CN115651890A

CN115651890A - Method for transdifferentiation of fibroblasts into papilla-like cells and application of method

Info

Publication number: CN115651890A
Application number: CN202211164118.9A
Authority: CN
Inventors: 李小龙; 张辉; 王永
Original assignee: Beijing Yonghe Medical Investment Management Co ltd
Current assignee: Beijing Yonghe Medical Investment Management Co ltd
Priority date: 2022-09-23
Filing date: 2022-09-23
Publication date: 2023-01-31

Abstract

The invention provides a method for transdifferentiation of fibroblasts into papilla-like cells and application thereof, and relates to the technical field of biotechnology. The transdifferentiation method comprises inducing fibroblasts by using Pefitinib to transdifferentiate the fibroblasts into hair papilla-like cells. The method can obtain a large amount of hair papilla-like cells with hair induction capability in vitro, and alleviates the problem that primary isolated hair papilla cells in the prior art are insufficient for developing an alternative therapy for treating alopecia.

Description

Method for transdifferentiation of fibroblasts into papilla-like cells and application of method

Technical Field

The invention relates to the technical field of biotechnology, in particular to a method for transdifferentiation of fibroblasts into papilla-like cells and application thereof.

Background

Alopecia is a common disease, caused by various factors including diseases, age, mental stress, etc., and is a sub-health problem which afflicts billions of men and women worldwide. Although hair loss does not cause immediate life risks, it can seriously affect the psychological state of the individual and reduce the quality of life. Drug therapy and hair transplantation are the major therapeutic approaches to date. Although hair transplantation is currently the most effective method, the scarcity of donor follicles has been a major limitation. In addition, drug therapy is of little consequence and has serious side effects. Thus, the limited efficacy and side effects of current treatments have prompted the search for alternative therapies that can be used in clinical practice.

In recent years, with the rapid development of tissue engineering and regenerative medicine, new alternative therapies for alopecia are provided through cell-based hair follicle regeneration. Hair papilla (DP) cells are a specialized mesenchymal cell population located at the base of the hair follicle, play an important role in embryonic development and the periodic cycle of the hair follicle, and are a potential source of cells for hair follicle regeneration. However, isolated and extracted DP cells quickly lost their hair-inducing ability in vitro culture. Primary isolated DP cells alone are not sufficient for the development of alternative therapies for treating alopecia and efficient and reliable methods are sought to produce large numbers of DP cells with hair-inducing ability.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first object of the present invention is to provide a method for transdifferentiation of fibroblasts into hair papilla-like cells, which alleviates the problem of insufficient primary isolated hair papilla cells for development of an alternative therapy for treating alopecia, which exists in the prior art.

The second object of the present invention is to provide hair papilla-like cells obtained by the above method.

The third object of the present invention is to provide a medium capable of inducing the transdifferentiation of fibroblasts into papilla-like cells.

The fourth object of the present invention is to provide the method for transdifferentiation of the fibroblast into a hair papilla-like cell, the use of the hair papilla-like cell or the induction medium.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the present invention, there is provided a method for transdifferentiating fibroblasts into papilla-like cells, comprising inducing fibroblasts using Pefitinib to transdifferentiate the fibroblasts into papilla-like cells.

Preferably, the method comprises culturing fibroblasts using an induction medium containing peicitinib;

preferably, the concentration of Pefitinib in the induction medium is 2-10. Mu.M, preferably 10. Mu.M;

preferably, the induction medium is a basal medium containing Pefitinib, and the basal medium is high-glucose DMEM containing serum.

Preferably, the fibroblast cells are cultured using the induction medium for 6 to 10 days;

preferably, the fibroblasts are cultured using the induction medium for 8 days;

preferably, the induction medium is refreshed every two days;

preferably, the induction medium is replaced 24h after fibroblast seeding.

Preferably, the fibroblasts are of human origin;

preferably, the fibroblasts are derived from skin tissue;

preferably, the fibroblasts are derived from scalp tissue or foreskin tissue.

Preferably, the fibroblasts are primary fibroblasts;

preferably, the method for isolating primary fibroblasts comprises: digesting the cleaned skin tissue by using dispase, separating true epidermis, digesting the true epidermis by using collagenase, and separating fibroblasts;

preferably, the 3D culture mode is adopted to continuously culture the hair papilla-like cells transformed and differentiated from the fibroblasts;

preferably, 3D culture is carried out for 24-48 h.

According to another aspect of the present invention, there is also provided a hair papilla-like cell obtained by the above method, which has a molecular tag specific to the hair papilla cell; the molecular label specific to the hair papilla cells comprises alpha-SMA and VCAN;

preferably, the molecular tag specific for dermal papilla cells further comprises NOG.

According to another aspect of the present invention, there is also provided an induction medium for inducing the transdifferentiation of fibroblasts into papilla-like cells, the induction medium comprising Pefitinib at a working concentration of 2-10 μ M;

preferably, pefitinib is contained at a working concentration of 10 μ M;

According to another aspect of the present invention, there is also provided a method for transdifferentiation of the above-mentioned fibroblasts into hair papilla-like cells, use of the above-mentioned hair papilla-like cells, or the above-mentioned induction medium for hair follicle regeneration for non-diagnostic and therapeutic purposes.

According to another aspect of the present invention, the present invention also provides a method for transdifferentiation of the above-mentioned fibroblasts into hair papilla-like cells, use of the above-mentioned hair papilla-like cells, or the above-mentioned induction medium for the preparation of a product for hair follicle regeneration.

According to another aspect of the present invention, there is also provided a product for hair follicle regeneration, which comprises the above-mentioned hair papilla-like cells, or the above-mentioned induction medium.

Compared with the prior art, the invention has the following beneficial effects:

the method for transdifferentiating fibroblast cells into hair papilla-like cells, provided by the present invention, comprises inducing fibroblast cells using Pefitinib to transdifferentiate the fibroblast cells into hair papilla-like cells.

The method provided by the invention has the advantages that the transdifferentiated primary cells are fibroblasts, the fibroblasts have high availability and can be easily separated from skin tissues, and in addition, the culture, propagation and cryopreservation characteristics of the fibroblasts also have culture advantages in terms of nutrient requirements and culture viability.

The invention discloses a method for inducing the transdifferentiation of fibroblasts to hair papilla-like cells, which is characterized in that Pecetinib is a novel oral micromolecule Janus kinase inhibitor. The peicitinib serving as a small molecule has the advantages of clear chemical components, high purity and small batch-to-batch difference, and also has the characteristics of quick action and dose-dependent biological activity. The method for inducing the fate transition differentiation of the fibroblast based on the micromolecule can obtain a large number of hair papilla-like cells with the capacity of inducing hair follicle regeneration and hair growth in vitro, and has important application value for replacing a hair follicle regeneration method based on the hair papilla cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1A is a diagram of the morphology of primary human foreskin fibroblasts extracted in example 1;

FIG. 1B is a diagram of the result of fluorescent staining identification of primary human foreskin fibroblasts extracted in example 1;

FIG. 2A shows the morphological changes and ALP staining results of human foreskin fibroblasts induced by the small molecule drug Pefitinib in example 2;

FIG. 2B shows the result of qRT-PCR detection of the expression of the DP marker gene ALPL in example 2;

FIG. 2C shows the results of qRT-PCR detection of the expression of the DP marker gene VCAN in example 2;

FIG. 2D shows the results of qRT-PCR detection of DP marker gene α -SMA expression in example 2;

FIG. 2E shows the result of immunofluorescence staining of cells after Pefitinib induction in example 2;

FIG. 3A is the morphology of primary human scalp hair follicle DP cells in example 3;

FIG. 3B shows the ALP staining results of DP cells of primary human scalp hair follicles in example 3;

FIG. 3C is the result of immunofluorescence staining identification of primary human scalp hair follicle DP cells in example 3;

FIG. 4A is a graph showing the results of qRT-PCR comparison of the expression of DP marker gene α -SMA in human foreskin fibroblasts induced by primary DP (P2) and Pefitinib in example 3;

FIG. 4B is a graph showing the results of comparing the expression of DP marker gene NOG in human foreskin fibroblasts induced by primary DP (P2) and Pefitinib by qRT-PCR in example 3;

FIG. 4C is a graph showing the results of comparing the expression of the DP marker gene VCAN in human foreskin fibroblasts induced by primary DP (P2) and Pefitinib by qRT-PCR in example 3;

FIG. 4D is a graph showing the results of comparing the expression of DP marker gene SOX2 in human foreskin fibroblasts induced by primary DP (P2) and Pefitinib by qRT-PCR in example 3;

FIG. 4E is a graph showing the result of comparing the expression of DP marker gene LEF1 in human foreskin fibroblasts induced by primary DP (P2) and Pefitinib by qRT-PCR in example 3;

FIG. 5A is a graph showing the results of 3D culture of human foreskin fibroblasts before and after Pefitinib induction, in vivo hair follicle reconstruction detection and hair induction ability of induced 3D cell balls in example 3;

FIG. 5B is a graph showing the results of in vivo hair follicle reconstruction testing for the hair-inducing ability of the 3D cell balls after Pefitinib induction;

FIG. 6A is the morphology of primary human scalp fibroblasts extracted in example 5;

FIG. 6B is a graph of immunofluorescence staining identification results of primary human scalp fibroblasts extracted in example 5;

FIG. 7A shows the morphological changes induced by Pefitinib and ALP staining of human scalp fibroblasts in example 6;

FIG. 7B shows the expression of the DP marker gene ALPL after inducing human scalp fibroblasts by Peficitinib with different concentrations in qRT-PCR in example 6;

FIG. 7C shows the DP marker gene VCAN expression after qRT-PCR detection of Pefitinib at different concentrations induces human scalp fibroblasts in example 6;

FIG. 7D shows DP marker gene α -SMA expression after qRT-PCR detection of Pefitinib at different concentrations induced human scalp fibroblasts in example 6;

FIG. 8 is a graph showing the results of in vivo reconstruction of hair follicles after 3D culture of human scalp fibroblasts before and after Pefitinib induction in example 7.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

According to one aspect of the present invention, there is provided a method for transdifferentiating fibroblasts into papilla-like cells, which comprises inducing fibroblasts using Pefitinib to transdifferentiate the fibroblasts into papilla-like cells. The method for transdifferentiation of fibroblasts into papilla-like cells according to the present invention is intended for non-diagnostic and therapeutic purposes.

Transdifferentiation refers to direct reprogramming of a cell of interest from a particular somatic cell, without going through the pluripotent stem cell stage. The method has the advantages of higher efficiency and better safety. Currently, small molecules have been successfully used to induce transdifferentiation of fibroblasts into functional cardiomyocytes and neural cells. Through cell-based methods, hair-like structures can be created in a laboratory setting, or cells can be manipulated in their primary niches (in vivo lineage reprogramming) to reconstitute hair follicles. However, in cases of loss of function of cultured human hair cells, shortage of donor hair, induction of non-hair cell sources and determination of development of culture conditions still present challenging problems. The high availability of fibroblasts, ease of isolation from skin tissue, and the culturing, propagation and cryopreservation characteristics of fibroblasts also provide culture advantages in terms of nutritional requirements and culture viability.

Pefitinib (Pecetitinib, CAS No.: 944118-01-8) is a novel oral small molecule Janus kinase inhibitor developed by Astellas Pharma for the treatment of rheumatoid arthritis (including prevention of structural joint damage) that is not responsive to conventional therapies. Pefitinib can inhibit JAK1, JAK2, JAK3 and Tyk2, and inhibit activation and proliferation of inflammatory cells involved in synovial inflammation and joint destruction in rheumatoid arthritis patients by inhibiting various inflammatory cytokine signaling pathways.

The invention screens and discovers that Pefitinib can induce fibroblast to transdifferentiate into hair papilla-like cells from an FDA approved drug library, and realizes the direct in vitro acquisition of the hair papilla-like cells with the capacity of inducing hair follicle regeneration and hair growth. The peicitinib serving as a small molecule has the advantages of clear chemical components, high purity and small batch-to-batch difference, and also has the characteristics of quick action and dose-dependent biological activity. In addition, the appropriately targeted delivery and controlled release of the small molecule pefitinib may modulate their in vivo effects spatially and temporally. The method for inducing fibroblast fate transition differentiation based on the micromolecule Pefitinib can obtain a large number of hair papilla-like cells with the capacity of inducing hair follicle regeneration and hair growth in vitro, and has important application value for replacing a hair follicle regeneration method based on hair papilla cells.

The cells obtained by the fibroblasts after induction have the capacity of inducing hair follicle regeneration and hair growth, can express specific marker genes alpha-SMA and VCAN of hair papilla cells, and also express specific marker genes NOG of the hair papilla cells in the cells transformed and differentiated from the human foreskin fibroblasts, but the expression level of SOX2 and LEF1 of the cells transformed and differentiated from the human foreskin fibroblasts is far lower than that of primary human hair papilla cells, so that although the cells induced by the application have the capacity of inducing hair follicle regeneration and hair growth, the cells also express the specific marker genes of partial hair papilla cells, but are not completely consistent with the primary hair papilla cells, and therefore, the cells transformed and differentiated from the fibroblasts induced by Peficitin are named as hair papilla-like cells.

While cells cultured in research are a simple and convenient tool to solve complex biological problems, cell lines of different origins, which are commonly used in research and drug development, are important models for the study of human health and disease, they may not necessarily be effective in reflecting what happens in vivo. Primary fibroblasts isolated directly from human specimens, thus, retain the morphological and functional characteristics of the tissue from which they originate, and without any genetic manipulation, help to resolve this limitation. Fibroblasts can be isolated from various tissues, and the invention does not limit the tissue from which the fibroblasts are derived, but human skin tissue is more accessible, less invasive and less ethical in comparison with other sources.

Fibroblasts isolated from skin tissue show a wide range of potential applications, providing abundant materials for different studies. The skin tissue may be, for example, but not limited to, scalp tissue or foreskin tissue. Circumcision is one of the most surgical procedures performed worldwide, and the removed foreskin is usually discarded mostly as biological waste. Foreskin contains many cells, but fibroblasts are the most abundant cells in its dermis. Therefore, foreskin is an important source for isolating human primary fibroblasts.

The isolation method of primary fibroblasts preferably comprises: the washed skin tissue was digested with dispase and then the epidermis was separated, and the epidermis was digested with collagenase and then fibroblasts were separated. Washing the skin tissue preferably with a buffer containing an antibiotic; isolating fibroblasts preferably comprises collecting cells by filtration using a 70 μm cell sieve.

Preferably 0.2 to 0.5% w/v of Dispase II, preferably digestion conditions of Dispase of from 2 to 6 ℃ for from 12 to 24h; the working concentration of the dispase may be, for example, but 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 not limited to, 2, 3, 4, 5, or 6 ℃ and the digestion time may be, for example, but not limited to, 16, 17, or 18 hours. Preferably the dispersive enzymatic digestion conditions are: 0.25% w/v Dispase II was digested in a refrigerator at 4 ℃ overnight for 16-18 h.

Collagenase is preferably 0.2 to 0.5% w/v collagenase type I, and the digestion conditions are preferably 35 to 38 ℃ for 2 to 4 hours. The working collagenase concentration may be, for example, but is not limited to, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5%; digestion temperatures may be, for example, but are not limited to, 35, 36, 37, or 38 ℃; the digestion time may be, for example, but not limited to, 2, 3, or 4 hours. Preferably the collagenase digestion conditions are: 0.25% w/v collagenase type I digested at 37 ℃ for 2-4 h.

The medium used for culturing fibroblasts may be, but is not limited to, a conventional cell culture medium acceptable in the art, and in some alternative embodiments, serum-containing high-glucose DMEM, for example, 10 vol% fetal bovine serum-containing high-glucose DMEM, is used. When fibroblasts are derived from human waste skin tissue, belonging to primary cells, antibiotics acceptable in the art, such as streptomycin and/or penicillin, may be added before the culture.

In the step of inducing the transdifferentiation of fibroblasts into hair papilla-like cells using pefitinib, it is preferable to culture the fibroblasts in an induction medium containing pefitinib to transdifferentiate the fibroblasts into hair papilla-like cells. The concentration of Pefitinib in the induction medium is preferably 2 to 10. Mu.M, and may be, for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Mu.M, preferably 10. Mu.M. The concentration of Peicitinib is obtained by experimental verification, the transdifferentiation effect is poor due to too low concentration, severe cytotoxicity is caused due to too high concentration, and the experiment shows that the induced transdifferentiation effect is optimal when the concentration is 10 mu M.

In addition to peicitinib, the induction medium may also contain conventional basal medium for culturing cells, which is acceptable in the art, but the invention is not limited thereto, and in some alternative embodiments, serum-containing high-glucose DMEM is used, for example, 10% by volume of fetal bovine serum-containing high-glucose DMEM. The preferred mode of induction culture is as follows:

(a) Inoculating fibroblast cells onto a cell culture dish, culturing in a basal medium, incubating the cells at 37 deg.C with 5% CO ₂ Culturing in an incubator;

(b) And after 24h, abandoning the basic culture medium, replacing the induction culture medium, continuously carrying out induction culture for 6-10 days, preferably 8 days, replacing a new induction culture medium every two days, and obtaining the papilla-like cells which are transformed and differentiated from the fibroblasts.

In some preferred embodiments, the preparation method further comprises determining whether the fibroblast cells are successfully transdifferentiated into the hair papilla-like cells by using alkaline phosphatase staining and detecting the expression of the related hair papilla-specific marker gene, and continuing the 3D culture of the hair papilla-like cells successfully transdifferentiated.

The use of alkaline phosphatase staining is due to the fact that hair follicles are highly regenerative and known to express ALP, and previous reports indicate that hair papillae exhibit a high level of ALP activity throughout the hair follicle cycle, and the high expression of ALP in hair papilla cells has been used as a simple and reliable method of distinguishing hair papillae from other hair follicle structures throughout the hair follicle cycle. McElwee et al found that low-generation hair papilla cells highly expressing ALP activity induced hair follicle formation when transplanted into recipient ear skin wounds, while high-generation hair papilla cells losing ALP activity failed to produce hair follicles. It follows that ALP activity is not only a specific hair papilla cell marker but also a key marker reflecting its hair-inducing ability in hair follicles. The basic characteristics of cells obtained after transdifferentiation can be identified by detecting the expression condition of the related papilla specific marker genes, and the qRT-PCR detection can be carried out by extracting total RNA.

One of the main objectives of culturing DP cells in vitro is currently to study their role in hair growth and regeneration. However, 2D cultured DP cells gradually lost their hair-inducing ability during in vitro subculture. And the formation of 3D spheres can obviously enhance the hair induction capability of DP cells cultured in vitro. Studies have shown that spheres formed from 3D cultures partially restore the gene expression characteristics of DP cells in vivo and thus their associated hair-inducing properties. Thus the peicitinib-induced cells were further cultured in 3D and their hair-inducing ability was verified by in vivo hair follicle reconstruction. The culture time for 3D culture is preferably 24 to 48 hours, and may be, for example, but not limited to, 24, 30, 36, 42 or 48 hours.

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

According to another aspect of the present invention, there is also provided an induction medium for inducing differentiation of fibroblasts into hair papilla-like cells, comprising Pefitinib at a working concentration of 2 to 10. Mu.M, preferably 10. Mu.M. The induction medium contains conventional substances acceptable in the art for culturing cells, such as basic substances for supplying nutrients to cells and promoting reproduction of cells, and substances for maintaining the growth environment of cells, in addition to peicitinib; specific examples may be, for example, but are not limited to, one or more of sugars, amino acids, buffer substances, antibiotics, growth factors, vitamins, inorganic ions, and serum. The induction medium provided by the invention can induce the fibroblast to transdifferentiate into hair papilla-like cells with the capacity of inducing hair follicle regeneration and hair growth.

Based on the beneficial effects of the method for transdifferentiating fibroblasts into hair papilla-like cells provided by the invention, the hair papilla-like cells and the induction medium obtained based on the inventive concept, the invention also provides the method for transdifferentiating the fibroblasts into the hair papilla-like cells, and the application of the hair papilla-like cells or the induction medium in hair follicle regeneration for non-diagnostic and therapeutic purposes or in the preparation of products for hair follicle regeneration. The hair papilla-like cells obtained by in vitro transdifferentiation can obtain a large amount of cells for hair follicle regeneration, and a large amount of raw materials are provided for research on hair follicle regeneration; the cost of preparing the product for hair follicle regeneration is reduced.

According to another aspect of the present invention, there is also provided a product for hair follicle regeneration, which comprises the above-described hair papilla-like cells, or the above-described induction medium.

The technical solutions and effects of the present invention will be further described below with reference to preferred embodiments.

In the following examples, the basal medium used was high-glucose DMEM containing 10% fetal bovine serum, unless otherwise specified; the induction culture medium is a basal culture medium added with Peficitinib with target concentration.

Example 1

Extracting and identifying human foreskin fibroblasts:

fresh human foreskin tissue is taken from a patient undergoing conventional circumcision, intraoperatively excised foreskin tissue is placed in a centrifuge tube containing normal saline or PBS, brought back to the laboratory on ice and immediately processed. After the foreskin tissue is repeatedly washed for 3-5 times by sterile PBS containing 2% double antibody, redundant adipose tissues are removed as much as possible under a body type dissecting mirror. Cutting foreskin tissue into small pieces with sterile scissors, adding 0.25% of Dispase II (Dispase II) to digest in a refrigerator at 4 ℃ overnight for 16-18 h, separating true epidermis, cutting dermis part into small pieces with sterile scissors, and digesting with 0.25% of I-type collagenase at 37 ℃ for 2-4 h. The cell-containing digest was filtered through a 70 μm cell sieve, centrifuged, washed 2-3 times with PBS, then the cells were resuspended using complete medium (DMEM +10% FBS +1% double antibody), the cells were transferred to a petri dish, placed at 37 ℃ 5% CO ₂ Culturing in an incubator, and changing the culture medium every 3 days until the cells grow full for passage. And (3) inoculating the primary human foreskin fibroblasts separated and extracted into a cell slide, and identifying the cells by using immunofluorescence staining.

The results are shown in FIG. 1A, and the isolated cells have a typical fibroblast morphology: the cells are slender, spindle-shaped and have obvious cell boundaries; growth was continued until complete confluence, with cells in the confluence region growing close to each other in a parallel arrangement. Fibroblasts synthesize extracellular matrix and Collagen, with type I and type III Collagen (Collagen I and Collagen III) highly expressed in skin fibroblasts. In addition, fibroblast specific protein 1 (FSP-1) is a fibroblast marker, also known as S100A4, a member of the S100 superfamily of intracellular proteins. Immunofluorescent staining (fig. 1B) showed that almost all cells expressed S100A4, collagen I and Collagen III at high levels, further verifying that the cells cultured separately from foreskin were fibroblasts.

Example 2

In order to explore the potential role of small molecules in inducing fibroblast to hair papilla cell fate changes, small molecules in FDA approved drug libraries on the market were systematically screened and analyzed. The primary human foreskin fibroblasts prepared in example 1 were inoculated into a 96-well plate, 24h after adherence, the medium containing the small molecule to be tested at a concentration of 10 μ M was replaced, the medium was changed every four days, and ALP staining was performed on day 8. Human foreskin fibroblasts treated with DMSO as a control did not show bluish violet color after staining, and small molecules that showed bluish violet color were screened out. In order to further confirm small molecules capable of inducing the transdifferentiation of human fibroblasts to hair papilla cells, the qRT-PCR method is used for detecting the expression conditions of a plurality of hair papilla cell marker genes, ALP dyeing positive small molecules obtained by primary screening can be rescreened, and finally the small molecule drug Pefitinib is obtained by screening, and the concentration of the Pefitinib is further screened.

The results are shown in fig. 2A, the morphology of the cells after peicitinib induction is changed, and strong alkaline phosphatase activity is shown; as shown in FIGS. 2B-2D, the expression of specific marker genes for hair papilla cells such as ALPL, VCAN and α -SMA is up-regulated; under the induction of 10 mu M concentration, the expression quantity of the hair papilla marker genes ALPL, VCAN and alpha-SMA is the highest, and the optimal induction concentration is obtained; as shown in FIG. 2E, the immunofluorescent staining result shows that Pefitinib induces the expression of the marker gene (a-SMA) cultured in vitro on papilla pili.

Example 3

In order to verify the efficiency of Pefitinib for inducing the transdifferentiation of human foreskin fibroblasts to DP cells, primary human scalp hair follicle DP cells are further separated and extracted to be used as a positive control. And (3) separating and extracting DP by adopting a method of enzyme digestion combined with microdissection. Taking scalp hair follicles on the occiput of a hair transplant operation patient for DP cell extraction, putting a hair follicle specimen into a centrifuge tube containing sterile normal saline, putting the hair follicle specimen on ice, and taking the hair follicle specimen back to a laboratory for immediate treatment. The skin tissue and adipose tissue surrounding the hair follicle were carefully removed under a physical dissecting scope. The hair bulb part including DP in the lower stage of the hair follicle was cut, transferred to a 15mL centrifuge tube containing 0.25% type I collagenase, and digested at 37 ℃ for 2 to 3 hours. After digestion was complete, the fully detached DP was collected under a stereodissector and if not completely detached, it was completely detached by gently peeling off the DP using a 1mL syringe needle. The separated DP was washed 2 times with PBS. 2mL of DP commercial medium was added to 6-well plates, and 2 to 3 DPs were placed in each well for culture. Standing for 3 days, observing DP adherence condition, and emigrating cells from DP after adherence. After the migrated DP cells were confluent, one well was stained for ALP, and the remainder was subcultured for immunofluorescent staining (P1) and RNA extraction (P2).

As shown in FIG. 3A, individual follicular ends prior to treatment had an intact blazed DP structure. After culturing for 3 days, DP adheres to the wall, and polygonal or short spindle cells begin to migrate out from the periphery; on day 6 of culture, cells grew radially centered on DP; DP cells grew aggregated in multiple layers after fusion at day 18 of culture. To further identify the isolated DP cells. The extracted DP cells were first ALP stained. As shown in fig. 3B, the DP cell center grown from cell explants (P0) showed strong ALP activity. The immunofluorescent staining results are shown in fig. 3C, and the isolated primary DP cells (P1) (cells after 1 passage of primary DP cells) expressed specific markers (a-SMA, VIM, VCAN, and SOX 2).

The expression of specific marker genes in human foreskin fibroblasts and P2 generation DP cells after Pefitinib induction is detected by qRT-PCR, and the transdifferentiation efficiency of the Pefitinib induction human foreskin fibroblasts to DP cells is further evaluated. As shown in FIGS. 4A-4E (wherein, hDP is primary human scalp hair follicle DP cell; con is primary human foreskin fibroblast; pecetitinib is primary human foreskin fibroblast induced by Pecetitinib in example 2), the expressions of a-SMA, NOG, VCAN, SOX2 and LEF1 in P2 generation DP cells are all significantly higher than that of primary human foreskin fibroblast. In human foreskin fibroblasts induced by Pecitinib, the expression of alpha-SMA and NOG is obviously higher than that of primary DP cells; the expression of VCAN is improved to a level equivalent to that of the primary DP cell, and has no significant difference with the primary DP cell; while the expression levels of SOX2 and LEF1 were still much lower than in primary DP cells. These results show that the Pefitinib induced human foreskin fibroblasts are not completely consistent with primary DP cells, so that the cells obtained after the Pefitinib induced human foreskin fibroblasts are called DP-like cells.

Example 4

Human foreskin fibroblasts induced by Pefitinib are collected and inoculated into a round bottom ultra-low attachment 96-well plate according to the cell density of 10000 cells/well for 3D culture, and the DMSO-treated human foreskin fibroblasts are similarly subjected to 3D culture as a control. To better form the cells into an intact sphere, the seeded cells were subjected to brief low speed centrifugation in 96-well plates. As a result, as shown in FIG. 5A, the centrifuged cells were collected in a large circle at the center of the well, and the cells were collected into a three-dimensional sphere with the time of culture.

Human foreskin fibroblasts are induced by Pefitinib and cultured in 3D, a Patch assay is adopted to carry out in-vivo hair follicle reconstruction experiments, cell balls cultured in 3D are collected and mixed with new-born mouse keratinocytes, and the cell mixture is transplanted to the back subcutaneous part of a nude mouse in a subcutaneous injection mode. The cell injection site can produce macroscopic bulges when the mixture of neonatal murine fibroblasts and neonatal murine keratinocytes serves as a positive control and the neonatal murine keratinocytes alone serves as a negative control. Since the hair follicle formed by this method is located inside the skin, it is not easy to observe the area where the hair grows. To follow the location of the transplanted cells, the cells after DMSO treatment and pefitinib induction were labeled with the fluorescent dye DiI before 3D culture. DiI, diIC18 (3), is commonly referred to as 1,1'-dioctadecyl-3, 3' -tetramethylenecarbacyanine perchlorate, and is one of the most commonly used fluorescent probes for cell membranes, exhibiting orange-red fluorescence. DiI, a lipophilic membrane dye, can diffuse laterally after entering the cell membrane to gradually stain the cell membrane of the whole cell. As shown in fig. 5A, diI-labeled cells appeared as a bright red sphere under a fluorescent microscope after 3D culture.

After the cells were transplanted for four weeks, the cell transplantation sites were taken and observed for hair growth under a physical dissecting mirror and a microscope. The results are shown in FIG. 5B (wherein the newtive group is neonatal mouse keratinocyte alone; positive is mixture of neonatal mouse fibroblast and neonatal mouse keratinocyte; control is mixture of DMSO-treated human foreskin fibroblast and neonatal mouse keratinocyte; pefitinib is mixture of Pefitinib-induced human foreskin fibroblast and neonatal mouse keratinocyte). Consistent with the expected results, the positive control transplanted with the neonatal mouse fibroblast mixture grew a large amount of hair, while the negative control transplanted with the isolated keratinocyte did not grow hair. Human foreskin fibroblasts treated by DMSO (dimethyl sulfoxide) grow hairless after 3D culture, and human foreskin fibroblasts induced by Pefitinib can be induced to generate a complete hair structure by combining with 3D culture.

Example 5

Extracting and identifying human scalp fibroblasts. Fresh human scalp specimens were taken from waste scalp tissue from hair transplantation surgery, collected in a centrifuge tube of normal saline, placed in an ice box, brought back to the laboratory and immediately disposed. The scalp tissue was repeatedly rinsed 3-5 times with sterile PBS containing 2% double antibody. Since the discarded scalp tissue is very small, the scalp tissue is washed and then directly placed into a 15mL centrifuge tube containing 0.25% Dispase II, and digested overnight at 4 ℃ for 16-18 h. Peeling off the epidermal layer of the scalp from the dermal layer under a split type dissecting mirror, leaving the dermal portion, placing into a 15mL centrifuge tube containing 0.25% of type I collagenase, and digesting in an incubator at 37 ℃ for 2-4 h. Digestion was observed every 30min and tubes were shaken, indicating digestion was complete when the digestive solution became turbid and dermal tissue was almost not digested, and digestion was stopped by addition of complete medium containing serum (DMEM +10% FBS +1% dual antibody). The cell-containing digest was filtered through a 70 μm cell sieve and transferred to a 15mL centrifuge tube. Centrifuging at 1000rpm for 5min, discarding supernatant, resuspending in PBS, centrifuging, and repeating for 2 times. Resuspend the cells using complete medium (DMEM +10% FBS +1% double antibody), transfer to petri dishes, place at 37 ℃, 5% CO ₂ Culturing in an incubator, and changing the culture medium every 3 days until the cells grow full for passage. Refining the primary scalp into fine fiberCells were seeded into cell slides and identified using immunofluorescence staining.

The results are shown in fig. 6, where the isolated cells are from the papillary dermis layer immediately below the epidermis around the hair follicle, showing a typical fibroblast morphology. The immunofluorescent staining results showed high level expression of S100A4, collagen I and Collagen III, identifying the tissue isolated from scalp tissue as fibroblasts from the morphological and immunofluorescent staining.

Example 6

In order to further evaluate the induction efficiency of the small molecule drug on human scalp fibroblasts, primary DP cells (P2) (cells obtained after the primary DP cells are passed for 2 times) are also used as a control, and the expression conditions of DP marker genes in the human scalp fibroblasts and the primary DP cells after the small molecule drug Peficitinib is induced are detected through qRT-PCR. The results are shown in fig. 7, the morphology of the cells after pefitinib induction is changed, the ALP activity is enhanced, the DP cell marker gene expression is obviously enhanced, and the expression level is higher compared with that of primary DP cells. The concentration of Pefitinib for inducing human scalp fibroblasts is further optimized, and the expressions of ALP, VCAN and alpha-SMA in the human scalp fibroblasts can be induced to be obviously higher than that of primary DP cells under the concentration of 10 mu M. Consistent with foreskin fibroblasts, a concentration of 10 μ M is also the optimal concentration of Pefitinib for inducing human scalp fibroblasts.

Example 7

In order to further explore the hair induction capability of Pefitinib for inducing human scalp fibroblasts to combine with 3D culture, human scalp fibroblasts after DMSO treatment and Pefitinib induction are respectively collected, labeled by a Dil cell membrane probe, and inoculated in 96 round bottom ultra-low adhesion wells according to the cell density of 10000 cells/well for 3D culture. The cell spheres and the neonatal mouse keratinocytes are collected within 48 hours and mixed and injected into the dorsal skin of a nude mouse, the neonatal mouse fibroblasts and the neonatal mouse keratinocytes are mixed to serve as a positive control, and the single keratinocytes are injected to serve as a negative control. After 4 weeks, the skin was removed to observe hair growth. The result of in vivo hair follicle reconstruction shows that a great amount of hair grows from the positive control, and no hair grows from the negative control injected with the keratinocyte alone, so that the reliability of the result of the experimental group is ensured. As shown in fig. 8, DMSO-treated human scalp fibroblasts induced a black-clumped hair follicle-like structure and pigmentation in nude mice after 3D culture, but no intact hair structure was produced, probably because the isolated scalp fibroblasts were papillary dermal fibroblasts that had a common fibroblast progenitor source with hair papilla cells. The human scalp fibroblast group induced by the combination of 3D culture and Pefitinib generates hair with an integral structure; indicating that the Pefitinib successfully induces the transdifferentiation of human scalp fibroblasts into DP-like cells with hair induction capability.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A method for transdifferentiating fibroblasts into papilla-like cells, which comprises inducing fibroblasts with Pefitinib to transdifferentiate the fibroblasts into papilla-like cells.

2. The method according to claim 1, comprising culturing fibroblasts in an induction medium containing Pefitinib;

preferably, the concentration of Pefitinib in the induction medium is 2-10 μ M, preferably 10 μ M;

3. The method of claim 2, wherein the fibroblast cells are cultured in the induction medium for 6 to 10 days;

preferably, the fibroblasts are cultured using the induction medium for 8 days;

preferably, the induction medium is renewed every second day;

preferably, the induction medium is replaced 24h after fibroblast seeding.

4. The method of claim 1, wherein the fibroblast cells are of human origin;

preferably, the fibroblasts are derived from skin tissue;

preferably, the fibroblasts are derived from scalp tissue or foreskin tissue;

preferably, the fibroblasts are primary fibroblasts;

preferably, the culture medium of the primary fibroblasts is the basal medium of claim 2;

preferably, the culture medium of the primary fibroblasts also contains antibiotics;

preferably, the antibiotic comprises streptomycin and/or penicillin;

preferably, the method for isolating primary fibroblasts comprises: digesting the cleaned skin tissue by using dispase, separating true epidermis, digesting the true epidermis by using collagenase, and separating fibroblast;

preferably, the W/v Dispase II is digested at 2-6 ℃ for 16-18 h using 0.2-0.5% w/v;

preferably, using 0.2-0.5% w/v of collagenase type I, digested at 35-38 ℃ for 2-4 h;

preferably, the isolating fibroblasts comprises collecting the cells using a 70 μm cell sieve filtration;

preferably, the skin tissue is washed with a buffer containing an antibiotic.

5. The method according to any one of claims 1 to 4, wherein the culturing of the hair papilla-like cells into which the fibroblasts are transdifferentiated is continued by means of 3D culture;

preferably, 3D culture is carried out for 24-48 h;

preferably, the method further comprises: and (3) adopting alkaline phosphatase staining and detecting the expression condition of the related papilla specific marker genes to judge whether the fibroblast is successfully transdifferentiated into papilla-like cells, and continuously carrying out 3D culture on the successfully transdifferentiated papilla-like cells.

6. Hair papilla-like cells obtained by the method according to any one of claims 1 to 5, wherein said hair papilla-like cells have a molecular tag specific to hair papilla cells; the molecular label specific to the hair papilla cells comprises alpha-SMA and VCAN;

7. An induction culture medium for inducing the transdifferentiation of fibroblasts into papilla-like cells, characterized by containing Pefitinib with a working concentration of 2-10 μ M;

preferably, pefitinib is contained at a working concentration of 10 μ M;

8. A method for transdifferentiation of fibroblasts into papilla-like cells according to any one of claims 1 to 5, the papilla-like cells according to claim 6, or the induction medium according to claim 7, for use in hair follicle regeneration for non-diagnostic and therapeutic purposes.

9. Use of the fibroblast transdifferentiation into hair papilla-like cells according to any one of claims 1 to 5, the hair papilla-like cells according to claim 6, or the induction medium according to claim 7 for the preparation of a product for hair follicle regeneration.

10. A product for hair follicle regeneration, which comprises the hair papilla-like cells according to claim 6 or the induction medium according to claim 7.