Method for transdifferentiation of fibroblasts into hair papilla-like cells and uses thereof

Abstract

The application provides a method for transdifferentiation of fibroblasts into hair papilla-like cells and application thereof, and relates to the technical field of biotechnology. The transdifferentiation method comprises inducing fibroblasts by using Peficitinib, so that the fibroblasts are transdifferentiated into hair papilla-like cells. A large number of hair papilla-like cells with hair induction capacity can be obtained in vitro by the method, and the problem that primary separated hair papilla cells are insufficient for developing alternative therapies for treating alopecia in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and in particular to a method for transdifferentiation of fibroblasts into dermal papilla-like cells and application thereof. BACKGROUND

[0002] Hair loss is a common disease caused by various factors including disease, age, mental stress, etc., and is a sub-health problem that plagues billions of men and women around the world. Although hair loss does not directly endanger life, it can seriously affect the psychological state of individuals and reduce the quality of life. Drug therapy and hair transplantation are the main treatment methods so far. Although hair transplantation is currently the most effective method, the scarcity of donor hair follicles has become a major limitation. In addition, drug therapy has little effect and has serious side effects. Therefore, the limited efficacy and side effects of current treatments have prompted people to seek alternative therapies that can be used in clinical practice.

[0003] In recent years, with the rapid development of tissue engineering and regenerative medicine, hair loss has been provided with a new alternative therapy through cell-based hair follicle regeneration. Dermal papilla (DP) cells are a group of specialized mesenchymal cells located at the bottom of the hair follicle, which play an important role in the embryonic development and cycle of the hair follicle, and are a potential cell source for hair follicle regeneration. However, the DP cells extracted by separation quickly lose their hair-inducing ability in in vitro culture. Primary isolated DP cells are not sufficient for the development of alternative therapies for treating hair loss, and effective and reliable methods are needed to produce a large number of DP cells with hair-inducing ability.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The first object of the present application is to provide a method for transdifferentiation of fibroblasts into dermal papilla-like cells, which alleviates the problem that primary isolated dermal papilla cells are not sufficient for the development of alternative therapies for treating hair loss in the prior art.

[0006] The second object of the present application is to provide a dermal papilla-like cell obtained by the above method.

[0007] The third object of the present application is to provide a culture medium capable of inducing transdifferentiation of fibroblasts into dermal papilla-like cells.

[0008] The fourth object of the present application is to provide the application of the above method for transdifferentiation of fibroblasts into dermal papilla-like cells, the above dermal papilla-like cell or the above induction culture medium.

[0009] To solve the above technical problems, the present application adopts the following technical solutions:

[0010] According to one aspect of the present application, the present application provides a method for transdifferentiating fibroblasts into hair papilla-like cells, comprising inducing fibroblasts using Peficitinib to transdifferentiate fibroblasts into hair papilla-like cells.

[0011] Preferably, the method comprises culturing fibroblasts using an induction medium containing Peficitinib;

[0012] Preferably, the concentration of Peficitinib in the induction medium is 2-10 μM, preferably 10 μM;

[0013] Preferably, the induction medium is a basic medium containing Peficitinib, and the basic medium is high-glucose DMEM containing serum.

[0014] Preferably, the fibroblasts are cultured using the induction medium for 6-10 days;

[0015] Preferably, the fibroblasts are cultured using the induction medium for 8 days;

[0016] Preferably, the induction medium is refreshed every two days;

[0017] Preferably, the induction medium is replaced 24 hours after the fibroblasts are inoculated.

[0018] Preferably, the fibroblasts are derived from humans;

[0019] Preferably, the fibroblasts are derived from skin tissue;

[0020] Preferably, the fibroblasts are derived from scalp tissue or prepuce tissue.

[0021] Preferably, the fibroblasts are primary fibroblasts;

[0022] Preferably, the method for isolating the primary fibroblasts comprises: separating the true epidermis from the cleaned skin tissue after digestion with dispersinase, and separating the fibroblasts from the true epidermis after digestion with collagenase;

[0023] Preferably, the hair papilla-like cells transdifferentiated from the fibroblasts are further cultured in a 3D culture mode;

[0024] Preferably, the 3D culture is performed for 24-48 hours.

[0025] According to another aspect of the present application, the present application also provides a hair papilla-like cell obtained by the above method, wherein the hair papilla-like cell has hair papilla cell-specific molecular markers; the hair papilla cell-specific molecular markers comprise α-SMA and VCAN.

[0026] Preferably, the molecular marker specific to the dermal papilla cell further comprises NOG.

[0027] According to another aspect of the present application, the present application further provides an induction medium for inducing fibroblasts to transdifferentiate into dermal papilla-like cells, wherein the induction medium contains Peficitinib at a working concentration of 2-10 μM.

[0028] Preferably, the Peficitinib is contained at a working concentration of 10 μM.

[0029] Preferably, the induction medium is a basic medium containing Peficitinib, and the basic medium is high-glucose DMEM containing serum.

[0030] According to another aspect of the present application, the present application further provides the use of the above-mentioned method for transdifferentiating fibroblasts into dermal papilla-like cells, the above-mentioned dermal papilla-like cells, or the above-mentioned induction medium in hair follicle regeneration for non-diagnostic and therapeutic purposes.

[0031] According to another aspect of the present application, the present application further provides the use of the above-mentioned method for transdifferentiating fibroblasts into dermal papilla-like cells, the above-mentioned dermal papilla-like cells, or the above-mentioned induction medium in the preparation of a product for hair follicle regeneration.

[0032] According to another aspect of the present application, the present application further provides a product for hair follicle regeneration, which comprises the above-mentioned dermal papilla-like cells or the above-mentioned induction medium.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] The present application provides a method for transdifferentiating fibroblasts into dermal papilla-like cells, which comprises inducing fibroblasts using Peficitinib to make the fibroblasts transdifferentiate into dermal papilla-like cells.

[0035] The present application provides a method, and the initial cell for transdifferentiation is fibroblasts, which have high availability and can be easily isolated from skin tissue, and in addition, the culture, propagation and cryopreservation characteristics of fibroblasts also have advantages in culture in terms of nutritional requirements and culture activity.

[0036] The efficacy component for inducing fibroblast transdifferentiation into hair papilla-like cells adopts Peficitinib, which is a new type of oral small molecule Janus kinase inhibitor. It is found in the present application that Peficitinib can induce fibroblast transdifferentiation into hair papilla-like cells, and realize the direct acquisition of hair papilla-like cells with the ability of inducing hair follicle regeneration and hair growth from in vitro. Peficitinib, as a small molecule, has the advantages of clear chemical composition, high purity, small batch difference, rapid action and dose-dependent biological activity. The method for inducing fibroblast fate transdifferentiation based on small molecules can obtain a large number of hair papilla-like cells with the ability of inducing hair follicle regeneration and hair growth in vitro, and has important application value for replacing the hair follicle regeneration method based on hair papilla cells. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0038] Figure 1A The morphology of the primary human foreskin fibroblasts extracted in Example 1;

[0039] Figure 1B The fluorescence staining identification result diagram of the primary human foreskin fibroblasts extracted in Example 1;

[0040] Figure 2A The morphological changes of human foreskin fibroblasts induced by the small molecule drug Peficitinib in Example 2 and the ALP staining results;

[0041] Figure 2B The results of qRT-PCR detection of DP marker gene ALPL expression in Example 2;

[0042] Figure 2C The results of qRT-PCR detection of DP marker gene VCAN expression in Example 2;

[0043] Figure 2D The results of qRT-PCR detection of DP marker gene alpha-SMA expression in Example 2;

[0044] Figure 2E The results of cell immunofluorescence staining after Peficitinib induction in Example 2;

[0045] Figure 3AMorphology of primary human dermal papilla DP cells in Example 3;

[0046] Figure 3B ALP staining results of primary human dermal papilla DP cells in Example 3;

[0047] Figure 3C Identification results of immunofluorescence staining of primary human dermal papilla DP cells in Example 3;

[0048] Figure 4A qRT-PCR comparison of DP marker gene a-SMA expression in primary DP (P2) and Peficitinib-induced human foreskin fibroblasts in Example 3;

[0049] Figure 4B qRT-PCR comparison of DP marker gene NOG expression in primary DP (P2) and Peficitinib-induced human foreskin fibroblasts in Example 3;

[0050] Figure 4C qRT-PCR comparison of DP marker gene VCAN expression in primary DP (P2) and Peficitinib-induced human foreskin fibroblasts in Example 3;

[0051] Figure 4D qRT-PCR comparison of DP marker gene SOX2 expression in primary DP (P2) and Peficitinib-induced human foreskin fibroblasts in Example 3;

[0052] Figure 4E qRT-PCR comparison of DP marker gene LEF1 expression in primary DP (P2) and Peficitinib-induced human foreskin fibroblasts in Example 3;

[0053] Figure 5A 3D culture of human foreskin fibroblasts before and after Peficitinib induction for in vivo hair follicle reconstruction detection of hair induction ability of 3D cell spheres after induction in Example 3;

[0054] Figure 5B In vivo hair follicle reconstruction detection of hair induction ability of 3D cell spheres after Peficitinib induction in Example 3;

[0055] Figure 6A Morphology of extracted primary human dermal papilla fibroblasts in Example 5;

[0056] Figure 6B Identification results of immunofluorescence staining of extracted primary human dermal papilla fibroblasts in Example 5;

[0057] Figure 7A To implement the results of Peficitinib-induced morphological changes and ALP staining of human scalp fibroblasts in Example 6;

[0058] Figure 7B To implement the results of qRT-PCR detection of DP marker gene ALPL expression in human scalp fibroblasts induced by different concentrations of Peficitinib in Example 6;

[0059] Figure 7C To implement the results of qRT-PCR detection of DP marker gene VCAN expression in human scalp fibroblasts induced by different concentrations of Peficitinib in Example 6;

[0060] Figure 7D To implement the results of qRT-PCR detection of DP marker gene α-SMA expression in human scalp fibroblasts induced by different concentrations of Peficitinib in Example 6;

[0061] Figure 8 To implement the results of 3D culture of human scalp fibroblasts before and after Peficitinib induction in Example 7. DETAILED DESCRIPTION

[0062] The technical solutions of the present application will be described below in conjunction with the embodiments, obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor belong to the scope of protection of the present application.

[0063] According to one aspect of the present application, the present application provides a method for transdifferentiating fibroblasts into hair papilla-like cells, which comprises using Peficitinib to induce fibroblasts to transdifferentiate fibroblasts into hair papilla-like cells. It should be noted that the method for transdifferentiating fibroblasts into hair papilla-like cells provided by the present application is for non-diagnostic and therapeutic purposes.

[0064] Transdifferentiation refers to the direct reprogramming of a certain specific somatic cell into a target cell without going through the pluripotent stem cell stage. It has the advantages of higher efficiency and better safety. At present, small molecules have been successfully used to induce the transdifferentiation of fibroblasts into functional cardiomyocytes and neural cells. Through cell-based methods, hair-like structures can be generated in a laboratory environment, or cells can be manipulated in their native niches (in vivo lineage reprogramming) to reconstruct hair follicles. However, in the case of cultured human hair cells, there are still challenging problems in the development of inducing non-hair cell sources and determining culture conditions. Fibroblasts are highly available and can be easily isolated from skin tissue, and in addition, the culture, propagation and cryopreservation characteristics of fibroblasts also have advantages in culture in terms of nutritional requirements and culture viability.

[0065] Peficitinib (CAS No.: 944118-01-8) is a new oral small molecule Janus kinase inhibitor developed by Astellas Pharma, which is used for the treatment of rheumatoid arthritis (including prevention of structural joint damage) that is insufficiently responsive to conventional therapy. Peficitinib can inhibit JAK1, JAK2, JAK3 and Tyk2, and inhibit the activation and proliferation of inflammatory cells involved in synovial inflammation and joint destruction in rheumatoid arthritis patients by inhibiting various inflammatory cytokine signaling pathways.

[0066] The present application screens Peficitinib from the FDA-approved drug library and finds that Peficitinib can induce the transdifferentiation of fibroblasts into hair papilla-like cells, realizing the direct acquisition of hair papilla-like cells with the ability to induce hair follicle regeneration and hair growth in vitro. Peficitinib, as a small molecule, has the advantages of clear chemical composition, high purity, small batch-to-batch difference, rapid action and dose-dependent biological activity. In addition, appropriate targeted delivery and controlled release of small molecules Peficitinib can regulate their in vivo effects in space and time. The method of the present application based on the small molecule Peficitinib-induced transdifferentiation of fibroblasts can obtain a large number of hair papilla-like cells with the ability to induce hair follicle regeneration and hair growth in vitro, which has important application value for replacing hair papilla cell-based hair follicle regeneration methods.

[0067] The cells obtained after inducing the fibroblasts have the ability to induce hair follicle regeneration and hair growth, and can express the specific marker genes a-SMA and VCAN of hair papilla cells, and also express the specific marker gene NOG of hair papilla cells in the cells transdifferentiated from human foreskin fibroblasts, but the expression levels of SOX2 and LEF1 of the cells transdifferentiated from human foreskin fibroblasts are much lower than those of primary human hair papilla cells. Therefore, although the cells transdifferentiated from fibroblasts induced by the present application have the ability to induce hair follicle regeneration and hair growth, and also express part of the specific marker genes of hair papilla cells, they are not completely consistent with primary hair papilla cells. Therefore, the cells transdifferentiated from fibroblasts induced by Peficitinib are named as hair papilla-like cells.

[0068] Cells cultured in research are a simple and convenient tool to solve complex biological problems. Cell lines of different origins are usually used for research and drug development, and are important models for studying human health and diseases, but they may not necessarily effectively reflect in vivo conditions. Therefore, the starting cells for transdifferentiation are preferably primary fibroblasts, which are directly isolated from human samples, retain the morphological and functional characteristics of their original tissues, and have not been subjected to any genetic manipulation, which helps to solve this limitation. Fibroblasts can be isolated from a variety of tissues, and the present application does not limit the tissue from which the fibroblasts are derived, but compared with other sources, human skin tissue is easier to obtain, less invasive, and less controversial in ethics.

[0069] Fibroblasts isolated from skin tissue show extensive application potential and provide rich materials for different researches. The skin tissue can be, but is not limited to, scalp tissue or foreskin tissue. Circumcision is one of the most performed surgical procedures worldwide, and the removed foreskin is usually mostly discarded as biological waste. The foreskin contains a variety of cells, but fibroblasts are the most abundant cells in its dermis. Therefore, the foreskin is an important source for isolating human primary fibroblasts.

[0070] The method for isolating primary fibroblasts preferably comprises: separating the true epidermis after digesting the washed skin tissue using dispersion enzyme, and separating the fibroblasts after digesting the true epidermis using collagenase. The skin tissue is preferably washed using a buffer containing antibiotics; the isolation of fibroblasts preferably comprises filtering the collected cells using a 70 μm cell strainer.

[0071] The dispersion enzyme is preferably 0.2-0.5% w / v Dispase II, and the digestion condition of the dispersion enzyme is preferably 2-6°C for 12-24h; the working concentration of the dispersion enzyme may be, for example, but is not limited to, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5% w / v; the digestion temperature may be, for example, but is not limited to, 2, 3, 4, 5 or 6°C, and the digestion time may be, for example, but is not limited to, 16, 17 or 18h. Preferably, the digestion condition of the dispersion enzyme is 0.25% w / v Dispase II at 4°C in the refrigerator for 16-18h of digestion.

[0072] The collagenase is preferably 0.2-0.5% w / v collagenase type I, and the digestion condition is preferably 35-38°C for 2-4h. The working concentration of the collagenase may be, for example, but is not limited to, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5% w / v; the digestion temperature may be, for example, but is not limited to, 35, 36, 37 or 38°C; and the digestion time may be, for example, but is not limited to, 2, 3 or 4h. Preferably, the digestion condition of the collagenase is 0.25% w / v collagenase type I at 37°C for 2-4h of digestion.

[0073] The culture medium for culturing the fibroblasts may be, for example, but is not limited to, a conventional cell culture medium acceptable in the art, and the present application does not limit the culture medium, and in some alternative embodiments, high-glucose DMEM containing serum is used, and a specific example is high-glucose DMEM containing 10% fetal bovine serum by volume. When the fibroblasts are derived from human waste skin tissue and are primary cells, an antibiotic acceptable in the art, such as streptomycin and / or penicillin, may be added in the early stage of culture.

[0074] In the step of inducing the fibroblasts to transdifferentiate into the hair papilla-like cells using Peficitinib, the fibroblasts are preferably cultured in an induction medium containing Peficitinib to induce the fibroblasts to transdifferentiate into the hair papilla-like cells. The concentration of Peficitinib in the induction medium is preferably 2-10μM, and may be, for example, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9 or 10μM, and is preferably 10μM. The concentration of Peficitinib is obtained through experiments, and a lower concentration will result in a poor transdifferentiation effect, and a higher concentration will cause serious cytotoxicity, and it is found through experiments that the concentration of 10μM is optimal for inducing the transdifferentiation.

[0075] In addition to Peficitinib, the induction medium may also contain a conventional base medium for culturing cells acceptable in the art, and the present application does not limit the base medium, and in some alternative embodiments, high-glucose DMEM containing serum is used, and a specific example is high-glucose DMEM containing 10% fetal bovine serum by volume. The preferred mode of induction culture is as follows:

[0076] (a) fibroblasts are seeded on cell culture dishes, cultured with basal medium, and placed in a 37°C, 5% CO2 incubator;

[0077] (b) 24h later, the basal medium is discarded and replaced with induction medium, and the induction culture is continued for 6-10 days, preferably 8 days, wherein the induction medium is replaced with fresh induction medium every two days, to obtain papilla-like cells transdifferentiated from fibroblasts.

[0078] In some preferred embodiments, the preparation method further comprises using alkaline phosphatase staining and detecting the expression of papilla-specific marker genes to determine whether the fibroblasts have successfully transdifferentiated into papilla-like cells, and the transdifferentiated papilla-like cells are further subjected to 3D culture.

[0079] Alkaline phosphatase staining is because the hair follicle is highly regenerative and known to express ALP. Previous reports have shown that the papilla has high levels of ALP activity throughout the hair follicle cycle, and the characteristic of high expression of ALP in papilla cells has been used as a simple and reliable method to distinguish papilla from other hair follicle structures throughout the hair follicle cycle. McElwee et al. found that low-generation papilla cells with high ALP activity could induce hair follicle formation when transplanted into the wound of the recipient's ear skin, while high-generation papilla cells lost ALP activity and could not produce hair follicles. Therefore, in the hair follicle, ALP activity is not only a specific marker for papilla cells, but also a key marker reflecting its hair induction ability. Detecting the expression of papilla-specific marker genes can identify the basic characteristics of the cells obtained after transdifferentiation, and total RNA extraction can be used to detect the expression of papilla-specific marker genes by qRT-PCR.

[0080] One of the main purposes of culturing DP cells in vitro is to study their role in hair growth and regeneration. However, 2D cultured DP cells gradually lose their hair induction ability during in vitro subculture. The formation of 3D spheroids can significantly enhance the hair induction ability of in vitro cultured DP cells. Studies have shown that 3D cultured spheroids can partially restore the gene expression characteristics of DP cells in vivo, and thus restore their related hair induction properties. Therefore, the cells induced by Peficitinib are further subjected to 3D culture, and their hair induction ability is verified by in vivo hair follicle reconstruction. The culture time of 3D culture is preferably 24-48h, for example, but not limited to, 24, 30, 36, 42 or 48h.

[0081] According to another aspect of the present application, the present application also provides a dermal papilla-like cell obtained by the above method, the dermal papilla-like cell prepared by the above method, having the ability to induce hair growth, and expressing the specific marker genes of dermal papilla cells, alpha-SMA and VCAN. The specific marker gene of dermal papilla cells, NOG, is also expressed in the dermal papilla-like cell transdifferentiated from the human foreskin fibroblast.

[0082] According to another aspect of the present application, the present application also provides an induction medium for inducing fibroblasts to differentiate into dermal papilla-like cells, wherein the induction medium contains Peficitinib at a working concentration of 2-10 μM, preferably 10 μM. In addition to Peficitinib, the induction medium also contains conventional substances acceptable in the art for culturing cells, such as one or more of sugars, amino acids, buffering substances, antibiotics, growth factors, vitamins, inorganic ions, and serum, for supplying nutrients to cells and promoting the reproductive proliferation of cells, and for maintaining the growth environment of cells; specific examples can be, but are not limited to, sugars, amino acids, buffering substances, antibiotics, growth factors, vitamins, inorganic ions, and serum. The induction medium provided by the present application can induce fibroblasts to transdifferentiate into dermal papilla-like cells having the ability to induce hair follicle regeneration and hair growth.

[0083] Based on the beneficial effects of the method for transdifferentiating fibroblasts into dermal papilla-like cells provided by the present application, and based on the dermal papilla-like cells and the induction medium obtained from the inventive concept, the present application also provides the above-mentioned method for transdifferentiating fibroblasts into dermal papilla-like cells, the above-mentioned dermal papilla-like cells, or the above-mentioned induction medium for use in hair follicle regeneration for non-diagnostic and therapeutic purposes, or for use in the preparation of products for hair follicle regeneration. The dermal papilla-like cells obtained by transdifferentiation in vitro can obtain a large number of cells for hair follicle regeneration, providing a large amount of raw material for the study of hair follicle regeneration; and reducing the cost of preparing products for hair follicle regeneration.

[0084] According to another aspect of the present application, the present application also provides a product for hair follicle regeneration, wherein the product contains the above-mentioned dermal papilla-like cells, or the above-mentioned induction medium.

[0085] The technical solutions and technical effects of the present application will be further described below in conjunction with preferred embodiments.

[0086] In the following examples, the base medium used is high-sugar DMEM containing 10% fetal bovine serum; the induction medium is the base medium with the addition of Peficitinib at the target concentration.

[0087] Example 1

[0088] Extraction and identification of human foreskin fibroblasts:

[0089] Fresh human foreskin tissues were obtained from patients undergoing routine circumcision surgery. The excised foreskin tissues were placed in a centrifuge tube containing normal saline or PBS and brought back to the laboratory on ice and immediately processed. The foreskin tissues were washed repeatedly 3-5 times with sterile PBS containing 2% D-PBS and excess fat tissue was removed under a dissecting microscope. The foreskin tissues were cut into small pieces using sterile scissors and digested with 0.25% Dispase II overnight at 4°C for 16-18h. The epidermis was separated and the dermis was cut into small pieces using sterile scissors and digested with 0.25% collagenase type I at 37°C for 2-4h. The cell-containing digest was filtered through a 70μm cell strainer, centrifuged and washed 2-3 times with PBS. The cells were resuspended with complete medium (DMEM + 10% FBS + 1% D-PBS) and transferred to culture dishes and incubated at 37°C in a 5% CO2 incubator. The medium was changed every 3 days until the cells were confluent and passaged. The isolated primary human foreskin fibroblasts were seeded onto cell culture slides and identified using immunofluorescence staining.

[0090] The results, as shown in Figure 1A , showed that the isolated cells had the typical morphology of fibroblasts: elongated, spindle-shaped, with distinct cell boundaries. The cells continued to grow until they were confluent, with the cells in the confluent area growing in close proximity to each other in parallel rows. Fibroblasts synthesize extracellular matrix and collagen, with high expression of collagen I and collagen III in skin fibroblasts. In addition, fibroblast-specific protein-1 (FSP-1), also known as S100A4, is a marker for fibroblasts and is a member of the intracellular protein S100 superfamily. Immunofluorescence staining Figure 1B showed that almost all the cells expressed S100A4, collagen I and collagen III at high levels, further confirming that the cells isolated and cultured from the foreskin were fibroblasts.

[0091] Example 2

[0092] To explore the potential role of small molecules in inducing fibroblasts to change the fate of hair papilla cells, small molecules in the FDA-approved drug library were systematically screened and analyzed. The primary human foreskin fibroblasts prepared in Example 1 were inoculated in 96-well plates, and after 24 h of adhesion, the medium containing 10 μM concentration of the small molecules to be tested was replaced, and the medium was replaced every four days. ALP staining was performed on the 8th day. The DMSO-treated control human foreskin fibroblasts showed no blue-purple color after staining, while small molecules that could show blue-purple color were screened out. To further confirm the small molecules that can induce human fibroblasts to transdifferentiate into hair papilla cells, qRT-PCR method was used to detect the expression of multiple hair papilla cell marker genes. The ALP staining positive small molecules obtained by primary screening were rescreened, and the small molecule drug Peficitinib was finally screened out, and its concentration was further screened.

[0093] The results are shown in Figure 2A , Peficitinib induced changes in cell morphology and showed strong alkaline phosphatase activity; as Figures 2B-2D shown, ALPL, VCAN and a-SMA, and other hair papilla cell-specific marker genes were up-regulated; and at a concentration of 10 μM, the expression of hair papilla marker genes ALPL, VCAN and a-SMA was the highest, which was the optimal induction concentration; as Figure 2E immunofluorescence staining results showed that the expression of hair papilla marker genes (a-SMA) in vitro culture was induced by Peficitinib.

[0094] Example 3

[0095] To verify the efficiency of Peficitinib-induced transdifferentiation of human foreskin fibroblasts into DP cells, primary human scalp hair follicle DP cells were further isolated and extracted as a positive control. DP was isolated and extracted by enzyme digestion combined with microdissection. The scalp hair follicles at the back of the head of patients undergoing hair transplantation were used for DP cell extraction. The hair follicle specimens were placed in a centrifuge tube containing sterile normal saline and taken to the laboratory on ice for immediate processing. The skin tissue and adipose tissue around the hair follicle were carefully removed under a body dissecting microscope. The lower segment of the hair follicle containing the DP was cut and transferred to a 15 mL centrifuge tube containing 0.25% collagenase type I and digested at 37°C for 2-3 h. After digestion was complete, the completely detached DP was collected under a stereoscopic dissecting microscope. If the DP was not completely detached, a 1 mL syringe needle was used to gently peel off the DP to completely separate it. The separated DP was washed twice with PBS. 2 mL of DP commercial culture medium was added to a 6-well plate, and 2-3 DPs were placed in each well for culture. After 3 days of standing, the DP adhered to the wall, and cells migrated out of the DP. After the DP cells migrated out and grew, one well was taken for ALP staining, and the rest were subcultured for immunofluorescence staining (P1) and RNA extraction (P2).

[0096] As shown in Figure 3A , the single hair follicle end before treatment was accompanied by an intact flame-shaped DP structure. After 3 days of culture, the DP adhered to the wall, and polygonal or short spindle-shaped cells began to migrate around it; on the 6th day of culture, the cells grew radially around the DP; on the 18th day of culture, the DP cells fused and grew in a multilayered and aggregated manner. To further identify the isolated DP cells, the extracted DP cells were first subjected to ALP staining. As shown in Figure 3B , the DP cells in the center of the cell explant growth (P0) showed strong ALP activity. The results of immunofluorescence staining are shown in Figure 3C , the isolated primary DP cells (P1) (cells after the primary DP cells were subcultured once) expressed specific markers (a-SMA, VIM, VCAN, and SOX2).

[0097] The expression of specific marker genes in human foreskin fibroblasts and P2 generation DP cells induced by Peficitinib was detected by qRT-PCR to further evaluate the efficiency of Peficitinib-induced transdifferentiation of human foreskin fibroblasts into DP cells. As shown in Figures 4A-4EThe expression of a-SMA, NOG, VCAN, SOX2 and LEF1 in P2 passage of DP cells were all significantly higher than that in primary human foreskin fibroblasts. While in Peficitinib-induced human foreskin fibroblasts, the expression of a-SMA and NOG were significantly higher than that in primary DP cells; the expression of VCAN was increased to a level comparable to that in primary DP cells, with no significant difference from primary DP cells; while the expression levels of SOX2 and LEF1 were still much lower than that in primary DP cells. These results showed that Peficitinib-induced human foreskin fibroblasts were not completely consistent with primary DP cells, therefore, the cells obtained after Peficitinib-induced human foreskin fibroblasts can be referred to as DP-like cells.

[0098] Example 4

[0099] Peficitinib-induced human foreskin fibroblasts were collected and seeded in round-bottom ultra-low attachment 96-well plates at a cell density of 10000 cells / well for 3D culture, and DMSO-treated human foreskin fibroblasts were also subjected to 3D culture as a control. In order to better make the cells form a complete sphere, the cells after seeding were subjected to a short low-speed centrifugation in the 96-well plate. The results are shown in Figure 5A As shown, the cells after centrifugation were aggregated in the center of the well to form a huge circle, and with the culture time, the cells were aggregated into a three-dimensional spherical shape.

[0100] Human foreskin fibroblasts, induced by Peficitinib and cultured in 3D, were used for in vivo hair follicle reconstruction experiments using a patch assay. Cell spheres from the 3D culture were collected and mixed with neonatal mouse keratinocytes. The cell mixture was then subcutaneously injected into the back of nude mice. The mixture of neonatal mouse fibroblasts and neonatal mouse keratinocytes served as a positive control, while neonatal mouse keratinocytes alone served as a negative control. Visible bulges appeared at the cell injection sites. Because the hair follicles formed by this method are located on the inner side of the skin, it is difficult to observe the location of hair growth. To track the location of transplanted cells, cells treated with DMSO and induced by Peficitinib were labeled with the fluorescent dye DiI before 3D culture. DiI, also known as DiIC18(3), is 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, one of the most commonly used fluorescent probes for cell membranes, exhibiting orange-red fluorescence. As a lipophilic membrane dye, DiI can diffuse laterally after entering the cell membrane, gradually staining the entire cell membrane. Figure 5A As shown, the DiI-labeled cells, after 3D culture, appear as a bright red sphere under a fluorescence microscope.

[0101] Four weeks after cell transplantation, hair growth was observed at the transplantation site under a dissecting microscope and a stereomicroscope. The results were as follows: Figure 5B As shown (where the negative group consisted of isolated neonatal mouse keratinocytes; the positive group consisted of a mixture of neonatal mouse fibroblasts and neonatal mouse keratinocytes; the control group consisted of a mixture of DMSO-treated human foreskin fibroblasts and neonatal mouse keratinocytes; and the peficitinib group consisted of a mixture of peficitinib-induced human foreskin fibroblasts and neonatal mouse keratinocytes), consistent with the expected results, the positive control group transplanted with the neonatal mouse fibroblast mixture grew abundant hair, while the negative control group transplanted with isolated keratinocytes showed no hair growth. DMSO-treated human foreskin fibroblasts showed no hair growth after 3D culture, while peficitinib-induced human foreskin fibroblasts, combined with 3D culture, were able to induce the formation of complete hair structures.

[0102] Example 5

[0103] Extraction and identification of human dermal fibroblasts. Fresh human scalp specimens were obtained from discarded tissue during hair transplantation surgery and collected in a centrifuge tube with normal saline and brought back to the laboratory in an ice box and processed immediately. The scalp tissue was washed with sterile PBS containing 2% D-PBS for 3-5 times. Since the discarded tissue was small, it was directly placed in a 15 mL centrifuge tube containing 0.25% Dispase II and digested overnight at 4°C for 16-18 h. The epidermis layer of the scalp was peeled off from the dermis layer under a dissecting microscope and the dermis part was placed in a 15 mL centrifuge tube containing 0.25% collagenase type I and incubated at 37°C for 2-4 h. The digestion was observed every 30 min and the tube was shaken. When the digestion solution became cloudy and the dermis tissue was almost digested, the digestion was stopped by adding complete medium (DMEM + 10% FBS + 1% D-PBS) containing serum. The digestion solution containing cells was filtered through a 70 μm cell strainer and transferred to a 15 mL centrifuge tube. The supernatant was discarded after centrifugation at 1000 rpm for 5 min and the cells were resuspended in PBS and centrifuged twice. The cells were resuspended in complete medium (DMEM + 10% FBS + 1% D-PBS) and transferred to a culture dish and incubated at 37°C in a 5% CO2 incubator. The medium was changed every 3 days until the cells were confluent and passaged. The primary human dermal fibroblasts isolated were seeded onto cell slides and identified by immunofluorescence staining.

[0104] The results are shown in Figure 6. The isolated cells were from the papillary dermis layer next to the subcutaneous epidermis and exhibited typical fibroblast morphology. The immunofluorescence staining results showed high levels of S100A4, Collagen I and Collagen III expression. The isolated cells from the scalp tissue were identified as fibroblasts from morphology and immunofluorescence staining.

[0105] Example 6

[0106] To further evaluate the efficiency of small molecule drugs in inducing human scalp fibroblasts, the expression of DP marker genes in human scalp fibroblasts and primary DP cells after Peficitinib induction was detected by qRT-PCR, with primary DP cells (P2) (cells after 2 passages of primary DP cells) as a control. The results are shown in Figure 7. After Peficitinib induction, the cell morphology changed, ALP activity increased, and the expression of DP cell marker genes was significantly enhanced, with a higher expression level compared to primary DP cells. Further optimization of the concentration of Peficitinib in inducing human scalp fibroblasts showed that the expression of ALP, VCAN, and a-SMA in human scalp fibroblasts induced by Peficitinib at a concentration of 10 μM was significantly higher than that in primary DP cells. Consistent with foreskin fibroblasts, a concentration of 10 μM was also the optimal concentration for Peficitinib to induce human scalp fibroblasts.

[0107] Example 7

[0108] To further explore the hair-inducing ability of Peficitinib-induced human scalp fibroblasts combined with 3D culture, human scalp fibroblasts treated with DMSO and Peficitinib were collected, respectively, and labeled with Dil cell membrane probe at a cell density of 10000 cells / well. The cells were seeded in round-bottom ultra-low attachment 96-well plates for 3D culture. Within 48 hours, cell spheres were collected and injected intradermally into the back of nude mice mixed with neonatal mouse keratinocytes. The mixture of neonatal mouse fibroblasts and neonatal mouse keratinocytes was used as a positive control, and keratinocytes alone were used as a negative control. After 4 weeks, the skin was taken for observation of hair growth. The results of in vivo hair follicle reconstruction showed that the positive control grew a large number of hair, while the negative control of keratinocytes alone had no hair growth, ensuring the reliability of the experimental results. As shown in Figure 8, human scalp fibroblasts treated with DMSO after 3D culture induced black cluster-like follicle structures and pigmentation in nude mice, but no complete hair structure was produced, which may be due to the fact that the isolated scalp fibroblasts are papillary dermal fibroblasts with common fibroblast progenitor cell origin with hair papilla cells. The human scalp fibroblasts induced by Peficitinib after 3D culture produced hair with complete structure; indicating that Peficitinib successfully induced human scalp fibroblasts to transdifferentiate into DP-like cells with hair-inducing ability. Figure 8

[0109] ​It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for transdifferentiation of fibroblasts into hair papilla-like cells, characterized in that, The method comprises inducing fibroblasts using Peficitinib to transdifferentiate fibroblasts into hair papilla-like cells. The method comprises culturing fibroblasts using an induction medium containing Peficitinib at a concentration of 10 μM.

2. The method of claim 1, wherein, The induction medium is a basic medium containing Peficitinib, and the basic medium is high-glucose DMEM containing serum.

3. The method of claim 1, wherein, The fibroblasts are cultured using the induction medium for 6-10 days.

4. The method of claim 3, wherein, The fibroblasts are cultured using the induction medium for 8 days.

5. The method of claim 3, wherein, The induction medium is replaced every two days.

6. The method of claim 1, wherein, The induction medium is replaced 24 hours after the fibroblasts are inoculated.

7. The method of claim 1, wherein, The fibroblasts are derived from human.

8. The method of claim 1, wherein, The fibroblasts are derived from skin tissue.

9. The method of claim 8, wherein, The fibroblasts are derived from scalp tissue or prepuce tissue.

10. The method of claim 1, wherein, The fibroblasts are primary fibroblasts.

11. The method of claim 10, wherein, The culture medium of the primary fibroblasts is the basic medium described in claim 2.

12. The method of claim 11, wherein, The culture medium of the primary fibroblasts further contains antibiotics.

13. The method of claim 12, wherein, The antibiotics include streptomycin and / or penicillin.

14. The method of claim 10, wherein, The method for isolating the primary fibroblasts comprises: separating the true epidermis from the washed skin tissue after digestion with dispase, and separating the fibroblasts from the true epidermis after digestion with collagenase.

15. The method of claim 14, wherein, The digestion is performed at 2-6°C for 16-18 hours using 0.2-0.5% w / v dispase II.

16. The method of claim 14, wherein, The digestion is performed at 35-38°C for 2-4 hours using 0.2-0.5% w / v collagenase type I.

17. The method of claim 14, wherein, The isolation of the fibroblasts comprises filtering and collecting the cells using a 70 μm cell strainer.

18. The method of claim 14, wherein, The skin tissue is washed using a buffer containing antibiotics.

19. The method according to any one of claims 1 to 18, characterized in that, The fibroblasts transdifferentiated into hair papilla-like cells are further cultured in a 3D culture.

20. The method of claim 19, wherein, The 3D culture is performed for 24-48 hours.

21. The method of claim 19, wherein, The method further comprises: determining whether the fibroblasts are successfully transdifferentiated into hair papilla-like cells by alkaline phosphatase staining and detecting the expression of hair papilla-specific marker genes, and further culturing the successfully transdifferentiated hair papilla-like cells in a 3D culture.

Images