Method for inversely differentiating human scalp fibroblasts into dermal papilla-like cells and application

Abstract

The invention relates to the technical field of biology, and particularly discloses a method for inversely differentiating human scalp fibroblasts into dermal papilla-like cells and application of the method. Ribociclib hydrochloride and vitamin D2 are added for combined induction, so that the human scalp fibroblasts are directly converted into the dermal papilla-like cells; the dermal papilla-like cells have high biomolecular expression similarity with primary human dermal papilla cells, and the problems that proliferation of the dermal papilla-like cells is limited and the hair induction potential of the dermal papilla-like cells is gradually reduced within a long time are solved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method and application of reverse differentiation of human scalp fibroblasts into dermal papillary cells. Background Art

[0002] Currently, the treatment of hair loss mainly adopts two methods: drug therapy and autologous hair follicle transplantation. Although the drugs available on the market for the treatment of hair loss have certain effects, they cannot promote the formation of new hair follicles, so they only treat the symptoms and not the root cause. With the development of regenerative medicine, cell-based hair follicle regeneration provides an alternative therapy. Human dermal papilla cells are a group of special dermal fibroblasts that originate from the base of hair follicles. They can restart the epithelial-mesenchymal interaction with functional keratinocytes and are essential for hair follicle morphogenesis, regulation of hair cycle and maintenance of hair follicle homeostasis. Therefore, human hair follicle regeneration depends largely on cultured and expanded dermal papilla cells.

[0003] However, conventional in vitro culturing techniques for dermal papilla cells face great challenges, including limited proliferation of dermal papilla cells and a gradual decrease in the hair-inducing potential of dermal papilla cells over a long period of time. Therefore, obtaining a large number of dermal papilla cells with hair-inducing ability is essential for effective hair follicle regeneration.

[0004] Direct reprogramming technology is the conversion of one somatic cell type into another. This method provides a new way to generate functional cells and has been widely used in the field of regenerative medicine. Chemical small molecule-mediated reprogramming has many advantages over reprogramming methods such as non-integrating vectors and non-viral gene delivery systems, such as convenience, economy, stability, rapidity, reversibility, and dose- or duration-dependent effects.

[0005] However, in the field of hair follicle regeneration, there are few reports on the use of small molecules to transform human scalp fibroblasts into dermal papilla-like cells that can induce hair growth. Summary of the invention

[0006] The purpose of the present application is to overcome the deficiencies of the above-mentioned prior art and to provide a method and application of reverse differentiation of human scalp fibroblasts into dermal papillary cells.

[0007] To achieve the above purpose, the technical solution adopted by this application is:

[0008] The present application provides a method for reversely differentiating human scalp fibroblasts into dermal papillary-like cells, comprising the following steps:

[0009] Human scalp fibroblasts were extracted, expanded and cultured. When the cell density of human scalp fibroblasts was 80% to 90%, an induction medium containing ribociclib hydrochloride and vitamin D2 was added to induce the human scalp fibroblasts to reversely differentiate into dermal papillary cells.

[0010] After extensive research and experiments, the inventors of the present application found that the present application, by adding Ribociclib hydrochloride and Vitamin D2 for combined induction, reversely differentiated human scalp fibroblasts into dermal papilla-like cells. These dermal papilla-like cells have a high similarity in biomolecular expression with primary human hair follicle dermal papilla cells, which solves the problem of limited proliferation of dermal papilla cells and a gradual decrease in the hair induction potential of dermal papilla cells over a long period of time, which is crucial for effective hair follicle regeneration.

[0011] Preferably, the cell density of the human scalp fibroblasts is 85%.

[0012] As a preferred embodiment of the method for reverse differentiation of human scalp fibroblasts into dermal papillary-like cells described in the present application, the ribociclib hydrochloride is dissolved in dimethyl sulfoxide to form a ribociclib hydrochloride solution; and the vitamin D2 is dissolved in dimethyl sulfoxide to form a vitamin D2 solution.

[0013] As a preferred embodiment of the method for reverse differentiation of human scalp fibroblasts into dermal papillary cells described in the present application, the concentration of ribociclib hydrochloride in the ribociclib hydrochloride solution is 1 to 2 μM; the concentration of vitamin D2 in the vitamin D2 solution is 10 to 20 μM.

[0014] The present application uses the above-mentioned concentration range of ribociclib hydrochloride and vitamin D2 for combined induction, which can reversely differentiate human scalp fibroblasts into dermal papilla-like cells. The obtained dermal papilla-like cells have a high similarity in biomolecular expression with primary human hair follicle dermal papilla cells, effectively promoting the regeneration of hair follicles.

[0015] As a preferred embodiment of the method for reverse differentiation of human scalp fibroblasts into dermal papillary cells described in the present application, the concentration of ribociclib hydrochloride in the ribociclib hydrochloride solution is 1 μM; the concentration of vitamin D2 in the vitamin D2 solution is 10 μM.

[0016] The present application adopts the above-mentioned preferred concentration of ribociclib hydrochloride and vitamin D2 for combined induction, which can better induce human scalp fibroblasts to reversely differentiate into dermal papilla-like cells with hair induction ability. The obtained dermal papilla-like cells have a high degree of biomolecular expression similarity with primary human hair follicle dermal papilla cells, which is crucial for effective hair follicle regeneration.

[0017] As a preferred embodiment of the method for reversely differentiating human scalp fibroblasts into dermal papillary-like cells described in the present application, the induction culture medium is a DMEM / F12 culture medium containing fetal bovine serum at a concentration of 15-25%.

[0018] As a preferred embodiment of the method for reversely differentiating human scalp fibroblasts into dermal papillary-like cells described in the present application, the human scalp fibroblasts are derived from scalp tissue discarded in hair transplantation surgery.

[0019] As a preferred embodiment of the method for reversely differentiating human scalp fibroblasts into dermal papillary-like cells described in the present application, the induction time is 5 days, and the induction culture medium is replaced once a day.

[0020] As a preferred embodiment of the method for reversely differentiating human scalp fibroblasts into dermal papillary cells described in the present application, the steps of extracting, amplifying and culturing human scalp fibroblasts include:

[0021] 1) The scalp tissue discarded during the hair transplantation operation was centrifuged, rinsed, and centrifuged and digested with dispase II to separate the dermis tissue;

[0022] 2) Digesting the dermal tissue with collagenase type I, and after digestion is complete, adding complete culture medium to terminate digestion and obtain a digestion solution containing cells;

[0023] 3) The digestion solution containing cells was filtered and centrifuged, the supernatant was discarded, the solution was resuspended in PBS and centrifuged again, and then the cells were resuspended in complete culture medium and cultured at 37° C. and 5% CO 2 to obtain human scalp fibroblasts.

[0024] As a preferred embodiment of the method for reversely differentiating human scalp fibroblasts into dermal papillary cells described in the present application, the mass concentration of the dispase II is 0.2-0.3%; the mass concentration of the collagenase type I is 0.2-0.3%.

[0025] Preferably, the mass concentration of the dispase II is 0.25%; the mass concentration of the collagenase type I is 0.25%.

[0026] The present application also provides the use of the above-mentioned method of reversely differentiating human scalp fibroblasts into dermal papilla-like cells in the preparation of hair follicle regeneration products.

[0027] Compared with the prior art, this application has the following beneficial effects:

[0028] The present application provides a method and application of reverse differentiation of human scalp fibroblasts into dermal papilla-like cells. The present application directly converts human scalp fibroblasts into dermal papilla-like cells by adding Ribociclib hydrochloride and Vitamin D2 for combined induction. These dermal papilla-like cells have a high similarity in biomolecule expression with primary human hair follicle dermal papilla cells, which solves the problem of limited proliferation of dermal papilla cells and gradual decrease in the hair induction potential of dermal papilla cells over a long period of time, and is crucial for effective hair follicle regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a morphological diagram of human scalp fibroblasts obtained in Example 1;

[0030] Figure 2 The morphological diagram and alkaline phosphatase staining result diagram of the dermal papilla cells of the human hair follicle obtained in Example 2;

[0031] Figure 3 The figure is a graph showing the morphology and alkaline phosphatase staining results of dermal papillary cells obtained by the method of Example 3;

[0032] Figure 4 This is a graph showing the expression of marker genes in dermal papillary-like cells obtained by the method of Example 3. DETAILED DESCRIPTION

[0033] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0034] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified, and the components and raw materials used in each parallel experiment are of the same type.

[0035] The examples of the present application are described in detail below. Unless otherwise specified, the experimental animals in the following examples are immunodeficient mice (BALB / c-nu / nu) and mice (C57BL / 6), purchased from Guangdong Medical Experimental Animal Center. Neonatal mouse fibroblasts and epidermal cells were isolated using 1-3 day old C57BL / 6 mice.

[0036] DMEM medium, penicillin / streptomycin (P / S), and fetal bovine serum (FBS) were purchased from Gibco, USA, with catalog numbers 25200072, C11995500BT, 15140122, and 10270106, respectively;

[0037] Dispase II and collagenase type I were purchased from Sigma with catalog numbers D4693 and C2674, respectively;

[0038] Ribociclib hydrochloride and Vitamin D2 were purchased from MCE with catalog numbers HY-15777A and HY-76542 respectively:

[0039] Human dermal papilla cell commercial culture medium was purchased from Promocell, Germany, with the catalog number C26501; QuickBlock TM Immunostaining blocking buffer and BCIP / NBT alkaline phosphatase colorimetric kit were purchased from Bio-Tech, with catalog numbers P0260 and C3206, respectively; reverse transcription kit and TB Premix Ex Taq TM II were purchased from Takara Co., Ltd., Japan, with item numbers RR047A and RR820A respectively; Matrigel was purchased from Corning Incorporated, USA, with item number 354234.

[0040] Example 1: Extraction, proliferation and cultivation of human scalp fibroblasts

[0041] The present application provides a method for extracting, amplifying and culturing human scalp fibroblasts, comprising the following steps:

[0042] (1) Fresh human scalp specimens were obtained from discarded scalp tissue during hair transplantation surgery. They were collected in centrifuge tubes containing physiological saline, placed in an ice box, brought back to the laboratory, and processed immediately.

[0043] (2) Rinse the scalp tissue repeatedly 3-5 times using sterile PBS containing 2% penicillin / streptomycin (P / S).

[0044] (3) Since the discarded scalp tissue is very small, it is directly placed in a 15 mL centrifuge tube containing 0.25% dispase (Dispase II) after washing and digested overnight in a 4°C refrigerator for 16 to 18 hours.

[0045] (4) Under a dissecting microscope, the epidermis of the scalp was torn off from the dermis, leaving the dermis tissue, which was placed in a 15 mL centrifuge tube containing 0.25% collagenase type I and digested in a 37°C incubator for 2 to 4 hours.

[0046] (5) Observe the digestion process every 30 minutes and shake the centrifuge tube. When the digestion solution becomes turbid and the dermal tissue is almost digested and disappears, it means that the digestion is complete. Add an equal volume of complete culture medium (DMEM+10% FBS+1% double antibody) to terminate the digestion and obtain the digestion solution containing cells.

[0047] (6) Filter the digestion solution containing cells through a 70 μm cell sieve and transfer it to a 15 mL centrifuge tube.

[0048] (7) Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend in PBS, and centrifuge. Repeat twice.

[0049] (8) Resuspend the cells in complete medium (DMEM+10% FBS+1% double antibody), transfer to a culture dish, and culture in an incubator at 37°C and 5% CO2. Change the medium every 3 days until the cells are fully grown and then passaged to obtain human scalp fibroblasts. The morphology is shown in the figure. Figure 1 shown.

[0050] Example 2: Extraction, amplification and culture of human hair follicle dermal papilla cells and ALP staining verification

[0051] This embodiment provides specific steps for extracting, amplifying and culturing human hair follicle dermal papilla cells, including:

[0052] 1) First, the hair follicles were subjected to a rigorous cleaning process, including three consecutive washes with PBS buffer containing 2% penicillin / streptomycin (P / S). Subsequently, the surrounding skin and fat tissue surrounding the hair follicles were carefully removed using a dissecting microscope.

[0053] 2) The lower part of the hair follicle, including the dermal papilla and hair bulb, was then excised and digested with 0.25% type I collagenase at 37°C for 3 hours.

[0054] 3) After the tissues surrounding the dermal papillae are completely digested and the dermal papillae themselves are exposed, DMEM complete medium supplemented with 10% FBS and 1% penicillin / streptomycin (P / S) is immediately added to stop the enzyme activity. The completely detached dermal papillae are carefully collected.

[0055] 4) After collection, the separated dermal papillae were thoroughly rinsed three times with PBS. After rinsing, 2 ml of human dermal papillae commercial culture medium was added to each well of a 6-well plate, and 2-3 dermal papillae were placed in each well for subsequent culture.

[0056] 5) In approximately one week, cells will migrate from the dermal papillae and adhere to the surface of the culture plate.

[0057] 6) Until the cells reach confluence for passaging.

[0058] 7) Fix the cells with 4% paraformaldehyde at room temperature for 20 minutes, and then wash three times with PBS. Then, stain the fixed cells using BCIP / NBT alkaline phosphatase colorimetric kit according to the manufacturer's instructions.

[0059] The results are as follows Figure 2 As shown, Figure 2 The left side shows the morphology of the dermal papilla cells of the hair follicles on the human head. Figure 2 This is the result of alkaline phosphatase staining of dermal papilla cells of hair follicles on human head.

[0060] Example 3: A method for reverse differentiation of human scalp fibroblasts into dermal papillary cells

[0061] This embodiment provides a method for reversely differentiating human scalp fibroblasts into dermal papillary-like cells, comprising the following steps:

[0062] 1) The human scalp fibroblasts obtained in Example 1 were seeded into a 10 cm cell culture dish at a seeding density of 85%.

[0063] 2) On the second day (day 0), the original culture medium (DMEM / F12 medium containing 20% ​​FBS) was replaced with the induction medium, which was DMEM / F12 medium supplemented with 1 μM ribociclib hydrochloride and 10 μM vitamin D2 (dissolved in DMSO) and 20% FBS.

[0064] 3) The induction medium was replaced every day, and the dermal papillary-like cells were harvested after 5 days of induction.

[0065] 4) ALP staining The dermal papillary cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes and then washed three times with PBS. Subsequently, the fixed dermal papillary cells were stained using the BCIP / NBT alkaline phosphatase colorimetric kit according to the manufacturer's instructions; the morphology of the dermal papillary cells and the alkaline phosphatase staining results are shown in Figure 3 shown.

[0066] Experimental Example 1: Detection of the expression of marker genes in dermal papillary-like cells obtained by the method of Example 3

[0067] The experiment of detecting the expression of the marker genes of the dermal papillary-like cells obtained by the method of Example 3 specifically comprises the following steps:

[0068] 1) Total RNA from the dermal papillary cells obtained by the method of Example 3 was extracted using TRIzol reagent according to the manufacturer's instructions.

[0069] 2) Then, use PrimeScript with gDNA Eraser TM 1 μg RNA was reverse transcribed into cDNA using the RT kit. The reverse transcription reaction was performed according to the manufacturer's protocol.

[0070] 3) Then use TB Premix Ex Taq TMII The obtained cDNA was subjected to qRT-PCR according to the manufacturer's instructions, and the primer sequences are shown in Table 1. The relative fold change was calculated using the 2^(-ΔΔCT) method, and each experiment was performed three times to achieve three biological replicates.

[0071] GraphPad Prism software (GraphPad 9.0) was used for statistical tests. The differences between the two groups were analyzed using unpaired t-test. All data were expressed as the mean ± SD of multiple samples (n ≥ 3). The significance level was P < 0.05, which was considered statistically significant.

[0072] Table 1

[0073]

[0074] like Figure 4 As shown, the expression of the marker genes (α-SMA, VCAN, ALPL) of the dermal papilla-like cells obtained by inducing human scalp fibroblasts using ribociclib hydrochloride and vitamin D2 is highly consistent with the human hair follicle dermal papilla cells obtained in Example 2.

[0075] The present application directly converts human scalp fibroblasts into dermal papilla-like cells by adding Ribociclib hydrochloride and Vitamin D2 for combined induction. These dermal papilla-like cells have a high similarity in biomolecular expression with primary human hair follicle dermal papilla cells, which solves the problem of limited proliferation of dermal papilla-like cells and gradual decrease in the hair induction potential of dermal papilla-like cells over a long period of time, which is crucial for effective hair follicle regeneration.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A method for reverse differentiation of human scalp fibroblasts into dermal papillary cells, characterized in that: The following steps are involved: Human scalp fibroblasts were extracted, expanded and cultured. When the cell density of human scalp fibroblasts was 80% to 90%, an induction medium containing ribociclib hydrochloride and vitamin D2 was added to induce the human scalp fibroblasts to reversely differentiate into dermal papillary cells.

2. The method for reversely differentiating human scalp fibroblasts into dermal papillary cells according to claim 1, characterized in that: The ribociclib hydrochloride is dissolved in dimethyl sulfoxide to form a ribociclib hydrochloride solution; the vitamin D2 is dissolved in dimethyl sulfoxide to form a vitamin D2 solution.

3. The method for reverse differentiation of human scalp fibroblasts into dermal papillary cells according to claim 2, characterized in that: The concentration of ribociclib hydrochloride in the ribociclib hydrochloride solution is 1 to 2 μM; the concentration of vitamin D2 in the vitamin D2 solution is 10 to 20 μM.

4. The method for reversely differentiating human scalp fibroblasts into dermal papillary cells according to claim 3, characterized in that: The concentration of ribociclib hydrochloride in the ribociclib hydrochloride solution is 1 μM; the concentration of vitamin D2 in the vitamin D2 solution is 10 μM.

5. The method for reverse differentiation of human scalp fibroblasts into dermal papillary cells according to claim 1, characterized in that: The induction culture medium is a DMEM / F12 culture medium containing 15-25% fetal bovine serum.

6. The method for reversely differentiating human scalp fibroblasts into dermal papillary cells according to claim 1, characterized in that: The human scalp fibroblasts are derived from scalp tissue discarded during hair transplantation surgery.

7. The method for reversely differentiating human scalp fibroblasts into dermal papillary cells according to claim 1, characterized in that: The induction period was 5 days, and the induction medium was replaced once a day.

8. The method for reverse differentiation of human scalp fibroblasts into dermal papillary cells according to claim 1, characterized in that: The steps of extracting, amplifying and culturing human scalp fibroblasts include: 1) The scalp tissue discarded during the hair transplantation operation was centrifuged, rinsed, and centrifuged and digested with dispase II to separate the dermis tissue; 2) Digesting the dermal tissue with collagenase type I, and after digestion is complete, adding complete culture medium to terminate digestion and obtain a digestion solution containing cells; 3) Filter the digestion solution containing cells, centrifuge, discard the supernatant, resuspend in PBS and centrifuge again, then resuspend the cells in complete culture medium, and culture at 37° C. and 5% CO 2 to obtain human scalp fibroblasts.

9. The method for reversely differentiating human scalp fibroblasts into dermal papillary cells according to claim 1, characterized in that: The mass concentration of the dispase II is 0.2-0.3%; the mass concentration of the collagenase type I is 0.2-0.3%.

10. Use of the method for reversely differentiating human scalp fibroblasts into dermal papilla-like cells according to any one of claims 1 to 9 in preparing hair follicle regeneration products.

Images