Improvement of artificial hair follicle germ

Abstract

An artificial hair follicle germ 10 has a three-dimensional structure in which a first block 11 including hair follicle epithelial stem cells, a second block 12 including hair papilla cells, and a third block 13 including dermal-derived mesenchymal cells (DDMCs) are stacked in this order. A method for artificially preparing a hair follicle germ 10 includes three-dimensionally culturing a first block 11 including hair follicle epithelial stem cells, a second block 12 including hair papilla cells, and a third block 13 including dermal-derived mesenchymal cells (DDMCs), these blocks being stacked in this order.

Description

Improvement of artificial hair follicle primordia The present invention relates to the improvement of artificial hair follicle primordia. The paragraph of Patent Document 1

[0012] discloses a method for manufacturing a tissue. The method is a method for manufacturing a tissue constructed by the interaction between mesenchymal cells and epithelial cells, in which a first cell aggregate consisting essentially of only one of mesenchymal cells and epithelial cells and a second cell aggregate consisting essentially of only the other are arranged in contact with each other inside a support carrier. Paragraph

[0083] discloses that hair follicle epithelial cells and hair follicle mesenchymal cells were each applied to a collagen gel drop to prepare cell aggregates of each, and the hair follicles were reconstituted so that both were in close contact with each other. International Publication No. 2006 / 129672 The present invention provides an improvement of artificial hair follicle primordia. <1> An artificial hair follicle primordium having a three-dimensional structure in which a first block composed of hair follicle epithelial stem cells, a second block composed of dermal papilla cells, and a third block composed of DDMCs (dermis-derived mesenchymal cells) overlap in this order. Artificial hair follicle primordium. <2> Use of the hair follicle primordium according to <1>, generating a hair follicle from the hair follicle primordium in vitro and causing down growth in the hair follicle. Use. <3> Use of the hair follicle primordium according to <1>, generating a hair follicle from the hair follicle primordium in vitro, forming a dermal root sheath of the hair bulb surrounding the second block from the dermal papilla cells in the second block, and further forming an upper dermal root sheath surrounding the first block from the cells in the third block. Use. <4> A method for manufacturing a skin equivalent having a hair follicle, generating a hair follicle from the hair follicle primordium according to <1> in vitro and optionally causing down growth in the hair follicle in vitro. The hair follicles are incorporated in vitro into a skin equivalent having an epidermal layer and a dermal layer. method. <5> A method for producing a skin equivalent having hair follicles, The hair follicle primordia described in <1> are incorporated in vitro into a skin equivalent having an epidermal layer and a dermal layer. In vitro, hair follicles are generated from the incorporated hair follicle primordia, and optionally, the hair follicles are subjected to down-growth in vitro. method. <6> A method for manufacturing biopharmaceuticals, In vitro, hair follicles are generated from the hair follicle primordia described in <1>, and optionally, down-growth is induced in vitro in the said hair follicles. A biopharmaceutical containing the aforementioned hair follicles is prepared. method <7> A biopharmaceutical containing the hair follicle primordium described in <1>. <8> A method for manufacturing a device for evaluating the effects of a pharmaceutical product or a candidate substance thereof on hair follicles, In vitro, hair follicles are generated from the hair follicle primordia described in <1>, and optionally, down-growth is induced in vitro in the said hair follicles. A device comprising the aforementioned hair follicle is fabricated. method <9> A device for evaluating the effect of a pharmaceutical product or a candidate substance thereof on hair follicle primordia, A device containing the hair follicle primordium described in <1>. <10> A three-dimensional culture of blocks consisting of a first block made of hair follicle epithelial stem cells, a second block made of dermal papilla cells, and a third block made of DDMCs (dermal-derived mesenchymal cells), in which these blocks are stacked in this order. A method for artificially creating hair follicle primordia. <11> From a group of cells that make up the dermis, a group is selected in which the cell surface is PDGFRα+ and CD31 / CD45-. When the cells in the selected population are classified by the expression levels of Sca1 and CD34 on their cell surface, specific mesenchymal cells are obtained by selecting a population with higher expression levels of both Sca1 and CD34 from among the populations that are both Sca1+ and CD34+. This method involves culturing three-dimensionally a first block consisting of hair follicle epithelial stem cells, a second block consisting of dermal papilla cells, and a third block consisting of the aforementioned specific mesenchymal cells, in a state in which these blocks are stacked in this order. A method for artificially creating hair follicle primordia. <12> The second block is formed by accumulating hair papilla cells on the third block in a droplet. The first block is formed by accumulating hair follicle epithelial stem cells on the second block, and then These blocks are cultured in three dimensions. The method according to either <10> or <11>. This invention provides an improvement to artificial hair follicle primordia. Schematic diagram of vibrissa, schematic diagram of three-dimensional culture, schematic diagram of regenerated hair follicle, schematic diagram of downgrowth, perspective view of skin equivalent, photographic observation of hair follicle primordium growth, dot plot of FACS analysis of cells, photographic observation of stained cells, incidence of hair bulb and downgrowth, photographic observation of hair follicle development and mesenchymal cells, three-dimensional distribution of mesenchymal cells within a hair follicle. <Tactile hairs and hair follicles> In this embodiment, we utilize cell types contained in the tissue that constitutes the whisker (or vibrissa), which consists of large hair follicles. Therefore, Figure 1 provides an overview of the tissue from which these cell types originate. Figure 1 is a schematic diagram of a whisker. The hair shaft of the whisker arises from the hair follicle. The hair follicle penetrates the subcutaneous layer and dermis to the epidermis. At the base of the hair follicle are the hair bulb and the dermal papilla (DP). The hair bulb has a DS cup (Dermal Sheath Cup). The DS cup encloses the dermal papilla (DP) and the hair matrix (HM). In addition, there is a bulge region at the top of the hair follicle, which consists of bulge epithelial cells (BE). Bulge epithelial cells are a type of epithelial stem cell. Furthermore, above the hair bulb, there is mesenchymal tissue called the upper dermal sheath (Upper DS) or connective tissue sheath (CTS). UpDS and CTS are synonymous. This upper DS surrounds the bulge as a series of structures. The inventors have confirmed that the cells surrounding the bulge region of the upper DS are not stained by immunohistochemistry for αSMA (smooth muscle actin). They have also confirmed that cells other than those surrounding the bulge region of the upper DS are stained by the same immunohistochemistry. These structures are not limited to the large hair follicles that make up the whiskers of rodents, but are commonly found in the large hair follicles that make up human scalp hair. To prepare single cells from these tissues, known enzymes, buffers, culture media, and protocols can be used. The cells may be primary cultured cells or passaged cultured cells. The cells may also be cells obtained by differentiation induction from pluripotent stem cells such as ES cells or iPS cells. <Method for creating hair follicle primordia> Figure 2 is a schematic diagram of a three-dimensional culture performed to artificially create hair follicle primordia. The first block 11, the second block 12, and the third block 13 are stacked in this order and these blocks are cultured in three dimensions. Hair follicle primordia 15 are obtained by three-dimensional culture. Three-dimensional culture may be performed, for example, in a droplet 18. The droplet 18 may be a droplet made of sol. The sol may be made of collagen and buffer. The cells that make up these blocks may be human cells or mammalian cells other than human cells. As shown in Figure 2, the first block 11 consists of hair follicle epithelial stem cells. In this embodiment, bulge epithelial cells BE are used as hair follicle epithelial stem cells. The number of bulge epithelial cells BE may be 100 to 1,000,000, 1,000 to 10,000, or 10,000 to 100,000. As shown in Figure 2, the second block 12 consists of dermal papilla cells DP. The number of dermal papilla cells DP may be 100 to 1,000,000, 1,000 to 10,000, or 10,000 to 100,000. As shown in Figure 2, the third block 13 consists of DDMCs (Dermal-derived mesenchymal cells, DMCs) or equivalent specific mesenchymal cells. In one embodiment, the DDMCs are derived from the mesenchymal tissue of the hairy skin of a living organism. The number of cells constituting the third block 13 may be 100 to 1,000,000, 1,000 to 10,000, or 10,000 to 100,000. As shown in Figure 2, the hair follicle primordium 15 has a three-dimensional structure in which a first block consisting of hair follicle epithelial stem cells, a second block consisting of dermal papilla cells, and a third block are stacked in this order. In Figure 2, the stacking of each block is carried out as follows, for example. First, a third block 13 is formed by accumulating DDMCs in the droplet 18. Next, a second block 12 is formed by accumulating dermal papilla cells DP on top of the third block 13. Furthermore, a first block 11 is formed by accumulating bulge epithelial cells BE on top of the dermal papilla cell DP block. In this way, a reconstructed body consisting of at least three types of cells is created. By culturing this reconstructed body in the droplet 18, its structure stabilizes and it becomes a hair follicle primordium 15. The specific mesenchymal cells used in Block 3, Item 13 can be prepared as follows: First, a population of cells constituting the dermis is selected, specifically those whose cell surface is PDGFRα+ and CD31 / CD45-. Next, the cells within the selected population are classified according to the expression levels of Sca1 and CD34 on their cell surface. At this time, from the population that is Sca1+ and CD34+, a population with higher expression levels of both Sca1 and CD34 is selected. Through these steps, specific mesenchymal cells suitable for Block 3 are obtained. <Hair follicle primordia and their use> One embodiment of the present invention is the hair follicle primordium 15 shown in Figure 2 and its use. Figure 3 is a schematic diagram of a hair follicle arising from the hair follicle primordium. The upper panel shows the spatial arrangement of these mesenchymal cell types in a plan view of the hair follicle 20, and the lower panel shows the hair follicle 20 in a side view. The hair follicle 20 is regenerated from the hair follicle primordium. The hair follicle 16 arises within the transplanted skin. A hair bud-like structure is formed by the interaction between dermal papilla cells DP and bulge epithelial cells BE in the second block 12. At the base of the hair follicle 16, the dermal root sheath of the hair bulb, i.e., the DS cup 22, which surrounds the second block 12, is formed from the second block 12. Also, at the upper part of the hair follicle 16, the dermal root sheath (UpDS) which surrounds the first block 11, is formed from the third block 13. Figure 4 is a schematic diagram of a down-grown hair follicle 25. The upper panel shows the hair follicle 25 in a plan view, and the lower panel shows the hair follicle 25 in a side view. The hair follicle 20 shown in Figure 3 undergoes down-growth to become a long, mature hair follicle like hair follicle 25. By culturing the mature hair follicle as an organ in vitro, it can be grown until it becomes a hair shaft. Also, hair regenerates on skin to which a mature hair follicle has been transplanted. In the hair follicle 25, the region of the first block 11 not surrounded by the DS cup 22 becomes the invariant region 32 derived from bulge epithelial cells BE. The region of the first block 11 surrounded by the DS cup 22 becomes the derived block 31. The derived block 31 becomes a variable region including the hair matrix. The hair matrix in the variable region creates the hair shaft and inner root sheath. Furthermore, the invariant region 32 eventually forms the outermost layer of the hair follicle epithelial tissue as the outer root sheath. A part of the outer root sheath, separated from the hair bulb, becomes the bulge region. A portion of the second block 12 gives rise to the DS cup 22, while another portion remains as the hair papilla (DP). Also, the UpDS 23 is generated from the third block 13 shown in Figure 2. <Skin equivalent> Figure 5 is a perspective view of the skin equivalent 30. Down-growth hair follicles 25 can be used in the manufacture of the skin equivalent 30. The hair follicles 25 are transplanted onto a structure 29 in which an epidermal layer 27 consisting of at least epidermal cells and a dermal layer 28 consisting of dermal cells are artificially laminated. The cells constituting the structure 29 may be human cells or non-human mammalian cells. As shown in Figure 5, a skin equivalent 30 having hair follicles 25 is obtained by transplantation. The hair follicles 25 may be replaced with hair follicles 20 before downgrowth as shown in Figure 3, or with hair follicle primordia 15 as shown in Figure 2. If hair follicles before downgrowth are transplanted onto the structure 29, hair follicles 25 that have undergone downgrowth may be formed on the structure 29. If hair follicle primordia 15 are transplanted onto the structure 29, hair follicles 20 before downgrowth may be formed on the structure 29 as shown in Figure 3, or hair follicles 25 that have undergone downgrowth may be formed on the structure 29. <Biopharmaceuticals> One embodiment of the present invention is a biopharmaceutical containing hair follicle primordia 15 as shown in Figure 2. The biopharmaceutical contains buffers and additives necessary for maintaining the hair follicle primordia 15. The pH and osmotic pressure necessary for maintaining the hair follicle primordia 15 are maintained within the biopharmaceutical. The biopharmaceutical may also be a personalized medicine. For example, at least one of the cells constituting the first block 11, the second block 12, and the third block 13 may be cells grown from cells taken from a subject receiving the transplant of hair follicle primordia 15. The hair follicle primordium 15 in the biopharmaceutical is transplanted into the target skin. The target may be a human or a non-human mammal. The transplantation site may be the head or eyebrows. The transplantation may regenerate hair such as head hair or eyebrows. In other embodiments of the biopharmaceutical, instead of the hair follicle primordium 15 shown in Figure 2, the biopharmaceutical may include a hair follicle 20 before downgrowth shown in Figure 3 or a hair follicle 25 after downgrowth shown in Figure 4. <Evaluation of pharmaceuticals, etc.> One embodiment of the present invention is an evaluation system for pharmaceuticals, etc. In one embodiment, the evaluation system is provided as a device for evaluating the effect of a pharmaceutical or candidate substance on hair follicles. In one embodiment, the device includes a hair follicle primordium 15 as shown in Figure 2. In another embodiment, the device includes a hair follicle 20 before downgrowth as shown in Figure 3, and a downgrowthed hair follicle 25 as shown in Figure 4. <Summary of Examples> Conventionally, it has been known that a combination of epithelial stem cells and dermal papilla cells has the function of inducing hair follicle development. In this example, specific third block cells are added to this combination. The added third block supports the down-growth of the hair follicle. The third block further forms a stem cell niche. Such a hair follicle primordium functions well even in vitro. The hair follicle primordium of this example can induce hair follicle development. The third block of the hair follicle primordium in this embodiment consists of at least one of cells derived from the mesenchymal tissue of the hairy part of the skin of a living organism and cells derived from the dermal root sheath (DS), an invariant region of the hair follicle of a living organism. Preferably, the dermal root sheath (DS) is the dermal root sheath that underlines the bulge region. A hair bud-like structure is formed through the interaction of the cells of the third block, the dermal papilla cells, and the epithelial cells. After this process, the cells of the third block enclose the epithelial tissue other than the hair bud-like structure. As shown in the experimental results, cells of the dermal root sheath derived from dermal papilla cells form the hair bulb. Furthermore, cells derived from the third block form the upper dermal root sheath (DS). Thus, through experiments to regenerate hair follicles in vitro, it became clear that cells derived from adipose tissue are not necessarily essential for the expression of these functions. Therefore, it was found that suitable cell types for use in the third block can be obtained from mesenchymal cells of the hairy skin or from cells of the dermal root sheath belonging to the unchanging part of the hair follicle. The cell surface marker expression status of the cell types suitable for use in the third block was PDGFRα+ / Sca1++ / CD34++ and CD31- / CD45-. These cells also form the dermal root sheath in the dermal region excluding the hair bulb. The dermal papilla (DP) cells used in the second block and the cells used in the third block first induce the hair bulb. Furthermore, they induce the variable region of the down-growth phase. Finally, they underline the invariant region containing the epithelial stem cell niche. <Background of the Example> Fur plays important roles such as physical insulation between the body and the outside world, regulating body temperature, providing sensitivity to harmful external stimuli, and facilitating social communication between individuals. Fur is able to perform these roles because it has various morphologies that allow it to be distinguished from one another, and because it is widely distributed on the body surface. Hair is formed by hair follicles. Hair follicle formation is induced by the interaction of epithelial cells and mesenchymal cells. This interaction is regulated by signaling molecules such as Shh, Wnt, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF). After the hair follicle morphology is formed, multiple stem cell niches are created within the follicle. These stem cell niches include epithelial stem cell niches, pluripotent mesenchymal stem cell niches, and pigmented stem cell niches. The epithelial stem cell niche can differentiate into hair follicle epithelium, sebaceous gland, and skin epithelium. The pluripotent mesenchymal stem cell niche can differentiate into dermal papilla cells (DPs), dermal root sheath cells, skin fibroblasts, and adipocytes. These stem cell niches maintain their respective stem cells throughout the individual's lifespan. Hair follicles are divided into a constant region and a variable region. The variable region repeats the hair cycle, consisting of growth, regression, and resting phases, throughout the individual's life. In the variable region, hair follicle epithelial stem cells in the bulge region and dermal papilla (DP) are activated during the transition from the resting phase to the growth phase. This activation induces growth of the variable region from the lower end of the dermis into the subcutaneous layer. This induction phenomenon is similar to the organogenesis of hair follicles observed during embryogenesis. Organogenesis of most organs other than hair follicles is induced only once during embryogenesis. However, hair follicles continue to regenerate even after the individual's birth. The inventors used a technique to reconstruct the organoid of the hair follicle. This organoid can reproduce the organogenesis of the hair follicle. This organoid was reconstructed from epithelial stem cells isolated and saturated from the bulge region of a living hair follicle, and growing DPs cells. The hair follicle primordium thus regenerated produces hair follicles that function well not only in vitro but also in vivo. Prior to their research on hair follicle regeneration, the inventors had artificially reconstructed various ectodermal organs other than hair follicles using the basic principles of organoid generation. In the process, they demonstrated that ectodermal genitalia could be induced in an in vitro culture system. However, they were unable to develop and grow hair follicles in vitro using stem cells derived from living organisms. The organogenesis of hair follicles and their regeneration during the hair cycle have been shown to be regulated by microenvironmental factors provided by the surrounding tissues within the hairy skin. Various stem cells and progenitor cells are involved in these microenvironmental factors. In recent years, it has been reported that the adipose tissue surrounding hair follicles, called dermal white adipose tissue (dWAT), has properties different from other adipose tissues. DWAT plays an important role in hair follicle morphogenesis and maintenance of the hair cycle. Furthermore, mesenchymal cell interactions and the complex tissue structure of the skin, including adipose tissue, are closely related to hair follicle regeneration. However, artificial reproduction of skin structure and in vitro hair follicle morphogenesis have not yet been achieved. Moreover, the detailed mechanisms behind the development of complex skin structure and function remain unclear. Reconstructing organs and organ systems by artificially arranging the cells and tissues that make up living organisms has long been a fundamental technological challenge. Of these, skin was the first organ whose reconstruction has been demonstrated. 3D artificial human skin equivalents (HSEs) have been shown to partially reproduce the physiological phenomena of natural skin. These physiological phenomena include cell proliferation, cell signaling, extracellular matrix production, and responses to various skin stimuli. Artificial skin is a medical application of HSE (Hypercutaneous Epiphysitis) by conceptually developing it. Artificial skin is used to treat severe burns and genetic skin diseases. This reconstructed skin contributes to improving human well-being. On the other hand, HSEs are also useful in the fields of basic skin research, cosmetics, drug discovery, and drug safety testing. The application of HSEs to these fields is highly valuable because they can replace animal testing. For this reason, cell culture techniques have been improved through various studies aimed at improving HSE models. Such studies include basic biological research, substrate improvements, the application of 3D bioprinting, and the use of pluripotent stem cells in skin-on-chip systems called organoids. The inventors recently developed a new HSE that has the same tension homeostasis as natural skin in vitro. Based on this development result, it was reported that the tension homeostasis of the skin plays an important role in the tissue structure and function control of the skin. This HSE has a structure consisting only of the epidermal layer and the dermal layer. Therefore, the inventors have not yet been able to reproduce a 3D epidermal organ system that has skin appendages such as hair follicles, sebaceous glands, sweat glands, and subcutaneous tissue. The inventors aim to reconstruct the epidermal system and partially reproduce skin functions. For this reason, the inventors aim to reproduce an HSE having one or more types of skin appendages among hair follicles, sebaceous glands, and sweat glands as an in vitro model. In this example, a fully functional hair follicle was generated in a 3D culture system, and down growth was induced in this hair follicle. Thus, we successfully constructed an in vitro culture system for generating hair follicles. The hair follicles cultured in vitro can be incorporated into natural skin or artificial skin. Also, this hair follicle becomes one of the elements for reproducing a skin organ system that includes organs other than the epidermal layer and the dermal layer in vitro. These technical achievements not only contribute to future basic research in dermatology but also to the development of various pharmaceuticals, cosmetics, and topical medications. <Materials and Methods / Animals> Each of the C57BL / 6N, C57BL / 6-Tg (CAG-EGFP), and BALB / cSlc-nu / nu mice was purchased from Japan SLC Inc. (Shizuoka, Japan). The R26-Lyn-mCherry mice were provided by the Laboratory Animal Facility of the RIKEN (Japan). The handling of experimental animals in this study was carried out in accordance with the guidelines of the National Institutes of Health. The animal experiments in this study were approved by the Institutional Animal Care and Use Committee of the RIKEN Kobe Branch (approval number A2014-02-17). <Separation of DDMCs from the Skin on the Back of Mice and Cell Culture> To synchronize the hair growth phase, depilatory agents were used to remove hair from the dorsal side of mice in the hair resting phase. Five days after depilation, the entire dorsal skin of the mice was cut into 3 mm strips. The cut skin was incubated in a solution of 50 U / mL dispase (Corning, NY, USA) at 37°C for 15 minutes. The epidermis was removed from the incubated skin. The dermis, including adipose tissue, was dissected. The cells were incubated in DMEM (Nacalai Tesque, Kyoto, Japan) containing 910 U / mL collagenase, supplemented with 10% FB, 100 U / mL penicillin, and 100 μg / mL streptomycin (Thermo Fisher Scientific, Massachusetts, USA) at 37°C for 1 hour. This resulted in the unicellularization of mesenchymal cells. The resulting cell suspension was filtered through a 40 μm mesh cell strainer (Corning) and a 15 μm mesh cell strainer (PluriSelect Life Science, DE, Germany). <Isolation of hair follicle tissue from mouse cheek whiskers> Full-thickness facial skin was collected from each of C57BL / 6N, C57BL / 6-Tg (CAG-EGFP), and R26-Lyn-mCherry mice. Growing hair follicles were separated from this skin together with the collagen sheath. The hair bulb and ring bulge of the vibrissae were dissected using sharp scissors. The hair papilla and the lower dermal root sheath were obtained from the hair bulb. The hair papilla and the lower dermal root sheath were separated from the hair bulb under a stereomicroscope using sharp forceps and an ophthalmic scalpel (Mani, MANI, INC., Tochigi, Japan). In addition, each tissue of the bulge epithelium and the upper dermal root sheath was obtained from the ring bulge. The ring bulge was reacted in DMEM medium containing 50 U / mL dispase (Corning, NY, USA) and 910 U / mL collagenase at 37°C for 15 minutes to separate the mesenchymal component and the epithelial component from each other. Further, the collagen sheath was removed from the mesenchymal component. Furthermore, the epithelial tissue residue was removed from the mesenchymal component. The ring bulge treated in this way was minced to a size of about 400 - 1000 μm square to obtain the upper dermal root sheath tissue. On the other hand, the epithelial component was washed with PBS(-) and then digested with trypsin to obtain single cells of the bulge epithelial tissue. <Primary and Subculture of Mesenchymal Cells Derived from Mouse Vibrissa Hair Follicles> Dermal papillae, upper dermal root sheaths, and lower dermal root sheaths derived from mouse whiskers were placed on plastic dishes. Primary cultures of cells from these tissues were then performed using the outgrowth method. Primary cultures of dermal papilla cells were performed using a primoria culture dish (Corning). Subcultures of dermal papilla cells were performed using a standard cell culture dish. Standard cell culture dishes were also used for primary and subcultures of other cells. Dermal papilla cells were cultured using RegenOrgan-DP medium (Organtec Co., Ltd.) supplemented with 1% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin (Thermo Fisher Scientific, Massachusetts, USA). Upper and lower dermal root sheaths were cultured using RegenOrgan-MSC medium (Organtec Co., Ltd.) supplemented with 1% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Approximately 10 days after the start of the initial culture, these cells were unicellularized using enzymes according to standard procedures and then subjected to subculture or the production of regenerated hair follicle primordia. <Culture of epithelial stem cells derived from hair follicles of mouse cheek tissue> Bulge epithelial cells, homogenized by trypsin digestion, were cultured primaryally in either a three-dimensional or monolayer culture. Three-dimensional culture was performed under conditions described in prior art, such as Patent Document 1, including the use of collagen gel drops. Monolayer culture was performed under the same culture medium conditions, except that collagen gel was not used. In an E-cadherin Fc culture plate (Somar Co., Ltd.), 4.2 x 10⁻³ cells were cultured. 3 pieces / cm 2 Seeds were seeded to the specified density. The culture medium was changed every three days. After culturing the cells until they reached 50% complete or confluent, they were used for reconstitution and gene analysis experiments. <Preparation of regenerated hair follicle primordia and cultivation of organs> The basic manufacturing method for the regenerated hair follicle primordia followed existing methods. The regenerated hair follicle primordia consisted of a first block made of bulge epithelial stem cells derived from living facial hair, a second block made of dermal papilla cells derived from living facial hair, and a third block made of DDMCs derived from living hair tissue. Each layer was compartmentalized by densely packing 10,000 single-cell molecules into each layer. The regenerated hair follicle primordia were cultured using an existing semi-gastric culture method. The culture conditions were as follows: Culture was started in stem cell medium (Advanced DMEM / F12 containing 50 ng / mL FGF7, 50 ng / mL FGF10, 50 ng / mL Noggin, 50 ng / mL SHH agonist (SAG, Cayman), and 10 μM Rock inhibitor (Y27632, WAKO), supplemented with 1x B-27, 1x GlutaMAX, and 10 μM HEPES). After two days of culture, the medium was changed to mature medium (Advanced DMEM / F12 containing 1x N2, 1x B-27, and 1x GlutaMAX supplements, NFFSE medium). The oxygen partial pressure during the culture period was maintained under atmospheric conditions. A 5% CO2 atmosphere was used. The culture medium was changed every 2-3 days. <Flow Cytometry (FACS)> The cells were washed once with FACS buffer consisting of D-PBS (Nacalai Tesque), 0.2 mM EDTA (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and 0.05% BSA (Merck KgaA, Darmstadt, Germany). FACS buffer containing various antibodies was added to the cells and reacted at 4°C for 15 minutes. The cells were then washed twice with FACS buffer. Further cell analysis and sorting were performed using BD FACS Aria III. Population analysis was performed using FACS Diva (BD Biosciences, NJ, USA) or Weasel version 3.5. <H&E staining, oil red O staining, and fluorescence 3D imaging> For histological analysis, regenerated hair follicles were fixed in 4% paraformaldehyde. Regenerated hair follicles were embedded using paraffin or Tissue-Tek OCT (Sakura Finetek Japan Co., Ltd., Tokyo, Japan). H&E staining was performed according to standard procedures. Paraffin sections (5 μm thick) were stained with H&E. Stained sections were observed using Axio Scan Z1 (Carl Zeiss, Oberkochen, Germany). To obtain three-dimensional data of the regenerated hair follicle primordia and the cell arrangement within the in vitro regenerated hair follicles, fluorescently labeled live cells were imaged using LSM780 and LSM880 confocal microscopes (Carl Zeiss). Cross-sections necessary for obtaining three-dimensional data were extracted as engineering slices. Oil Red O staining was performed by immersing the fixed and washed samples in Oil Red O staining solution. After removing the staining solution, excess staining solution was separated by washing the samples with PBS before imaging. <Results> Figure 6 shows photographs of hair follicle primordia cultured in vitro. The photographs capture hair follicle development and downgrowth from day 0 to day 17, and in some cases up to day 14, after the creation of the hair follicle primordia. As explained using Figure 2, the hair follicle primordia consist of a first block made of epithelial stem cells (BE) derived from the bulge region of a living hair follicle, a second block made of dermal papilla cells (DP) derived from a living hair follicle, and a third block made of specific mesenchymal cells. For the third block, either mesenchymal cells (DDMCs) derived from the hairy skin or dermal root sheath cells (UpDS) derived from the invariant region of a living hair follicle can be used. The hair follicle primordia were regenerated by adding cells from the third block. Reconstruction of hair follicle primordia can be carried out by various methods in accordance with the basic principles of organ primordium synthesis. As shown in Figure 6, when hair follicle primordia regenerated by the improved organ primordium method of this embodiment were cultured using a semi-gas phase culture method, the structure of the hair follicle was formed by day 9 from the start of culture. Furthermore, down-growth of the variable region including the hair bulb began. Down-growth was observed until at least day 17. After that, hair shaft growth was observed. On the other hand, when hair follicle primordia were regenerated using only BE and DP, following the conventional period primordium method, the formation of aggregates of dermal papilla cells and epithelial envelopment were observed. However, subsequent formation of the hair bulb and down-growth were not observed. As another example, BE / DP / DDMCs cells were mixed and aggregated evenly, as in the DDMCs gel mixture row and the cell mixture row. In this case, small hair bulbs were formed randomly, but an orderly hair follicle structure was not formed. Hair shaft growth also did not continue. In this embodiment, adipose-derived cells were not necessarily essential for hair follicle regeneration in vitro. The scale bar represents 100 μm. Figure 7 is a dot plot showing the expression status of mesenchymal cell markers involved in hair follicle development and downgrowth in vitro, as determined by FACS analysis. FACS analysis was performed using cell surface markers involved in mesenchymal cell development and differentiation. FACS analysis was used to identify mesenchymal cells involved in hair follicle development and downgrowth in vitro. Single-celled mesenchymal cells were obtained by enzymatic treatment of the hairy skin of living mice. First, as shown in the dot plot on the left, mesenchymal cells were classified using PDGFRα, a mesenchymal cell marker in skin, and CD31 / CD45, a hematopoietic cell marker. Of these, the fraction that was PDGFRα+ and CD31- / CD45- was separated. Next, as shown in the dot plot on the right, fractionation was performed using the mesenchymal stem cell markers Sca1 and CD34. For comparison in the next test, not only PDGFRα+ cells but also PDGFRα- cells were isolated. Furthermore, cells with the characteristics PDGFRα+ / Sca1+ / CD34+ (middle) and PDGFRα+ / Sca1++ / CD34++ (high) were isolated from PDGFRα+ cells. These isolated cells were used in the third block, and the formation of hair follicles by the reconstruction method according to this embodiment was evaluated as described below. Furthermore, the involvement of the reconstruction method in down-growth was evaluated. Figures 8A and 8B show the results of testing the function of cells involved in hair follicle development and downgrowth in vitro. PDGFRα(+) cells are mesenchymal cells derived from the hairy skin of living mice. After hair bulb development from these cells, downgrowth could be induced in these cells. On the other hand, although hair bulb formation was observed in PDGFRα(-) cells, downgrowth was not induced. Next, it was revealed that Sca1++ / CD34++ cells (high) had a high function of promoting downgrowth, similar to α+ cells. Sca1+ / CD34+ cells (middle) did not show such a tendency. F-DDMC, used for comparison, represents the entire population of mesenchymal cells obtained from mouse hairy skin, without any culture procedures. Figure 8A shows photographs of regenerated hair follicle primordia after culture, in which each cell fraction was incorporated into the third block using the organoid preparation method of this embodiment. From left to right, the images show a stereomicroscope image (microscopy), a H&E histological image (H&E), and a stained image of mature adipocytes with Oil Red O. The scale bar is 100 μm. Figure 8B shows the quantification of the induction rate of hair bulb formation and the incidence of down growth for each cell fraction used in the third block. The vertical axis shows the proportion of the total number of reconstructed primordia. Figure 9 is an observational photograph showing the change in the distribution of mesenchymal cells in a hair follicle primordium reconstructed using the hair follicle primordium preparation method according to this embodiment. The behavior of DDMCs cells and DP cells during hair follicle development from the hair follicle primordium of this embodiment was analyzed using fluorescently labeled cells. Epidermal cells (Epi) were included in the cells to be fluorescently labeled. As shown in Figure 9, by day 4 of organ culture, dermal papilla cells formed aggregates while maintaining contact with epithelial cells. At this time, DDMCs cells (arrows) rearranged to surround the regenerated hair follicle primordia. By day 6 of organ culture, dermal papilla cells (DPs) transformed into denser aggregates, forming the dermal papilla structure. The epithelium also expanded to envelop the dermal papilla and form a hair bud. Part of the dermal papilla began to envelop part of the hair bud (arrowhead). The hair bud increased in size, and by days 8-9 of culture, it constructed an immature hair follicle structure. DDMCs were also distributed to surround the hair follicle. From day 8 to day 9 of culture, the DDMCs took up a position lining the epithelium of the hair follicle (Day 9 arrow). By day 11 of culture, the hair follicle structure was complete. A newly formed hair shaft was observed. The cup of the dermal root sheath was derived from DP cells. The upper dermal root sheath was derived from DDMCs cells. The upper middle panel of Figure 9 (Epi / DP / DDMCs) is a merged image of all fluorescence images. In the middle panel (Epi / DP), cells derived from DP cells are represented in bright white. In the lower panel (Epi / DDMCs), cells derived from DDMCs are represented in bright white. The scale bar is 50 μm. Figure 10 is a reconstruction of the three-dimensional distribution and cell morphology of mesenchymal cells in a hair follicle primordium according to this embodiment. Mature hair follicles were regenerated by culturing the hair follicle primordium according to this embodiment. The distribution of cells in the mature hair follicles in vitro was analyzed three-dimensionally along with the morphology of those cells. The cells analyzed were cells of the upper dermal root sheath derived from DDMCs and cells of the dermal root sheath derived from dermal papilla cells. The upper dermal root sheath has a transversely elongated structure characteristic of down-growing hair follicles. Although this structure is not dense, it encloses the portion of the regenerated hair follicle above the hair bulb. The scale bar is 50 μm. This application is based on Japanese Patent Application No. 2024-220831, filed on 17 December 2024, entitled “Improvement of an Artificial Hair Follicular Primordial,” and enjoys the benefit of priority from this Japanese Patent Application. The entire contents of this Japanese Patent Application are incorporated herein by reference. The entire contents of Patent Document 1 are also incorporated herein by reference. 11: Block 1, 12: Block 2, 13: Block 3, 15: Hair follicle primordium, 16: Hair follicle, 18: Droplet, 20: Hair follicle, 22: DS cup, 23: UpDS, 25: Hair follicle, 27: Epidermal layer, 28: Dermal layer, 29: Structure, 30: Skin equivalent, 31: Derived block, 32: Unchanged part

Claims

1. An artificial hair follicle primordium having a three-dimensional structure in which a first block composed of hair follicle epithelial stem cells, a second block composed of dermal papilla cells, and a third block composed of DDMCs (dermal-derived mesenchymal cells) are stacked in this order.

2. Use of the hair follicle primordium according to claim 1, wherein a hair follicle is generated from the hair follicle primordium in vitro and the hair follicle is given down growth.

3. Use of the hair follicle primordium according to claim 1, wherein a hair follicle is generated from the hair follicle primordium in vitro, and the dermal root sheath of the hair bulb portion enclosing the second block is formed from the dermal papilla cells in the second block within the hair follicle, and the upper dermal root sheath enclosing the first block is formed from the cells in the third block.

4. A method for producing a skin equivalent having hair follicles, comprising generating hair follicles from hair follicle primordia described in claim 1 in vitro, optionally causing down-growth of the hair follicles in vitro, and incorporating the hair follicles into a skin equivalent having an epidermal layer and a dermal layer in vitro.

5. A method for producing a skin equivalent having hair follicles, comprising: incorporating the hair follicle primordia described in claim 1 into a skin equivalent having an epidermal layer and a dermal layer in vitro; generating hair follicles from the incorporated hair follicle primordia in vitro; and optionally causing the hair follicles to downgrow in vitro.

6. A method for producing a biopharmaceutical, comprising generating a hair follicle from a hair follicle primordium described in claim 1 in vitro, and optionally causing down-growth of the hair follicle in vitro, thereby preparing a biopharmaceutical containing the hair follicle.

7. A biopharmaceutical comprising the hair follicle primordium described in claim 1.

8. A method for manufacturing a device for evaluating the effects of a pharmaceutical product or a candidate substance thereof on hair follicles, comprising generating hair follicles from hair follicle primordia described in claim 1 in vitro, optionally causing down-growth of the hair follicles in vitro, and manufacturing a device comprising the hair follicles.

9. A device for evaluating the effect of a pharmaceutical product or a candidate substance thereof on a hair follicle primordium, comprising the hair follicle primordium described in claim 1.

10. A method for artificially creating a hair follicle primordium, comprising three-dimensional culture of blocks in which a first block consisting of hair follicle epithelial stem cells, a second block consisting of dermal papilla cells, and a third block consisting of DDMCs (dermal-derived mesenchymal cells) are stacked in that order.

11. A method for artificially producing a hair follicle primordium, comprising: selecting a population of cells constituting the dermis in which the cell surface is PDGFRα+ and CD31 / CD45-; classifying the cells in the selected population by the expression levels of Sca1 and CD34 on their cell surface, and obtaining specific mesenchymal cells by selecting a population with higher expression levels of both Sca1 and CD34 from among the population that is Sca1+ and CD34+; and culturing these blocks in three dimensions in which a first block consisting of hair follicle epithelial stem cells, a second block consisting of dermal papilla cells, and a third block consisting of the specific mesenchymal cells are stacked in this order.

12. The method according to either claim 10 or 11, wherein the second block is formed by accumulating hair papilla cells on the third block in a droplet, the first block is formed by accumulating hair follicle epithelial stem cells on the second block, and then these blocks are cultured in three dimensions.

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