregate). Neural crest cell migration was observed from Day 3 onward. As neural crest cells migrated to the outer surface of the epidermal ectoderm, the epidermal ectoderm membrane became covered by a layer of neural crest cells and positioned inside the cell aggregate. As a result, the epidermal ectoderm membrane, which had formed a membranous (balloon-like) membrane enveloping the neural crest cell cluster, formed a lumen inside the superficial cell aggregate by Day 12. The epidermal ectoderm expressed TFAP2 and p63 and formed the epidermal cell layer (epidermis), which would eventually become the epidermis. KRT5 expression was confirmed in the epidermal cell layer (epidermal ectoderm membrane) on Days 10–17.

[0111] (5) Initiation of maturation culture (Day 12) On Day 12, cell aggregates were removed from the 96-well U-bottom plate and transferred to a 60-mm Petri dish containing 6 ml of D-PBS. After washing, they were transferred to a 60-mm Petri dish containing 6 ml of OMM (organoid maturation medium). The maturation culture was initiated at 37°C in a CO2 incubator while rotating at 65 rpm on an orbital shaker. OMM was a 1:1 mixture of Advanced-DMEM / F12 (Gibco) and Neurobasal medium (Gibco), supplemented with B-27 minus vitamin A supplement (Gibco, final concentration 0.5x), N-2 supplement (Gibco, final concentration 0.5x), GlutaMax (Gibco, final concentration 1x), 2-Mercaptoethanol (Wako, final concentration 0.1 mM), and Normocin (InvivoGen, final concentration 100 μg / ml). Half of the medium was replaced every 3 days.

[0112] Figure 5-1 shows a bright-field microscope image of the cell aggregates (skin organoids) on day 30, and Figure 5-2 shows a fluorescent immunostained image of the epidermal cell layer of the cell aggregates (skin organoids).

[0113] Between days 17 and 30, the epidermal cell layer gradually thickened, and the lumen formed by the epidermal cell layer expanded, resulting in the appearance of a region with an expanded lumen (head portion) that was brightly observed under a bright-field microscope (Figure 5-1). By day 30, the epidermal cell layer exhibited a layered structure characteristic of the embryonic epidermis, consisting of a basal layer (expressing KRT5, p63, and TFAP2) and a perithelium (expressing E-cadherin) (Figure 5-2). KRT5 is a basal cell marker, p63 is an epidermal stem cell marker, and E-cadherin is an epithelial cell marker, indicating that the epidermal cell layer is the tissue region that will eventually form the epidermis.

[0114] Furthermore, on days 17-30, a region containing neural crest cell-derived dermal fibroblasts (the tail portion) formed on the opposite side of the head portion (Figure 5-1). The dermal organoids exhibited a gourd-like three-dimensional structure consisting of two regions: the head portion and the tail portion. On day 30, a fibroblast layer expressing the fibroblast marker PDGFRa formed adjacent to the epidermal cell layer in the head portion (Figure 5-2). This fibroblast layer, together with the fibroblasts in the tail portion, is thought to represent a mesenchymal cell layer that will eventually form the dermis and subcutaneous tissue. Based on these findings, it was determined that on day 30, the cell aggregates contained tissue regions that would differentiate into the epidermis, dermis, and subcutaneous tissue, forming dermal organoids.

[0115] (6) Selection of Skin Organoids 1 (Day 30) On Day 30, skin organoids were selected based on the formation of the head portion. Specifically, skin organoids with a head portion accounting for 40% or more of the total were selected.

[0116] One selected skin organoid was transferred to each well of a 12-well plate (Nunc, low-adhesion treatment) containing 1.5 ml of OMM per well, and maturation culture was continued (rotating culture in a CO2 incubator at 37°C). Half of the medium was replaced every three days.

[0117] Figure 6-1 shows a bright-field microscope image of the skin organoids on day 70, and Figure 6-2 shows fluorescent immunostained and hematoxylin-eosin stained images of the epidermal cell layer of the skin organoids.

[0118] The epidermal cell layer contained cells expressing the spinous layer marker KRT10 and cells expressing the granular layer marker Loricrin. The epidermal cell layer showed a layered structure characteristic of the epidermis during embryonic development, consisting of the basal layer (expressing KRT5 and p63), spinous layer, and granular layer (Figure 6-2).

[0119] Furthermore, hair pegs, which are the initial hair follicle tissue, are formed in the epidermal cell layer. The hair pegs invaginate from the epidermal cell layer into the mesenchymal cell layer (fibroblast layer) and form papilla-like structures (Figure 6-2). The hair pegs that invaginate into the mesenchymal cell layer extend to the head portion that connects to the mesenchymal cell layer, reaching the surface of the head portion and protruding from that surface (see arrowhead in Figure 7). As the hair pegs extend, they develop a hair papilla and hair bulb. The protruding hair pegs are observed as multiple protruding structures on the surface of the head portion.

[0120] (7) Skin organoid selection 2 (Day 70) On Day 70, skin organoids for subsequent cranial window transplantation were selected based on the formation of hair piles. Specifically, skin organoids with hair piles formed in the epidermal cell layer and a boundary width between the head and tail portions (H in the left panel of Figure 10) of 2 mm or more were selected.

[0121] [Test Example 2: Examination of the effect of activation of the Wnt signaling pathway on the formation of the epidermal cell layer] Skin organoids were prepared in the same manner as in Test Example 1, except that activation of the Wnt signaling pathway was not performed on Day -1, and the effect of activation of the Wnt signaling pathway on the formation of the epidermal cell layer was examined.

[0122] Figure 8-1(A) shows bright-field microscopy images of cell aggregates untreated (-) and treated (+) with CHIR99021 on Day 30. Head portions were confirmed in 67% (16 / 20) of the cell aggregates treated with CHIR99021. In contrast, head portions were only formed in 45% (9 / 20) of the cell aggregates untreated with CHIR99021. Image analysis of hematoxylin-eosin-stained tissue sections of the cell aggregates (Figure 8-1(C)) was performed to measure the area of ​​the lumen formed by the epidermal cell layer. Figure 8-1(B) shows the results (n=5). The lumen area tended to be significantly larger (p<0.001) in CHIR99021-treated (+) cell aggregates compared to untreated (-) cell aggregates. These results demonstrated that treatment of iPSC aggregates to activate the Wnt signaling pathway before the start of differentiation induction of skin organoids (Day -1) can promote the development of the epidermal cell layer.

[0123] Furthermore, the optimal concentration of the Wnt signaling pathway activator was investigated. In addition to the condition in Test Example 1, the concentration of the Wnt signaling pathway activator (CHIR99021) on Day -1 was set to 1.5 μM, and skin organoids were prepared in the same manner as in Test Example 1, with the concentration set to untreated (0 μM), 1 μM, or 3 μM.

[0124] Figure 8-2 shows bright-field microscopy images of cell aggregates on Day 3 using human iPS cell line A (771-3G). Compared to untreated, the formation of epidermal ectoderm, observed as a bright membrane, was improved at concentrations of 1 μM and 1.5 μM. However, the efficiency of epidermal ectoderm formation was reduced at 3 μM.

[0125] Figure 8-3 shows brightfield microscopy images of cell aggregates on Day 3 using the human iPS cell line BF at a CHIR99021 concentration of 1 μM. Cell line B is 1383D6, cell line C is WTC-11, cell line D is iPSCs derived from a healthy individual, and cell line E is iPSCs derived from a patient with progeria syndrome, in which the causative gene WRN mutation has been repaired. At a CHIR99021 concentration of 1 μM, all of the BF cell lines successfully formed epidermal ectoderm, with no differences between the lines.

[0126] Figure 8-4 shows a fluorescent immunostained image of cell aggregates on Day 3 using human iPS cell line A (771-3G) at a CHIR99021 concentration of 1 μM. Epidermal ectoderm is confirmed as membrane structures positive for E-cadherin and TFAP2.

[0127] Test Example 3: Induction of differentiation of full-thickness skin tissue by cranial window transplantation The process for inducing differentiation of full-thickness skin tissue was initiated using the skin organoids selected on Day 70 of Test Example 1. An overview of the process is shown in Figure 9.

[0128] (1) Pretreatment: Skin organoids selected on Day 70 of Test Example 1 were removed from the culture medium and transferred to a 60 mm Petri dish containing 6 ml of cold D-PBS (4°C) for pre-transplantation treatment. The head portion of the skin organoid exhibits a layered structure with the epidermal cell layer (apical side) on the lumen side and the mesenchymal cell layer (basal side) on the outside, representing a tissue structure in which the polarity of normal skin is reversed. Therefore, slits were made on all four sides of the head portion to open the lumen, and the incision was continued until the epidermal cell layer was autonomously exposed to the outside. Furthermore, the tail portion was cut in a straight line parallel to the interface with the head portion, and two-thirds or complete excision was performed. Figure 10 shows the incision line of the head portion and the cut line between the tail portion and the head portion. This resulted in a sheet-shaped skin organoid (transplant sheet) with a layered structure in which the epidermal cell layer was backed by a mesenchymal cell layer.

[0129] (2) Cranial window transplantation (0 dpt) A cranial window was created in an 8-9-week-old immunodeficient mouse model (NOD / ShiJic-scidJcl, female). Pretreated skin organoids (transplant sheets) were transferred from cold D-PBS to the brain surface of the cranial window. Excess D-PBS was removed, and the skin organoids were positioned so that the mesenchymal cell layer was in contact with the brain surface and the epidermal cell layer was exposed (Figure 11). After confirming that the skin organoids were in a stable position on the brain surface, an 8-9 mm diameter coverslip was placed over the skin organoids, contacting the surface of the epidermal cell layer.

[0130] Next, 100-500 μL of saline was injected between the cover glass and the brain surface, allowing the skin organoids to be held in place between the cover glass and the brain surface. A 1:1 mixture of bone cement (Coe Tray Plastic) and instant adhesive (Aron Alpha) was applied to the cover glass and the mouse skull, aligning it to secure the cover glass in place. This transplantation day was designated 0 days post-transplantation (dpt), and the growth of the skin organoids after transplantation was observed. Brightfield observation confirmed that mouse-derived blood vessels had invaded the skin organoids at 14 dpt. This vascular infiltration suggests the potential use of the full-thickness skin tissue disclosed herein as a wound healing model.

[0131] (3) Coverage with Dressing (28 dpt) At 28 dpt, the epidermal cell layer was observed as a white membrane-like tissue. Hair follicle pigmentation was observed in the epidermal cell layer (Figure 12), and hair follicles extended toward the surface of the epidermal cell layer. Once this hair follicle extension was confirmed, the cover glass was replaced with a transparent film dressing. The cover glass was removed, and the fascia-like tissue covering the entire dermal organoid was excised using a microscissor. A circular piece of Tegaderm (3M, transparent film dressing) with a diameter of 1.0–1.5 cm was attached to the remaining mixed adhesive frame on the cranial window to cover the dermal organoid. To maintain moisture, a saline-soaked cutter was placed in a position that would not obstruct observation of the epidermal cell layer. The outer periphery of the Tegaderm was again glued and fixed with mixed adhesive (Figure 13).

[0132] (4) Aeration (49 dpt) At 49 dpt, the epidermal cell layer was covered with a white substance similar to the vernix caseosa found in prenatal fetuses, and the hair follicles showed pigmented hair bulbs and roots. At this stage, the Tegaderm covering the epidermal cell layer of the skin organoids was removed, creating an environment where the epidermal cell layer could directly contact the outside air. Histological analysis indicated that at this stage, the "skin organoids" had developed into "full-thickness skin tissue."

[0133] When Tegaderm was removed, the full-thickness skin tissue dried and a scab formed ( FIG. 14 , top panel), so the scab was excised using a microciser.

[0134] (5) Re-dressing (63 dpt) At 63 dpt, the full-thickness skin tissue was re-covered with Tegaderm (Figure 14, bottom panel) and allowed to grow for an additional 14 days. Tegaderm covering and adhesive fixation were performed as in step (3).

[0135] (6) Histological analysis of full-thickness skin tissue (77 dpt). Full-thickness skin tissue with a moisturized surface was harvested at 70-77 dpt. Partial tissue fusion was observed at the interface between the full-thickness skin tissue and the cranial window, so the fusion site was cut with a microscissor and removed from the cranial window. Although blood vessels invaded the full-thickness skin tissue from the surface of the mouse brain, there was little evidence of attachment to the brain tissue. The harvested full-thickness skin tissue was washed with cold D-PBS, fixed in 4% PFA at 4°C for 2-3 days, and then used for histological analysis.

[0136] A photograph of the recovered full-thickness skin tissue is shown in Figure 15. Hair-bearing follicles grew at intervals on the surface of the full-thickness skin tissue. "Spaced hair follicles" refer to hair follicles that grow hair at regular intervals, as observed in living skin, rather than hair follicles that form densely packed or ingrown hairs. The formation of "spaced hair follicles" suggests that the full-thickness skin tissue disclosed herein may be useful as a model for analyzing hair growth and wound healing, including hair regrowth.

[0137] The hematoxylin-eosin stained image of full-thickness skin tissue is shown in Figure 16-1, and the fluorescent immunostained image is shown in Figure 16-2. The full-thickness skin tissue consists of the epidermis, dermis, and subcutaneous tissue, and the formation of hair follicles (HF), sebaceous glands (SG), and sweat glands (SwG) was confirmed (Figure 16-1). Expression of fibronectin (a fibroblast marker) and PDGFRa (a fibroblast marker) was observed in the dermis, and lipid droplets were stained in the subcutaneous tissue (Figure 16-2).

[0138] Immunostained images of hair follicles and sweat glands are shown in Figures 17-1 and 17-2, respectively. Hair follicles are connected to the arrector pili muscles, which express SMA (smooth muscle actin) and ITAG8 (Figure 17-1). Sweat glands exhibited a morphology characteristic of early-stage sweat gland tissue, invaginating the epidermal cell layer to form a tubular structure, and expressed the sweat gland marker AQP5 (Figure 17-2). Furthermore, mouse-derived Langerhans cells were detected in the epidermal cell layer (Figure 17-3). Furthermore, application of imiquimod (IMQ), a drug used to induce skin inflammation and create a psoriasis model, to the surface of full-thickness skin tissue resulted in epidermal hypertrophy (Figure 17-4, upper right panel) and increased phosphorylation of the cell hyperproliferation marker STAT3 (middle panel), along with an increase in CD68-positive macrophages in the dermis (bottom panel).

[0139] Figure 18-1 shows a hematoxylin-eosin stained image of the epidermal cell layer of full-thickness skin tissue, and Figure 18-2 shows a fluorescent immunostained image. The epidermal cell layer of the skin organoid differentiated into four layers: the basal layer, spinous layer, granular layer, and a layer of enucleated cells (corresponding to the stratum corneum or stratum lucidum), and the mesenchymal cell layer differentiated into the dermis (Figure 18-1). Expression of loricrin (granular layer marker), KRT10 (spinous layer marker), CD49f (basal layer marker), and KRT5 (basal layer marker) was observed in the epidermis of the full-thickness skin tissue (Figure 18-2).

[0140] These histological findings indicate that the transplanted skin organoids developed into full-thickness skin tissue, including cells that make up the epidermis, dermis, and subcutaneous tissue. The full-thickness skin tissue contained enucleated cells that make up the stratum corneum or stratum lucidum, cells that make up the stratum granulosum (Loricrin-positive), cells that make up the stratum spinosum (KRT10-positive), and cells that make up the stratum basale (CD49f, KRT5-positive).

[0141] (7) Single-cell RNA sequencing analysis of full-thickness skin tissue (77 days after transplant). Full-thickness skin tissue with moisturized surfaces was collected at 70-77 days after transplant. Cells were isolated from the full-thickness skin tissue using a Gentle MACS dissociator (Miltenyi Biotec) and a Whole Skin Dissociation Kit, human (Miltenyi Biotec). Libraries were prepared using the Chromium Next GEM Single Cell 3' Library Kit v3.1 (10x Genomics). After quality check using a Bioanalyzer (Agilent), single-cell RNA sequencing analysis was performed using an Illumina NovaSeq 6000. The analyzed Fastq data were aligned to the human-mouse mixed, human, or mouse genome references using CellRanger analysis according to the Realignment- and Expression-based Multispecies Deconvolution for Single Cell (REMS) method, and data were separated into human- and mouse-derived cells. This data was analyzed using Seurat (version 4.3.0.1), and each cell was classified into a cell population based on the gene expression profile of each cell, and the cell types contained in each cell population were identified.

[0142] The classification of human-derived cell populations contained in full-thickness skin tissue is shown in Figure 18-3. Human-derived cells were separated into 13 cell populations (clusters). The papillary dermis is a cell population located just below the epidermis, and the reticular dermis is a cell population located just above the subcutaneous tissue. The presence of these cell populations indicates that the dermis in full-thickness skin tissue maintains a hierarchical structure consisting of two layers: the papillary dermis just below the epidermis and the reticular dermis just above the subcutaneous tissue. Furthermore, cell populations containing not only cells that make up the three layers of skin (epidermis, dermis, and subcutaneous tissue), but also cells containing neurons, muscle cells, pericytes, chondrocytes, dividing cells, and mesenchymal stem cells were also identified.

[0143] The classification of cell populations of mouse-derived cells contained in full-thickness skin tissue is shown in Figure 18-4. Mouse-derived cells were separated into 10 cell populations (clusters). Among the cell populations, cell populations containing vascular endothelial cells and cell populations containing innate immune cells were confirmed.

[0144] Test Example 4: Examination of the effect of dressing coverage on the formation of epidermal cell layers Skin organoids were grown up to 70 dpt in the same manner as in Test Example 3, except that the cover glass was not replaced with a transparent film dressing at 28 dpt, and the cover glass coverage was maintained until aeration began at 49 dpt.

[0145] Figure 19(A) shows full-thickness skin tissue that continues to be covered with a cover glass, and Figure 19(B) shows full-thickness skin tissue that has been replaced with a transparent film dressing. When replaced with a transparent film dressing, the epidermis forms to cover the entire outermost surface of the full-thickness skin tissue (left panel of B). As a result, the epidermal area is large and hair follicles can be arranged at regular intervals (right panel of B). On the other hand, when the cover glass is continued, the epidermis of the full-thickness skin tissue is partially buried (center panel of A). As a result, the epidermal area is small and hair follicles are overcrowded, and the regularly spaced hair follicles observed in living skin are not observed (left and right panels of A).

[0146] Test Example 5: Promotion of sweat gland differentiation by BMP. Beads were soaked in solutions of BMP2 and BMP4 (5 μg / ml or 50 μg / ml), and as a control, beads were soaked in PBS containing 0.1% BSA. Specifically, 100 μl of Affi-Gel beads (Bio-Rad) were placed in a 1.5 ml tube, 1 ml of D-PBS (Nacalai) was added, and the tube was centrifuged at 300 × g for 3 minutes, and the supernatant was removed. This washing process was repeated three times. After removing the D-PBS in the final step, BMP solution or PBS was added, mixed, and allowed to stand overnight at 4°C.

[0147] The skin organoids were covered with a transparent film dressing from 28 to 56 dpt, at which point the transparent film dressing covering the full-thickness skin tissue was removed. The vernix caseosa formed on the surface of the full-thickness skin tissue was removed, and any overgrown hairs were cut to ensure visibility. An incision of less than 1 mm deep was made on the surface of the full-thickness skin tissue using microscissors, and 20–100 beads were embedded in the incision, which was then re-covered with the transparent film dressing. For moisturization, a saline-soaked cutter was placed in a position that did not obstruct observation of the epidermal cell layer (Figure 20). The full-thickness skin tissue was grown until 70 dpt and then harvested.

[0148] Hair follicle formation was suppressed at the site where BMP beads (50 μg / ml) were implanted (position 2) compared to the site where BMP beads (5 μg / ml) were implanted (position 1) (see arrowheads in Figures 21(A) and (B)). Furthermore, sweat gland (AQP5-positive) differentiation was observed at both the site where BMP beads (5 μg / ml) were implanted (position 1) and the site where BMP beads (50 μg / ml) were implanted (position 2) (Figure 22). Sweat gland differentiation was more pronounced at position 2 (BMP 50 μg / ml) than at position 1 (BMP 5 μg / ml). BMP treatment suppressed hair follicle formation while promoting sweat gland formation. It was revealed that BMP treatment induces sweat glands more predominantly than hair follicles, suggesting that by changing the BMP treatment concentration, it may be possible to create full-thickness skin tissue with different ratios of hair follicles and sweat glands formed.

[0149] Test Example 6: Differentiation induction of skin organoids and full-thickness skin tissue from iPS cells from a patient with progeria and wound healing experiment As in Test Examples 1 and 3, differentiation induction of skin organoids and full-thickness skin tissue was performed from iPSCs from a patient with progeria and iPSCs from a healthy individual.

[0150] Full-thickness skin tissue with moisturized surfaces was harvested at 70 dpt. Photographs and hematoxylin-eosin stained images of the harvested full-thickness skin tissue are shown in Figure 23(A), and immunofluorescent stained images of the epidermis and dermis are shown in Figure 23(B). Compared to full-thickness skin tissue from healthy individuals, full-thickness skin tissue from progeria patients showed decreased expression of type 17 collagen (COL17A1) in the basal layer of the epidermis. COL17A1 is an epidermal stem cell marker, and its decreased expression is known to be an indicator of skin aging.

[0151] At 70 days after wound creation, Tegaderm was excised from full-thickness skin tissue induced through a mouse cranial window. A 1.5 mm diameter skin punch biopsy was performed on the exposed surface of the full-thickness skin tissue. The dotted area in the left photograph of the full-thickness skin tissue in Figure 24(A) is the biopsy site. The right photograph of Figure 24(A) shows an enlargement of the biopsy site and the wound. Figure 24(B) shows a photograph of the full-thickness skin tissue 10 days after wound creation. While the wound healed in the full-thickness skin tissue from a healthy subject, healing was delayed in the full-thickness skin tissue from a progeria patient, and the wound remained (the arrow in the enlarged image on the bottom right indicates the remaining wound). Figure 25 shows an immunostained image of the full-thickness skin tissue 10 days after wound creation. Expression of keratin 17 (KRT17) was confirmed in the full-thickness skin tissue from a healthy subject. KRT17 is known to be highly expressed in the epidermis during wound healing. On the other hand, in full-thickness skin tissue from progeria patients, the expression sites of KRT17 in the epidermis were discontinuous, indicating that epidermal regeneration was incomplete and epidermal continuity was not restored in full-thickness skin tissue from progeria patients.

Claims

1. A skin organoid induced from pluripotent stem cells and comprising cells capable of differentiating into cells that constitute skin, the skin organoid having an inner cavity, and an epidermal cell layer on its surface facing the inner cavity comprising cells capable of differentiating into the epidermis, and a mesenchymal cell layer on the outside of the epidermal cell layer comprising cells capable of differentiating into the dermis or subcutaneous tissue, the epidermal cell layer being composed of a granular layer comprising cells that express Loricrin, a spinous layer comprising cells that express KRT10, and a basal layer comprising cells that express KRT5 and p63, layered in this order from the inner cavity side, and having hair piles that invaginate into the mesenchymal cell layer.

2. The skin organoid of claim 1, wherein the mesenchymal cell layer comprises fibroblasts.

3. A transplant sheet comprising cells induced from pluripotent stem cells and capable of differentiating into cells that constitute full-thickness skin, the transplant sheet having a layered structure in which an epidermal cell layer comprising cells capable of differentiating into the epidermis is backed by a mesenchymal cell layer comprising cells capable of differentiating into the dermis or subcutaneous tissue, the epidermal cell layer being composed of a granular layer comprising cells that express loricrin, a spinous layer comprising cells that express KRT10, and a basal layer comprising cells that express KRT5 and p63, layered in this order, and having hair piles that invaginate into the mesenchymal cell layer.

4. A method for producing the skin organoid described in claim 1, comprising: (1) a step of culturing aggregates of human pluripotent stem cells in the presence of a substance that activates the Wnt signal pathway; (2) a step of culturing the cell aggregates obtained in step (1) in the presence of a bone morphogenetic protein (BMP), a TGFβ inhibitor, and basic fibroblast growth factor (bFGF); (3) a step of culturing the cell aggregates obtained in step (2) in the presence of an ALK inhibitor and bFGF; (4) a step of culturing the cell aggregates obtained in step (3) in the absence of a substance that activates the Wnt signal pathway, BMP, a TGFβ inhibitor, bFGF, and an ALK inhibitor, to obtain the skin organoid.

5. The manufacturing method described in claim 4, wherein the substance that activates the Wnt signal pathway is a GSK-3β inhibitor.

6. A method for producing the transplant sheet described in claim 3, comprising the steps of: incising the inner cavity of the skin organoid described in claim 1; and inverting the skin organoid so that the surface facing the inner cavity is exposed on the outer surface of the skin organoid, thereby obtaining the transplant sheet having a layered structure in which the epidermal cell layer is backed by the mesenchymal cell layer.

7. Full-thickness skin tissue derived from human pluripotent stem cells, having dermal polarity and containing functional arrector pili muscles, hair follicles and sweat glands.

8. The full-thickness skin tissue of claim 7, comprising immune cells.

9. A method for producing full-thickness skin tissue according to claim 7, comprising: (1) incising the inner cavity of the skin organoid according to claim 1 and inverting the skin organoid so that the surface facing the inner cavity is exposed on the outer surface of the skin organoid, thereby obtaining a transplant sheet having a layer structure in which the epidermal cell layer is backed by the mesenchymal cell layer; (2) transplanting the transplant sheet to a skin defect in a living body, wherein the transplant sheet is positioned so that the mesenchymal cell layer contacts the biological surface at the transplantation site and the epidermal cell layer is exposed; (3) covering the transplant sheet with a low gas-permeable substrate and maintaining the transplant sheet in a moist environment in the space between the biological surface and the low gas-permeable substrate; (4) removing the low gas-permeable substrate, covering the transplant sheet with a high gas-permeable substrate and maintaining the transplant sheet in a moist environment in the space between the biological surface and the high gas-permeable substrate; (5) removing the highly gas-permeable substrate and maintaining the transplant sheet at the transplant site while aerating it; and (6) covering the transplant sheet with the highly gas-permeable substrate again, thereby maintaining the transplant sheet in a moist environment in the space between the biological surface and the highly gas-permeable substrate.

10. The manufacturing method according to claim 9, wherein the transplant sheet is treated with BMP in step (5) or in steps (5) and (6).

11. The method of claim 10, wherein the BMPs are BMP2 and BMP4.

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