Method of generating hair follicles

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a method for generating hair follicles. The method for generating hair follicles comprises the step of carrying out mixed culture on hair follicle stem cells and hair follicle dermal stem cells. According to the method, through combined culture of the hair follicle stem cells and the hair follicle dermal stem cells, the regeneration efficiency of the hair follicles can be effectively improved, meanwhile, the generated hair follicles are more complete in structure and more sound in function, and the quality of regenerated hair is higher. Wherein the hair follicle dermal stem cells provide necessary signals and microenvironment support for the hair follicle stem cells, and proliferation, differentiation and hair follicle morphogenesis of the hair follicle stem cells are promoted. In addition, ethical problems caused by the adoption of embryo-derived cells are avoided, the operation process is simplified, and the cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for generating hair follicles. Background Art

[0002] The skin is a multi-layered organ that is essential for regulating body temperature and retaining body fluids, resisting external pressure, and regulating touch and pain sensations. Hair follicles are appendages of the skin. As a complex micro-organ with multiple functions, hair follicles have extraordinary regenerative abilities. This regenerative ability is essential for maintaining the homeostasis of hair follicles. In hair loss and skin diseases (such as androgenic alopecia AGA and alopecia areata AA), the regenerative ability of hair follicles is severely damaged. In recent years, human hair follicle regeneration technology has gradually become an important means of treating hair loss and skin diseases, but it still faces many challenges in terms of hair follicle regeneration efficiency and the formation of functional hair follicles.

[0003] Early studies on hair follicle regeneration focused mainly on human adult hair follicle stem cells. However, the results of transplanting human adult hair follicle stem cells alone were not ideal. It was difficult to effectively induce hair follicle regeneration, and the hair follicle formation rate was low and the function was incomplete. This shows that human adult hair follicle stem cells alone cannot completely replicate the complex process of hair follicle development in vivo. To this end, researchers began to explore the role of other parts of the hair follicle in hair follicle regeneration, such as dermal papilla cells located in the mesenchymal part at the bottom of the hair follicle. Early studies have shown that dermal papilla cells play a vital role in the development and regeneration of hair follicles. However, when human adult hair follicle stem cells and dermal papilla cells are mixed and transplanted, the regeneration efficiency of hair follicles is still unsatisfactory, suggesting that the signals provided by adult dermal papilla cells alone are not enough to fully activate the regeneration potential of human hair follicles.

[0004] Further studies have found that co-transplantation with embryonic mesenchymal cells can significantly improve the hair follicle regeneration efficiency of human hair follicle stem cells, successfully induce the formation of structurally complete and functional hair follicles, and grow normal hair. In addition, by gradually regulating the transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signaling pathways, cranial epithelial cells and neural crest cells can be co-induced in human embryonic stem cell spherical aggregates. After long-term culture, cystic skin organoid structures can be induced to differentiate, which are composed of stratified epidermis, fat-rich dermis, and pigmented hair follicles with sebaceous glands. However, the acquisition of embryonic-derived cells involves ethical issues, and the clinical application prospects of such methods are limited.

[0005] In addition to adult hair follicle stem cells, related technologies also attempt to use human pluripotent stem cells, mesenchymal stem cells and other cells for hair follicle regeneration. The researchers simulated the in vivo hair follicle development process and used a precisely regulated combination of growth factors, cytokines and extracellular matrix to direct the differentiation of induced pluripotent stem cells into hair follicle stem cells and dermal papilla cells. The hair follicle stem cells and dermal papilla cells obtained by in vitro differentiation were mixed in a specific ratio and transplanted into the back skin of immunodeficient mice. However, the probability of successfully inducing the formation of new hair follicles was very low, which was consistent with the results of co-transplantation of freshly isolated hair follicle stem cells and dermal papilla cells.

[0006] Therefore, it is necessary to provide a method for generating hair follicles with no ethical issues and high regeneration efficiency. Summary of the invention

[0007] The present invention provides a method for generating hair follicles, which method does not involve ethical issues and has high regeneration efficiency of hair follicles.

[0008] A first aspect of the present invention provides a method for generating hair follicles, the method comprising co-culturing hair follicle stem cells and hair follicle dermal stem cells.

[0009] In some embodiments of the present invention, the hair follicle dermal stem cells are derived from the supernatant of the hair bulb of the hair follicle after digestion with type I collagenase and centrifugation.

[0010] In some embodiments of the present invention, the ratio of the number of the hair follicle stem cells to the number of the hair follicle dermal stem cells is 1:(0.1-10).

[0011] In some embodiments of the present invention, the hair follicle stem cells and the hair follicle dermal stem cells are independently at least one of autologous cells, allogeneic cells, and xenogeneic cells.

[0012] In some embodiments of the present invention, at least one of the hair follicle stem cells and the hair follicle dermal stem cells is formed by differentiation of stem cells.

[0013] In some embodiments of the present invention, at least one of the hair follicle stem cells and the hair follicle dermal stem cells is pretreated before mixed culture, and the pretreatment includes at least one of growth factor pretreatment, small molecule compound pretreatment, and gene editing.

[0014] In some embodiments of the present invention, the mixed culture is 2D culture or 3D culture.

[0015] In some embodiments of the present invention, the mixed culture is performed in a delivery vehicle.

[0016] In some embodiments of the present invention, the mixed culture period is 10 to 60 days.

[0017] The second aspect of the present invention provides an artificial hair follicle, wherein the artificial hair follicle is obtained by the above-mentioned method.

[0018] The third aspect of the present invention provides a use of the aforementioned artificial hair follicle in preparing in vitro hair follicle-related products or screening and testing in vitro hair follicle-related products.

[0019] A fourth aspect of the present invention provides a composition comprising hair follicle stem cells and hair follicle dermal stem cells.

[0020] In some embodiments of the invention, the composition further comprises a delivery vehicle.

[0021] In some embodiments of the present invention, the delivery carrier includes at least one of a natural polymer, a synthetic polymer, and a solvent.

[0022] According to a fifth aspect of the present invention, there is provided a method for hair follicle transplantation, the method comprising the step of applying the aforementioned artificial hair follicles or the aforementioned composition to a site to be transplanted.

[0023] The sixth aspect of the present invention provides a use of the aforementioned artificial hair follicle or the aforementioned composition in the preparation of a product for reconstructing skin or hair follicles, promoting hair growth, or treating skin diseases.

[0024] The beneficial effects of the present invention are: The present invention can effectively improve the regeneration efficiency of hair follicles by combining the cultivation of hair follicle stem cells and hair follicle dermal stem cells, and at the same time, the generated hair follicles have a more complete structure, more complete functions, and higher quality of regenerated hair. Among them, the hair follicle dermal stem cells provide the necessary signals and microenvironmental support for the hair follicle stem cells, promoting the proliferation, differentiation and hair follicle morphogenesis of the hair follicle stem cells. In addition, it also avoids the ethical issues caused by the use of embryonic cells, simplifies the operation process, and reduces costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the immunofluorescence staining result in one embodiment of the present invention. Among them, A is the experimental process of different groups, B is the immunofluorescence staining result of human skin sample (hHF) and mouse skin sample (mHF), and C is the immunofluorescence staining result of mouse transplanted skin samples obtained by two different combined transplantation methods in Example 3 and Comparative Example 1.

[0026] Figure 2 This is a photograph of mouse transplanted skin tissue samples on the 35th day of transplantation using two different combined transplantation methods of Example 3 and Comparative Example 1 in one embodiment of the present invention.

[0027] Figure 3The following are the bioinformatics analysis results after single-cell RNA sequencing of normal human skin tissue and mouse transplanted skin tissue obtained by two different combined transplantation methods in Example 3 and Comparative Example 1 in one embodiment of the present invention. A is the clustering result (left) and integrated annotation result (right) of single-cell data of normal human hair follicles and skin tissue obtained by two different combined transplantation methods, and B is the comparison result of cell types obtained by annotation of different categories of samples. DETAILED DESCRIPTION

[0028] In the description of the present invention, the terms "first", "second", and "third" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first", "second", and "third" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0029] The first aspect of the present invention provides a method for generating hair follicles, comprising co-culturing hair follicle stem cells and hair follicle dermal stem cells.

[0030] The term "hair follicle" refers to the sheath-like structure surrounding the hair root, which is divided into two layers: the inner layer is a stratified epithelium that is continuous with the epidermis, called the epithelial sheath (including the inner root sheath and the outer root sheath), and the outer layer is a connective tissue that is continuous with the dermis, called the connective tissue sheath. The end of the hair follicle swells to form a hair bulb. The bottom surface of the hair bulb is concave to form a pit, in which there is connective tissue, and blood vessels and nerve endings protrude in a papilla shape, called the hair papilla. At the attachment point of the arrector pili muscle, the outer root sheath cells proliferate to form a bulge area, which contains different types of hair follicle stem cells (HFSCs), such as epidermal stem cells and melanin stem cells. It can be understood that HFSCs are distributed in multiple different anatomical regions of the hair follicle, such as the outer root sheath and the hair bulb, so HFSCs can be obtained from any one or more parts of the outer root sheath and the hair bulb, such as the bulge area. Hair follicle dermal stem cells (HFDSCs) refer to a type of stem cells that support the renewal of the dermis of the hair follicle. They are capable of self-renewal and have the ability to differentiate into dermal papilla (DP) cells and dermal sheath (DS) cells. Usually, HFDSCs are located at the base of the hair bulb of the hair follicle, near the dermal papilla. Therefore, hair follicle stem cells and hair follicle dermal stem cells belong to two different types of cells, and there are certain differences in their location and function.

[0031] In some embodiments, hair follicle stem cells are obtained by digesting the bulge of a hair follicle with dispase and collagenase. In some embodiments, the dispase comprises type I dispase. In some embodiments, the collagenase comprises type IV collagenase. In some embodiments, hair follicle stem cells are obtained by digesting the bulge of a hair follicle with dispase and collagenase in sequence. In some embodiments, hair follicle stem cells are obtained by digesting the bulge of a hair follicle with type I dispase and type IV collagenase in sequence. In some embodiments, the bulge of the hair follicle is first digested in an enzyme solution comprising a type I dispase at a working concentration of 0.1 to 10 mg / mL, and the working concentration of the type I dispase in the enzyme solution can specifically be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, and 10 mg / mL. In some embodiments, the temperature of the digestion reaction is 4-40°C, for example, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C. In some embodiments, the time of the digestion reaction is 10 min-10 h, for example, 10 min, 20 min, 30 min, 40 min, 45 min, 50 min, 55 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h. In some embodiments, the bulge of the hair follicle is subsequently digested in an enzyme solution comprising type IV collagenase at a working concentration of 0.1 to 10 mg / mL, and the working concentration of type IV collagenase in the enzyme solution can specifically be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, and 10 mg / mL.In some embodiments, the temperature of the digestion reaction is 4-40°C, for example, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C. In some embodiments, the time of the digestion reaction is 10 min-10 h, for example, 10 min, 20 min, 30 min, 40 min, 45 min, 50 min, 55 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h. In some embodiments, the digestion product is filtered. In some embodiments, a 20-60 μm filter is used to filter out impurities and larger cell clumps, for example, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm. In some embodiments, the filter is a cell sieve, and the specification of the cell sieve is 40 μm. In some embodiments, the liquid filtered by the filter is also centrifuged to collect the precipitate as hair follicle stem cells. In some embodiments, the centrifugal force of the centrifugation is 200 g or more, specifically 200-400 g, for example 200 g, 220 g, 240 g, 250 g, 260 g, 280 g, 300 g, 320 g, 340 g, 350 g, 360 g, 380 g, 400 g. In some embodiments, the centrifugation time is 2-10 min, for example, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min.

[0032] In some embodiments, the hair follicle dermal stem cells are derived from the supernatant after digestion and centrifugation of the hair bulb of the hair follicle with type I collagenase. In some embodiments, the hair bulb of the hair follicle is digested in an enzyme solution containing type I collagenase at a working concentration of 0.1 to 10 mg / mL, and the working concentration of type I collagenase in the enzyme solution can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL. In some embodiments, the hair bulb of the hair follicle is digested in an enzyme solution containing type I collagenase at a working concentration of 0.8 to 1.5 mg / mL. In some embodiments, the temperature of the digestion reaction is 4 to 40°C, for example, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C. In some embodiments, the temperature of the digestion reaction is 35 to 40°C. In some embodiments, the time of the digestion reaction is 10 min to 10 h, for example, 10 min, 20 min, 30 min, 40 min, 45 min, 50 min, 55 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h. In some embodiments, the time of the digestion reaction is 50 min to 60 min. Among them, type I collagenase is an enzyme mixture, the main components of which include clostripain, neutral protease and two collagenases, thereby degrading collagen and other intracellular substances. One or more enzymes in type I collagenase can be wild-type enzymes, or products modified by genetic engineering on the basis of wild-type enzymes, such as mutant enzymes obtained by random mutation, site-directed mutation, DNA shuffling, etc. for one or more purposes such as improving enzyme activity and thermal stability, and have similar or higher ability to effectively release cells from tissues. In some embodiments, after the hair bulb is digested with type I collagenase, the digestion product is further subjected to a first centrifugation step. In some embodiments, the centrifugal force of the first centrifugation is above 5 g, specifically 5 to 30 g, such as 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 18 g, 20 g, 25 g, 30 g, etc.When the centrifugal force of the first centrifugation is too low, the separation effect is not ideal, and it is difficult to clearly distinguish the supernatant containing HFDSC and the precipitate containing DP microspheres; when the centrifugal force is too large, it is helpful for separation, but it may damage the precipitate of DP microspheres. In some embodiments, the time of the first centrifugation is 1 to 5 minutes, for example, it can be 1 min, 2 min, 3 min, 4 min, 5 min. In some embodiments, it also includes filtering the supernatant after the first centrifugation. In some embodiments, after the first centrifugation, a 20-60 μm filter is used to filter and remove impurities and larger cell clumps, for example, it can be 20 μm, 30 μm, 40 μm, 50 μm, 60 μm. In some embodiments, the filter is a cell sieve, and the specification of the cell sieve is 40 μm. In some embodiments, the liquid filtered by the filter is also subjected to a second centrifugation to collect the precipitate as hair follicle dermal stem cells. In some embodiments, the centrifugal force of the second centrifugation is 200 g or more, specifically 200-400 g, such as 200 g, 220 g, 240 g, 250 g, 260 g, 280 g, 300 g, 320 g, 340 g, 350 g, 360 g, 380 g, 400 g. In some embodiments, the second centrifugation time is 2-10 min, such as 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min.

[0033] In some embodiments, the hair follicle stem cells and the hair follicle dermal stem cells are at least one of autologous cells, allogeneic cells, and xenogeneic cells. Among them, autologous, allogeneic, and xenogeneic refer to the type of individual intended to generate hair follicles. For example, when you want to generate hair follicles of a human individual, the autologous cells are hair follicle stem cells or hair follicle dermal stem cells derived from the human individual, the allogeneic cells are hair follicle stem cells or hair follicle dermal stem cells derived from other human individuals of the same genus and species, and the xenogeneic cells are hair follicle stem cells or hair follicle dermal stem cells derived from other species. When autologous cells are used to generate hair follicles, the generated hair follicles can completely avoid immune rejection when transplanted. In some embodiments, when the hair follicle stem cells or hair follicle dermal stem cells are allogeneic cells, the cells can be modified in advance to reduce their immunogenicity, such as using gene editing technology to modify allogeneic cells. In some embodiments, when the generated hair follicles are used for allogeneic transplantation, it is also necessary to perform local or systemic immunosuppression on the transplanted individual. In some embodiments, when the hair follicle stem cells or hair follicle dermal stem cells are xenogeneic cells, they are modified in advance to be suitable for the intended transplant species, such as by modifying their genotype, etc. In some embodiments, at least one of the hair follicle stem cells and the hair follicle dermal stem cells is an adult stem cell.

[0034] In some embodiments, the hair follicle stem cells and hair follicle dermal stem cells are cells from mammals, including animals of the order Carnivora, Perissodactyla, Artiodactyla, Rodentia, Lagomorpha, Primates, and more, such as any one of dogs, cats, pigs, sheep, cattle, horses, rabbits, mice, monkeys, orangutans, gorillas, chimpanzees, and humans.

[0035] In some embodiments, hair follicle stem cells and hair follicle dermal stem cells can be obtained by separation, culture or further passage (such as P1~P50 generations, such as P2 generations, P3 generations, P4 generations, P5 generations, P10 generations, P20 generations, P30 generations, P40 generations, and P50 generations), and can also be formed by differentiation of stem cells. In some embodiments, the stem cells are stem cells of non-embryonic origin. In some embodiments, the stem cells include induced pluripotent stem cells (iPSCs). In some embodiments, induced pluripotent stem cells are cultured in human embryonic stem cell culture medium to form embryoid bodies, which are then inoculated into 3T3 fibroblast feeder layers, and cultured successively with induction medium A, differentiation medium B, and differentiation medium C, and then digested and separated into single cells and inoculated on 3T3 fibroblasts treated with mitomycin C, and cultured with differentiation medium D to obtain hair follicle stem cells. Among them, the human embryonic stem cell culture medium includes DMEM / F12 culture medium and additives, and the additives include serum or serum substitutes, GlutaMAX, non-essential amino acids, β-mercaptoethanol and bFGF; the induction culture medium A includes DMEM / F12 culture medium and additives, and the additives include serum, insulin, hydrocortisone, cholera toxin, triiodothyronine, vitamin C, adenine and retinoic acid; the differentiation culture medium B includes DMEM / F12 culture medium and additives, and the additives include serum, insulin, hydrocortisone, cholera toxin, triiodothyronine, vitamin C, adenine, retinoic acid, BMP-4 and EGF; the differentiation culture medium C includes D-KSFM culture medium and additives, and the additives include serum, insulin, hydrocortisone, cholera toxin, triiodothyronine, vitamin C, adenine and retinoic acid; the differentiation culture medium D includes D-KSFM culture medium and additives, and the additives include BMP-4 and EGF.

[0036] In some embodiments, at least one of the hair follicle stem cells and the hair follicle dermal stem cells is pretreated before mixed culture, and the pretreatment includes at least one of growth factor pretreatment, small molecule compound pretreatment, and gene editing. By modifying them in at least one of the above ways, the proliferation, differentiation and hair follicle regeneration ability of the cells can be improved.

[0037] The term "culture" refers to the process in which cells grow and proliferate under certain conditions to produce a large number of cells. Accordingly, mixed culture means that hair follicle stem cells and hair follicle dermal stem cells are co-cultured for at least a period of time during the culture process, and there is spatial connectivity, material exchange or interaction between the two.

[0038] In some embodiments, the ratio of the number of hair follicle stem cells to hair follicle dermal stem cells in mixed culture is 1:(0.1~10), for example, it can be 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0039] In some embodiments, the mixed culture can be 2D culture or 3D culture. In some embodiments, the 3D culture method includes 3D culture with a scaffold or 3D culture without a scaffold. In some embodiments, the 3D culture method without a scaffold includes at least one of the low adhesion plane method, the hanging drop method, the stirring method, and the 3D culture dish. In some embodiments, the scaffold material of the 3D culture can be one or more of bioceramics, bioglass, metal, natural polymers (such as alginate, silk, hyaluronic acid, chitosan, silk fibroin, fibrin, collagen, pluronic acid, etc.) and synthetic polymers (such as polylactic acid, polyglycolic acid, polycaprolactone, polyacrylonitrile, polyethylene glycol, polyimide, polyvinyl alcohol, etc.). In some embodiments, the mixed culture is carried out in a delivery carrier. In some embodiments, the delivery carrier can be liquid, solid (regular or irregular shapes such as spheres). In some embodiments, the delivery carrier includes one or more of the above-mentioned natural polymers, synthetic polymers, solvents (such as water, PBS buffer, saline, etc.). In some embodiments, the mixed culture period is 10 to 60 days, for example, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, or 60 days.

[0040] In some embodiments, the method of generating hair follicles is not directly used to treat hair loss, skin diseases, etc. For example, after the hair follicles are generated, the generated hair follicles are transplanted, or the generated hair follicles are used for research, screening tests, etc.

[0041] The second aspect of the present invention provides an artificial hair follicle, which is obtained by the above method.

[0042] The third aspect of the present invention provides the use of the aforementioned artificial hair follicle in preparing in vitro hair follicle-related products or screening and testing in vitro hair follicle-related products.

[0043] In some embodiments, the in vitro hair follicle-related products include in vitro tissue engineering models, such as hair follicle models, hair models containing hair follicles, skin models containing hair follicles, etc. In some embodiments, screening and testing of in vitro hair follicle-related products include products used to screen drugs that can promote hair growth, test the effects of cosmetics on hair follicles and hair growth, and other related effects.

[0044] A fourth aspect of the present invention provides a composition comprising hair follicle stem cells and hair follicle dermal stem cells.

[0045] In some embodiments, the composition further comprises a delivery carrier. In some embodiments, the delivery carrier can be liquid or solid (regular or irregular shapes such as spheres). In some embodiments, the delivery carrier comprises at least one of natural polymers, synthetic polymers, and solvents. In some embodiments, natural polymers include alginate, silk, hyaluronic acid, chitosan, fibroin, fibrin, collagen, pluronic acid, etc. In some embodiments, synthetic polymers include polylactic acid, polyglycolic acid, polycaprolactone, polyacrylonitrile, polyethylene glycol, polyimide, polyvinyl alcohol, etc. In some embodiments, solvents include water, PBS buffer, saline, etc.

[0046] In some embodiments, the hair follicle stem cells in the composition are obtained by digesting the bulge of the hair follicle with dispase and collagenase. In some embodiments, the dispase includes type I dispase. In some embodiments, the collagenase includes type IV collagenase. In some embodiments, the hair follicle stem cells are obtained by digesting the bulge of the hair follicle with dispase and collagenase in sequence. In some embodiments, the hair follicle stem cells are obtained by digesting the bulge of the hair follicle with type I dispase and type IV collagenase in sequence. In some embodiments, the bulge of the hair follicle is first digested in an enzyme solution comprising a type I dispase at a working concentration of 0.1 to 10 mg / mL, and the working concentration of the type I dispase in the enzyme solution can specifically be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, and 10 mg / mL. In some embodiments, the temperature of the digestion reaction is 4-40°C, for example, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C. In some embodiments, the time of the digestion reaction is 10 min-10 h, for example, 10 min, 20 min, 30 min, 40 min, 45min, 50 min, 55 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h. In some embodiments, the bulge of the hair follicle is subsequently digested in an enzyme solution comprising type IV collagenase at a working concentration of 0.1 to 10 mg / mL, and the working concentration of type IV collagenase in the enzyme solution can specifically be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, and 10 mg / mL.In some embodiments, the temperature of the digestion reaction is 4-40°C, for example, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C. In some embodiments, the time of the digestion reaction is 10 min-10 h, for example, 10 min, 20 min, 30 min, 40 min, 45min, 50 min, 55 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h. In some embodiments, the digestion product is filtered. In some embodiments, a 20-60 μm filter is used to filter out impurities and larger cell clumps, for example, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm. In some embodiments, the filter is a cell sieve, and the specification of the cell sieve is 40 μm. In some embodiments, the liquid filtered by the filter is also centrifuged to collect the precipitate as hair follicle stem cells. In some embodiments, the centrifugal force of the centrifugation is 200 g or more, specifically 200-400 g, for example, 200 g, 220 g, 240 g, 250 g, 260 g, 280 g, 300 g, 320 g, 340 g, 350 g, 360 g, 380 g, 400 g. In some embodiments, the centrifugation time is 2-10 min, for example, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min.

[0047] In some embodiments, the hair follicle dermal stem cells are derived from the supernatant after digestion and centrifugation of the hair bulb of the hair follicle with type I collagenase. In some embodiments, the hair bulb of the hair follicle is digested in an enzyme solution containing type I collagenase at a working concentration of 0.1 to 10 mg / mL, and the working concentration of type I collagenase in the enzyme solution can be 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL. In some embodiments, the hair bulb of the hair follicle is digested in an enzyme solution containing type I collagenase at a working concentration of 0.8 to 1.5 mg / mL. In some embodiments, the temperature of the digestion reaction is 4 to 40°C, for example, 4°C, 6°C, 8°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C. In some embodiments, the temperature of the digestion reaction is 35 to 40°C. In some embodiments, the time of the digestion reaction is 10 min to 10 h, for example, 10 min, 20 min, 30 min, 40 min, 45 min, 50 min, 55 min, 60 min, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h. In some embodiments, the time of the digestion reaction is 50 min to 60 min. In some embodiments, after the hair bulb is digested with collagenase type I, the step of first centrifuging the digestion product is also included. In some embodiments, the centrifugal force of the first centrifugation is above 5 g, specifically 5-30 g, such as 6 g, 7 g, 8 g, 9 g, 10 g, 11 g, 12 g, 13 g, 14 g, 15 g, 16 g, 18 g, 20 g, 25 g, 30 g, etc. In some embodiments, the time of the first centrifugation is 1-5 min, such as 1 min, 2 min, 3 min, 4 min, 5 min. In some embodiments, the supernatant after the first centrifugation is filtered. In some embodiments, after the first centrifugation, a 20-60 μm filter is used to filter and remove impurities and larger cell clumps, such as 20 μm, 30 μm, 40 μm, 50 μm, 60 μm. In some embodiments, the filter is a cell sieve, and the specification of the cell sieve is 40 μm.In some embodiments, the liquid filtered by the filter is further subjected to a second centrifugation to collect and precipitate hair follicle dermal stem cells. In some embodiments, the centrifugal force of the second centrifugation is 200 g or more, specifically 200-400 g, such as 200 g, 220 g, 240 g, 250 g, 260 g, 280 g, 300 g, 320 g, 340 g, 350 g, 360 g, 380 g, 400 g. In some embodiments, the time of the second centrifugation is 2-10 min, such as 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min.

[0048] In some embodiments, the hair follicle stem cells and the hair follicle dermal stem cells are at least one of autologous cells, allogeneic cells, and xenogeneic cells. Among them, autologous, allogeneic, and xenogeneic refer to the type of individual intended to generate hair follicles. In some embodiments, when the hair follicle stem cells or the hair follicle dermal stem cells are allogeneic cells, they are pre-modified to reduce their immunogenicity, for example, the allogeneic cells are modified using gene editing technology. In some embodiments, the composition also includes an immunosuppressant. In some embodiments, when the hair follicle stem cells or the hair follicle dermal stem cells are xenogeneic cells, they are xenogeneic cells that have been pre-modified to be suitable for the intended species of transplantation, such as xenogeneic cells whose genotypes have been modified. In some embodiments, at least one of the hair follicle stem cells and the hair follicle dermal stem cells is an adult stem cell.

[0049] In some embodiments, the hair follicle stem cells and hair follicle dermal stem cells are cells from mammals, including animals of the order Carnivora, Perissodactyla, Artiodactyla, Rodentia, Lagomorpha, Primates, and more, such as any one of dogs, cats, pigs, sheep, cattle, horses, rabbits, mice, monkeys, orangutans, gorillas, chimpanzees, and humans.

[0050] In some embodiments, hair follicle stem cells and hair follicle dermal stem cells can be primary cells that are separated and cultured, or passaged cells that are further passaged (such as P1 to P50 generations, such as P2 generations, P3 generations, P4 generations, P5 generations, P10 generations, P20 generations, P30 generations, P40 generations, and P50 generations). In some embodiments, hair follicle stem cells and hair follicle dermal stem cells can also be hair follicle stem cells and hair follicle dermal stem cells formed by differentiation of stem cells. In some embodiments, stem cells are stem cells of non-embryonic origin. In some embodiments, stem cells include induced pluripotent stem cells (iPSCs). In some embodiments, induced pluripotent stem cells are cultured in human embryonic stem cell culture medium to form embryoid bodies, which are then inoculated into 3T3 fibroblast feeder layers, and cultured in induction medium A, differentiation medium B, and differentiation medium C in succession. Single cells are digested and separated and inoculated into 3T3 fibroblasts treated with mitomycin C, and cultured in differentiation medium D to obtain hair follicle stem cells. Among them, the human embryonic stem cell culture medium includes DMEM / F12 culture medium and additives, and the additives include serum or serum substitutes, GlutaMAX, non-essential amino acids, β-mercaptoethanol and bFGF; the induction culture medium A includes DMEM / F12 culture medium and additives, and the additives include serum, insulin, hydrocortisone, cholera toxin, triiodothyronine, vitamin C, adenine and retinoic acid; the differentiation culture medium B includes DMEM / F12 culture medium and additives, and the additives include serum, insulin, hydrocortisone, cholera toxin, triiodothyronine, vitamin C, adenine, retinoic acid, BMP-4 and EGF; the differentiation culture medium C includes D-KSFM culture medium and additives, and the additives include serum, insulin, hydrocortisone, cholera toxin, triiodothyronine, vitamin C, adenine and retinoic acid; the differentiation culture medium D includes D-KSFM culture medium and additives, and the additives include BMP-4 and EGF.

[0051] In some embodiments, at least one of the hair follicle stem cells and the hair follicle dermal stem cells is a pretreated cell, and the pretreatment includes at least one of growth factor pretreatment, small molecule compound pretreatment, and gene editing. By modifying it in at least one of the above methods, the proliferation, differentiation and hair follicle regeneration ability of the cell can be improved.

[0052] In some embodiments, the ratio of the number of hair follicle stem cells to the number of hair follicle dermal stem cells in the composition is 1:(0.1~10), for example, it can be 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0053] In some embodiments, the composition further includes a drug that promotes hair growth, such as minoxidil, finasteride, etc. In some embodiments, the various components in the composition can be applied simultaneously or sequentially in one or more different ways. In some embodiments, the method of application can be one or more of external or internal administration.

[0054] A fourth aspect of the present invention provides a method for hair follicle transplantation, comprising the step of applying the aforementioned artificial hair follicles or the aforementioned composition to a site to be transplanted.

[0055] In some embodiments, the site to be transplanted is the skin surface.

[0056] In some embodiments, the method of hair follicle transplantation is transplantation for non-therapeutic purposes, for example, hair follicles may be transplanted at a specific site for the purpose of constructing a model of hair follicles, hair, skin, etc.

[0057] In some embodiments, the transplantation method is minimally invasive transplantation, for example, the transplantation may be performed using minimally invasive techniques such as microneedles and lasers, which can reduce surgical trauma and shorten recovery time.

[0058] In some embodiments, the transplantation method further comprises the use of drug-assisted therapy, such as the combined use of drugs that promote hair growth (such as minoxidil, finasteride, etc.) to enhance the effect of hair follicle regeneration.

[0059] In some embodiments, the transplantation method further comprises combined physical therapy, such as combined use of low-energy laser therapy, scalp massage and other physical therapy methods to improve scalp blood circulation and promote hair growth.

[0060] A fifth aspect of the present invention provides a use of the aforementioned artificial hair follicle or the aforementioned composition in the preparation of a product for reconstructing skin or hair follicles, promoting hair growth, or treating skin diseases.

[0061] In some embodiments, skin diseases include vitiligo, scars, and the like.

[0062] In some embodiments of the present invention, adult hair follicle stem cells (HFSCs) and human hair follicle dermal stem cells (hHFDSCs) are transplanted together to successfully achieve a breakthrough in regenerating human hair follicles on nude mouse skin. Compared with the prior art, this technical solution brings the following beneficial effects: (1) Improvement of hair follicle regeneration efficiency: Compared with the combined transplantation of HFSCs and DPs, the combined transplantation of HFSCs and hHFDSCs significantly improved the hair follicle regeneration efficiency. With the same number of transplanted cells, more new hair follicles can be formed, thereby increasing the hair regeneration density. Hair follicle dermal stem cells provide the necessary signals and microenvironment support for hair follicle stem cells, promoting the proliferation, differentiation and hair follicle morphogenesis of hair follicle stem cells.

[0063] (2) Improvement of the quality of regenerated hair follicles: The hair follicles generated by combined transplantation have a more complete structure, more robust functions, and higher quality of regenerated hair. The length, diameter, pigmentation, growth rate, etc. of the hair are closer to natural hair. The addition of hair follicle dermal stem cells helps to build a hair follicle microenvironment that is closer to the physiological state, thereby promoting the normal development and functional maintenance of the regenerated hair follicles.

[0064] (3) Simplified process and cost savings: Compared with the use of embryonic stem cells or induced pluripotent stem cells for hair follicle regeneration, the use of adult stem cells avoids the complex cell reprogramming and differentiation process, simplifies the operation process, and reduces production costs and time. At the same time, adult stem cells can be obtained from autologous tissues, avoiding immune rejection reactions and eliminating the need for immunosuppressive therapy, further reducing treatment risks and costs.

[0065] (4) High safety: The use of adult stem cells avoids the ethical issues associated with embryonic stem cells, and autologous transplantation can also avoid immune rejection reactions and does not require the use of immunosuppressants, thereby reducing the risk of infection and other complications and improving the safety of treatment.

[0066] (5) Provide new solutions for the treatment of hair loss and skin diseases: It provides a new and more effective solution for the treatment of hair loss, scarring alopecia and other skin diseases, such as skin burns, skin scars, and surgical trauma. It is expected to solve the limitations of existing treatment methods, such as the limited effect of drug treatment for hair loss, insufficient donor area for hair transplantation, difficulty in repairing skin damage, and difficulty in treating stubborn skin scars.

[0067] (6) Promote the progress of basic research: The synergistic effect of hair follicle stem cells or hair follicle dermal stem cells in hair follicle regeneration has been confirmed, which has deepened the understanding of the mechanism of hair follicle development and regeneration, and provided an important theoretical basis for further hair regeneration research. In addition, the combination of hair follicle stem cells and hair follicle dermal stem cells can be used to generate hair follicle organoids for clinical treatment and drug screening of skin and hair loss diseases.

[0068] In summary, the combined transplantation scheme provided in the embodiments of the present invention has significant advantages, including higher hair follicle regeneration efficiency and hair quality, simpler operation procedures, lower costs and higher safety.

[0069] The present invention is further described in detail below through specific examples.

[0070] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0071] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The materials and reagents used in the present examples are commercially available unless otherwise specified. The names, manufacturers, and product numbers of some reagents are shown in Table 1.

[0072] Table 1. Reagent names, manufacturers, and product numbers

[0073] Example 1: Isolation and culture of primary hHFDSCs 1. Acquisition of hair follicles Full-thickness scalp samples were taken from the occipital region of the skull of patients undergoing craniotomy or hair transplantation in the hospital, and hair follicles were isolated. Immediately after the hair follicles were removed from the body, they were temporarily stored on ice at low temperatures to maintain cell activity. The follicles were then quickly transported to the hospital laboratory for further processing, and cold chain transportation equipment was used during transportation to maintain a low temperature environment. The blood stains on the hair follicles were washed with 1× PBS buffer, and then dried with dust-free paper to avoid residual reagents from affecting the subsequent cell isolation process. The entire process was performed aseptically to avoid contamination.

[0074] 2. Isolation and Purification of hHFDSCs The terminal bulb was cut 1 mm from the end of the hair follicle, and then the pigment and keratin components in the hair follicle were removed by mechanical force. The remaining dermis of the hair follicle in the terminal bulb was transferred to a 15 mL centrifuge tube and digested with 1 mg / mL type I collagenase at 37°C for 55 minutes. During the digestion process, the cell suspension obtained after digestion was centrifuged at 10 g for 2 minutes to separate the supernatant containing hHFDSCs and the precipitate containing DP microspheres. The supernatant was filtered through a 40 μm cell sieve to remove impurities and larger cell clumps. The filtered supernatant was centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The precipitate obtained was purified primary hHFDSCs.

[0075] 3. Cell Culture The isolated and purified hHFDSCs were cultured in high-glucose DMEM medium (supplemented with 20v / v% FBS, 1v / v% penicillin-streptomycin, 1v / v% non-essential amino acids, and 1v / v% GlutaMAX) at 37°C and 5% CO2. The medium was replaced every 3 days, and sterile operation was performed during replacement to avoid contamination.

[0076] 4. Cell identification and quality control The isolated and purified primary hHFDSCs were identified by immunofluorescence staining using antibodies against MMP11, COL3A1, RBP4 and COL11A1. The results showed that the isolated cells all expressed four markers, MMP11, COL3A1, RBP4 and COL11A1. Therefore, the isolated cells were hHFDSCs.

[0077] By regularly testing the morphology, proliferation ability and activity of hHFDSCs, the quality of the cells and the repeatability of the experiment are ensured. The cell morphology is recorded by microscopic photography, and the proliferation ability is evaluated by passage. The results show that the primary hHFDSCs and the hHFDSCs of the P3 generation are consistent in cell morphology after three days of culture in high-glucose DMEM medium, and the cell activity is high. Therefore, the hHFDSCs isolated in this embodiment can be amplified normally, and the cell quality is good. In addition, the relevant experimental results of immunofluorescence staining and morphological observation also show that the hHFDSC isolated in this way has the differentiation potential to differentiate into DP cells and DS cells.

[0078] Example 2: Isolation of primary HFSCs The hair follicles were obtained by the method of reference Example 1, and the bulge area of ​​the hair follicles was separated by dissection under a microscope, and then transferred to a 15 mL centrifuge tube, and 1 mL of 1 mg / mL Dispase I was added, and digested at 37 ° C for 2 hours. During the digestion process, the mixture was repeatedly blown and mixed with a pipette. Then 1 mL of 1.5 mg / mL type IV collagenase was added, and digested at 37 ° C for 30 minutes. During the digestion process, the cell suspension was filtered with a 40 μm cell sieve. The filtered supernatant was transferred to a clean 15 mL centrifuge tube, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The obtained precipitate was the primary HFSCs.

[0079] Example 3: hHFDSCs+HFSCs co-transplantation Take 3×10 6 6×10 primary hHFDSCs in Example 2 5 Primary HFSCs were mixed into 15 μL Matrigel, and then the cell-Matrigel mixture was incubated in a 37°C incubator for 30 minutes until the Matrigel solidified to obtain Matrigel matrix gel encapsulating the mixed cells.

[0080] Six-week-old BALB / c nu / nu mice were anesthetized with isoflurane, and a circular area with a diameter of 3 mm was marked on the back of the mouse. A full-thickness skin wound with a diameter of 3 mm was made in the marked area with sterile scissors. Matrigel matrix encapsulated with mixed cells was transplanted to the wound site on the back of the mouse, and then the wound was covered with 3M transparent dressing and self-adhesive elastic bandage. After 4 weeks, the wound tissue on the back of the mouse was subjected to histological analysis.

[0081] Comparative Example 1: This comparative example provides a method for combined transplantation of hDPCs+HFSCs, which is different from Example 3 in that 3×10 6 Primary hHFDSCs were replaced with an equal number of primary human dermal papilla cells (DP). The primary DPs were obtained as follows: The hair follicles were obtained by the method of reference Example 1, and the terminal bulb was cut from the end of the hair follicle 1 mm away, and then transferred to a 15 mL centrifuge tube, 1 mL of 1.5 mg / mL type IV collagenase was added, and digested at 37 ° C for 3 hours. During the digestion process, the cell suspension was repeatedly blown and mixed with a pipette. After digestion, the cell suspension was filtered with a 40 μm cell sieve. The filtered supernatant was transferred to a clean 15 mL centrifuge tube, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The obtained precipitate was the primary DP. The isolated and purified DP was cultured in high-glucose DMEM medium (supplemented with 10v / v% FBS, 1v / v% penicillin-streptomycin) at 37 ° C and 5% CO2. The culture medium was replaced every 3 days.

[0082] Example 4: Immunofluorescence staining to verify hair follicle regeneration Human skin samples, normal mouse skin samples, and skin samples from the transplanted parts of mice 4 weeks after the two different combinations of cells of Example 3 and Comparative Example 1 were transplanted were collected.

[0083] The samples were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 7 μm thick paraffin sections.

[0084] After dewaxing and antigen retrieval, the paraffin sections were blocked with 5% bovine serum albumin (BSA) for 1 hour. The blocked sections were incubated with primary antibody (anti-human mitochondrial antibody) at 4 °C overnight, then washed three times with PBS, and the sections were incubated with fluorescently labeled secondary antibody (goat anti-rabbit IgG antibody, Jackson ImmunoResearch, catalog number: 111-545-003) at room temperature for 1 hour.

[0085] Cell nuclei were stained with 10 mg / mL Hoechst 33342 for 15 min, and images were acquired using an OLYMPUS FV4000 confocal microscope.

[0086] The experimental results are as follows Figure 1 As shown in B and C, in B, the human skin sample (hHF) showed a red positive signal, while the mouse skin sample (mHF) showed no red signal and was negative, proving that the anti-human mitochondrial antibody can specifically recognize human cells. Further observation of the results in C showed that the mouse skin sample transplanted with HFSCs and hHFDSCs also had obvious positive red signals, indicating the presence of hair follicle structures of human cells, while the mouse skin sample transplanted with HFSCs and DPs had no positive hair follicle structure. The above results show that the combined transplantation of HFSCs and DPs basically has no hair follicle regeneration potential, while the combined transplantation of HFSCs and hHFDSCs has a higher hair follicle regeneration potential.

[0087] Example 6: Single-cell sequencing verifies hair follicle cell types in hair follicle structure The skin tissue of mice transplanted by the combined transplantation methods of Example 3 and Comparative Example 1 on the 35th day after transplantation was observed under a microscope. Figure 2 As shown in the figure, it can be seen that in Example 3, the HFSC+hHFDSCs combined transplantation method can observe obvious hair follicle structure in the mouse skin tissue, and there is obvious pigment deposition, indicating that the new hair follicles have certain biological functions. In contrast, in Comparative Example 1, the HFSC+DP combined transplantation method can not observe the hair follicle structure in the mouse skin tissue, and there is no pigment deposition phenomenon.

[0088] Take the mouse skin tissue of the two combined transplantation methods of Example 3 and Comparative Example 1 on the 35th day of transplantation, and cut the tissue into small pieces using sterile ophthalmic scissors. Digest the chopped tissue with 5 mL of preheated 4.0 mg / mL Dispase Ⅱ at 37 ° C for 50 minutes. Then digest with preheated 1.6 mg / mL type I collagenase and 2.0 mg / mL type IV collagenase at 37 ° C for 30 minutes. Add 20 mL of high-glucose DMEM medium for digestion, and filter the cell suspension using a 70 μm cell sieve. The filtered filtrate is centrifuged at 300 g for 5 minutes, and then the precipitate is resuspended in PBS buffer containing 2v / v% FBS. Then filter through a 30 μm cell sieve to remove the remaining debris. Finally, centrifuge at 300 g for 5 minutes and resuspend in PBS buffer containing 0.04% bovine serum albumin (BSA) to produce the final single cell suspension for single-cell RNA sequencing library preparation.

[0089] The single cell suspension was constructed and sequenced according to the instructions of MGI DNBelab C Series High-throughput Single Cell RNA Library Preparation Kit V2.5. The raw data of single cell RNA sequencing were processed, integrated, clustered and annotated. The results are as follows Figure 3 As shown. Figure 3It can be seen from B that the cells obtained by the combined transplantation of HFSCs+hHFDSCs in Example 3 include all the cell types of normal hair follicles, including: hHFDSCs, DS (the proportion of this type of cells is low and not shown in the figure), DP, qHFSC (activated hair follicle stem cells), aHFSC (quiescent hair follicle stem cells), ORSB (outer root sheath basal cells), ORSS (outer root sheath outer layer basal cells), TAC1 (transient proliferating cell 1), TAC2 (transient proliferating cell 2), IRS (inner root sheath cells), IRSC (inner root sheath keratinocytes), MCC (medullary cortical keratinocytes); while the combined transplantation of HFSCs+DPs in Comparative Example 1 cannot form all the cells of the epidermal lineage. The above results prove that the hair follicles generated by the combined transplantation of HFSCs+hHFDSCs have all the cell types that perform the functions of hair follicles.

[0090] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for generating hair follicles, characterized in that include: The hair follicle stem cells and the hair follicle dermal stem cells were co-cultured.

2. The method according to claim 1, characterized in that: The ratio of the number of the hair follicle stem cells to the number of the hair follicle dermal stem cells is 1:(0.1-10).

3. The method according to claim 1, characterized in that: The hair follicle stem cells and the hair follicle dermal stem cells are independently at least one of autologous cells, allogeneic cells, and xenogeneic cells; and / or, At least one of the hair follicle stem cells and the hair follicle dermal stem cells is formed by differentiation of stem cells; and / or, At least one of the hair follicle stem cells and the hair follicle dermal stem cells is pretreated before mixed culture, and the pretreatment includes at least one of growth factor pretreatment, small molecule compound pretreatment, and gene editing.

4. The method according to claim 1, characterized in that: The mixed culture is 2D culture or 3D culture; and / or, The mixed culture is carried out in a delivery vehicle; and / or, The mixed culture time is 10 to 60 days.

5. Artificial hair follicles, characterized in that: Obtained by the method according to any one of claims 1 to 4.

6. Use of the artificial hair follicle according to claim 5 in preparing in vitro hair follicle related products or screening and testing in vitro hair follicle related products.

7. A composition, characterized in that: Including hair follicle stem cells and hair follicle dermal stem cells.

8. The composition according to claim 7, characterized in that: Also included are delivery vehicles.

9. The composition according to claim 8, characterized in that: The delivery carrier includes at least one of natural polymers, synthetic polymers and solvents.

10. A method for hair follicle transplantation, characterized in that: The method comprises the step of applying the artificial hair follicle according to claim 5 or the composition according to any one of claims 7 to 9 to a site to be transplanted.

11. Use of the artificial hair follicle according to claim 5 or the composition according to any one of claims 7 to 9 in preparing products for reconstructing skin or hair follicles, promoting hair growth, and treating skin diseases.

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