Method for inversely differentiating human foreskin fibroblasts into human hair follicle 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 foreskin fibroblasts into human hair follicle dermal papilla-like cells and application. According to the application, pefiitinib and Vitamin D2 are added for combined induction, so that the human primary foreskin fibroblasts can be directly converted into the adult hair follicle dermal papilla-like cells, the expression of marker alkaline phosphatase and the expression of symbolic genes (SOX2, NOG, VCAN, LEF1 and alpha-SMA) of the human hair follicle dermal papilla-like cells are highly similar to those of the primary human hair follicle dermal papilla-like cells, and the human hair follicle dermal papilla-like cells can be directly converted into the adult hair follicle dermal papilla-like cells. Moreover, the hair follicle regeneration can be induced in vivo, and the composition is suitable for people with alopecia symptoms.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method and application of reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells. Background Art

[0002] Hair loss is a common skin disease associated with aging, disease, mental stress, medication, and severe wound damage. Current treatments include medication and autologous hair transplantation, but both methods are limited by adverse reactions or insufficient hair follicles.

[0003] With the development of regenerative medicine, cell-based hair follicle regeneration provides an alternative therapy. Human dermal papilla cells are a special group of 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 the hair cycle, and maintenance of hair follicle homeostasis. Therefore, human hair follicle regeneration largely relies on culture-expanded dermal papilla cells.

[0004] 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.

[0005] 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.

[0006] However, in the field of hair follicle regeneration, studies using a single small molecule to convert human fibroblasts into dermal papilla-like cells capable of inducing hair growth are still lacking. Summary of the invention

[0007] 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 reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells.

[0008] To achieve the above purpose, the technical solution adopted by this application is:

[0009] The present application provides a method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells, comprising the following steps:

[0010] Human primary foreskin fibroblasts were extracted, expanded and cultured. When the cell density of human primary foreskin fibroblasts was 80% to 90%, culture medium containing 1-15 μM peficitinib and 0.1-1 μM Vitamin D2 was added to induce the conversion of human primary foreskin fibroblasts into human hair follicle dermal papilla-like cells.

[0011] The present application can directly convert human primary foreskin fibroblasts into human hair follicle dermal papilla-like cells by adding peficitinib and Vitamin D2 for combined induction. The obtained human hair follicle dermal papilla-like cells have a high degree of similarity in biomolecular expression with primary human hair follicle dermal papilla cells. The expression of alkaline phosphatase, a marker of the human hair follicle dermal papilla-like cells of the present application, and the expression of marker genes (SOX2, NOG, VCAN, LEF1 and α-SMA) are highly similar to those of primary human hair follicle dermal papilla cells, and hair follicle regeneration can be induced in vivo.

[0012] As a preferred embodiment of the method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells described in the present application, when the effective concentration of peficitinib is 1-5 μM, the effective concentration of Vitamin D2 is 0.1-0.5 μM, the induction time is 8 days, and the culture medium is replaced every 4 days of induction.

[0013] As a preferred embodiment of the method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells described in the present application, when the effective concentration of Peficitinib is 5-10 μM, the effective concentration of Vitamin D2 is 0.5-0.75 μM, the induction time is 6 days, and the culture medium is replaced every 3 days of induction.

[0014] As a preferred embodiment of the method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells described in the present application, when the concentration of Peficitinib is 10-15 μM, the concentration of Vitamin D2 is 0.75-1 μM, the induction time is 4 days, and the culture medium is replaced every 2 days of induction.

[0015] Preferably, the effective concentration of Peficitinib is 10 μM, and the effective concentration of Vitamin D2 is 0.75 μM.

[0016] The present application adopts the above-mentioned concentration of Peficitinib and Vitamin D2 to convert human foreskin fibroblasts into human hair follicle dermal papilla-like cells, thereby achieving hair follicle regeneration, and is suitable for people with hair loss symptoms.

[0017] As a preferred embodiment of the method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells described in the present application, the human primary foreskin fibroblasts are human foreskin fibroblasts extracted from discarded human foreskin tissues.

[0018] As a preferred embodiment of the method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells described in the present application, the culture medium is a DMEM culture medium containing 10% to 20% FBS by mass concentration, preferably, the culture medium is a DMEM culture medium containing 10% FBS by mass concentration.

[0019] As a preferred embodiment of the method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells described in the present application, the peficitinib and Vitamin D2 are dissolved in a dimethyl sulfoxide solution.

[0020] As a preferred embodiment of the method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells described in the present application, the steps of extracting, amplifying and culturing primary human foreskin fibroblasts include:

[0021] 1) Rinse the foreskin tissue with sterile phosphate buffer, then cut the foreskin tissue into pieces and digest it in a neutral protease II solution to separate the dermis tissue;

[0022] 2) The dermal tissue was digested with type I collagenase, filtered, and the obtained cells were suspended in a DMEM culture medium containing fetal bovine serum and penicillin / streptomycin, and then cultured at 37° C. and 5% CO 2 .

[0023] The present application also provides the use of the above method of reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells in the preparation of hair follicle regeneration products.

[0024] Hair follicle regeneration refers to a minimally invasive method of in vivo hair follicle regeneration based on cell transplantation. Cell transplantation mainly involves the transplantation of stem cells or a mixture of epidermal and dermal components. Injecting a mixture containing epidermal stem cells and dermal papillae-like cells into nude mice can induce new hair follicles with the correct tissue structure.

[0025] Compared with the prior art, this application has the following beneficial effects:

[0026] The present application provides a method and application of reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells. The present application can directly convert human primary foreskin fibroblasts into human hair follicle dermal papilla-like cells by adding peficitinib and Vitamin D2 for induction. The expression of alkaline phosphatase, a marker of the human hair follicle dermal papilla-like cells, and the expression of marker genes (SOX2, NOG, VCAN, LEF1, and α-SMA) are highly similar to those of primary human hair follicle dermal papilla cells, and hair follicle regeneration can be induced in vivo, so the present application is suitable for people with symptoms of hair loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The first and third day status images of the human primary foreskin fibroblasts obtained in Example 1 and the immunofluorescence staining images of the human primary foreskin fibroblasts;

[0028] Figure 2 The human hair follicle dermal papilla cells extraction process and adhesion schematic diagram and ALP staining diagram obtained in Example 2;

[0029] Figure 3 This is a diagram of cell morphology changes and ALP staining after combined induction with peficitinib and Vitamin D2 in Example 3;

[0030] Figure 4 This is a result diagram of the expression of marker genes in human hair follicle dermal papilla-like cells obtained by the method of Example 3;

[0031] Figure 5 This is the experimental result of inducing hair follicle regeneration by using human primary foreskin fibroblasts induced by the combination of peficitinib and Vitamin D2. DETAILED DESCRIPTION

[0032] 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.

[0033] In the following examples and comparative 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.

[0034] The experimental animals in the following examples are immunodeficient mice (BALB / c-nu / nu) and mice (C57BL / 6), which were purchased from Guangdong Medical Experimental Animal Center.

[0035] Neonatal mouse fibroblasts and epidermal cells were isolated from 1-3 day old C57BL / 6 mice. Human foreskin tissue samples were collected from outpatients undergoing routine circumcision with the patients' consent, and scalp hair follicles were collected from the occipital region of patients undergoing hair transplantation.

[0036] EDTA-free trypsin solution, DMEM culture 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] Type I collagenase and type II neutral protease were purchased from Sigma, USA, with catalog numbers C2674 and D4693 respectively; commercial culture medium for human dermal papilla cells was purchased from Promocell, Germany, with catalog number C26501; peficitinib was purchased from MCE, USA, with catalog number HY-L022M; vitamin D2 was purchased from MCE, USA, with catalog number HY-76542; 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.

[0038] Example 1: Extraction, amplification and culture of primary human foreskin fibroblasts and immunofluorescence staining verification

[0039] This example provides specific steps for extracting, amplifying and culturing primary human foreskin fibroblasts, including:

[0040] 1) Rinse the human foreskin tissue sample thoroughly with sterile phosphate buffered saline (PBS) 3-5 times. After rinsing, the foreskin tissue should be carefully cut into small pieces using sterile scissors. These pieces are then immersed in a solution containing 0.25% dispase II and stored in a refrigerator at 4°C overnight.

[0041] 2) After overnight digestion, the dermis and epidermis were carefully separated from the human foreskin tissue sample using sterile forceps, making sure to keep the dermis. The dermis was then washed three times with PBS to remove any remaining debris.

[0042] 3) The dermal tissue was further minced using sterile scissors, treated with 0.25% type I collagenase, and digested in a 37° C. incubator until complete.

[0043] 4) After digestion, filter the cell suspension through a cell strainer and wash with PBS. Then suspend the cells in DMEM medium and add 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S). Transfer the suspended cells to a culture dish and culture them in a 37°C, 5% CO2 incubator.

[0044] 5) The culture medium was changed every 3 days until the cells reached confluence for passaging.

[0045] 6) The cells were fixed in 4% paraformaldehyde for 20 minutes and then stained with QuickBlock TM The cells were blocked with immunostaining blocking buffer for 30 minutes and then incubated with primary antibodies at 4°C overnight.

[0046] 7) After primary antibody incubation, allow cells to interact with goat anti-mouse or anti-rabbit IgG H&L pre-adsorbed secondary antibodies for 1 hour at room temperature.

[0047] 8) Nuclear DNA was counterstained blue with DAPI.

[0048] 9) Finally, image analysis was performed using confocal microscopy, e.g. Figure 1 ( Figure 1 The left side shows the state diagram of the first and third days of extraction; Figure 1 The right side shows the immunofluorescence staining of cells.

[0049] Figure 1 Conclusion: Firstly, primary human foreskin fibroblasts were isolated and expanded from foreskin tissue, and then identified by combining morphology and immunofluorescence staining.

[0050] like Figure 1 As shown in A, the isolated cells have a typical fibroblast morphology: the cells are slender and spindle-shaped with obvious cell borders; they continue to grow until they are fully confluent, and the cells in the confluent area grow close to each other in a parallel arrangement. Fibroblasts synthesize extracellular matrix and collagen, of which type I and type III collagen (Collagen I and Collagen III) are highly expressed in skin fibroblasts. In addition, fibroblast-specific protein 1 (FSP-1) is a fibroblast cytoplasmic marker, also known as S100A4, and is a member of the intracellular protein S100 superfamily.

[0051] Immunofluorescence staining ( Figure 1Middle B) shows that almost all cells express S100A4, Collagen I and Collagen III at high levels, further verifying that the cells isolated and cultured from the foreskin in the present application are fibroblasts, and these high-purity foreskin fibroblasts can be used for subsequent experiments.

[0052] Example 2: Extraction, amplification and culture of human hair follicle dermal papilla cells and ALP staining verification

[0053] This embodiment provides specific steps for extracting, amplifying and culturing human hair follicle dermal papilla cells, including:

[0054] 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.

[0055] 2) The lower part of the hair follicle, including the dermal papilla and the hair bulb, was then excised and digested with 0.25% type I collagenase at 37°C for 3 hours.

[0056] 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.

[0057] 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.

[0058] 5) In approximately one week, cells will migrate from the dermal papillae and adhere to the surface of the culture plate.

[0059] 6) Until the cells reach confluence for passaging.

[0060] 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.

[0061] The results are as follows Figure 2 As shown, Figure 2 The left side shows the extraction process and adhesion diagram of human hair follicle dermal papilla cells. Figure 2 This is the result of alkaline phosphatase staining of human hair follicle dermal papilla cells.

[0062] Figure 2 in conclusion:

[0063] The DP of the hair follicle dermal papilla was isolated by enzyme digestion combined with microdissection. Figure 2 As shown in the figure, the end of a single hair follicle after treatment has a complete flame-shaped DP structure. These DPs are only wrapped by the basement membrane. Collagenase can be used to digest the collagen fibers, adipose tissue, etc. around the DP, thereby freeing the DP. The free DP is excised to separate it completely, and the hair shaft and large pieces of undigested tissue are picked out under a microscope. At this time, the digested DP is mixed with a large number of digested collagen components and cells. Taking advantage of the clump-like characteristics of the DP, the DP is precipitated by low-speed centrifugation, while other fat, collagen and cell components are suspended in the supernatant and discarded. After repeated attempts, a relatively pure DP can be harvested. On the third day of culture, the DP adhered to the wall, and polygonal or short spindle-shaped cells began to migrate out of the surrounding area; on the sixth day of culture, the cells grew radially with the DP as the center; on the 18th day, after the DP cells fuse, they grow in multi-layer aggregates. The extracted DP cells were first stained with ALP. The results are shown in Figure 2 As shown, the center of the extracted DP cells showed strong ALP activity, while the peripheral migrated cells gradually weakened.

[0064] Example 3: A method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells

[0065] This embodiment provides a method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells, comprising the following steps:

[0066] 1) The primary human foreskin fibroblasts obtained in Example 1 were seeded into a 10 cm cell culture dish at a seeding density of 80%.

[0067] 2) On the second day (day 0), the original culture medium was replaced with a complete culture medium supplemented with 10 μM peficitinib and 0.75 μM Vitamin D2 (dissolved in dimethyl sulfoxide solution), wherein the complete culture medium was DMEM supplemented with 10% FBS.

[0068] 3) The culture medium containing small molecules was replaced every 4 days.

[0069] After an 8-day incubation period, human primary foreskin fibroblasts were converted into human hair follicle dermal papilla-like cells; fibroblasts were fixed with 4% paraformaldehyde for 20 minutes at room temperature and then washed three times with PBS. Subsequently, the fixed cells were stained using the BCIP / NBT alkaline phosphatase colorimetric kit according to the manufacturer's instructions.

[0070] Morphological changes of primary human foreskin fibroblasts induced by peficitinib and Vitamin D2 Figure 3As shown on the left, ALP staining of primary human foreskin fibroblasts induced by peficitinib Figure 3 Shown on the right.

[0071] Figure 3 in conclusion: Figure 3 The results showed that the combination of peficitinib and Vitamin D2 induced changes in the cell morphology of human foreskin fibroblasts. The cells became larger and rounder, and showed obvious ALP-positive results.

[0072] Example 4: Detection of the expression of marker genes in human hair follicle dermal papilla-like cells obtained by the method of Example 3

[0073] The experiment of detecting the expression of the marker genes of the human hair follicle dermal papilla-like cells obtained by the method of Example 3 specifically comprises the following steps:

[0074] 1) Total RNA from human hair follicle dermal papilla-like cells obtained by the method of Example 3 was extracted using TRIzol reagent according to the manufacturer's instructions.

[0075] 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.

[0076] 3) Then use TB Premix Ex Taq TM II 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.

[0077] 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). P < 0.05 was considered statistically significant.

[0078] Table 1

[0079]

[0080] like Figure 4 As shown, the expression of marker genes of human hair follicle dermal papilla-like cells obtained from human primary foreskin fibroblasts after combined induction with peficitinib and Vitamin D2 is highly consistent with the human hair follicle dermal papilla cells obtained in Example 2.

[0081] Example 5: Experiment on hair follicle regeneration induced by human primary foreskin fibroblasts induced by peficitinib and Vitamin D2

[0082] The experiment of inducing hair follicle regeneration by human primary foreskin fibroblasts according to the method of Example 3 (after combined induction with peficitinib and Vitamin D2) specifically includes the following steps:

[0083] 1. Use 5-6 week old BALB / c-nu / nu nude mice for in vivo hair follicle reconstruction.

[0084] 2. Use a 4 mm diameter skin biopsy punch to create a symmetrical full-thickness skin wound in the back area. Mix 1 million human primary foreskin fibroblasts induced by peficitinib and Vitamin D2 with 1 million newborn mouse keratinocytes and add them to 30 μl of Matrigel to obtain a mixture.

[0085] 3. The mixture was then incubated in a 37°C incubator for 30 minutes until the matrix gel solidified. Subsequently, the solidified cell-matrix gel mixture was transplanted into the skin incision and covered with 3M transparent dressing and self-adhesive elastic bandage to prevent infection.

[0086] After 4 weeks, the mice were euthanized and the skin sites of the transplanted cells were harvested. In the positive control group, isolated newborn mouse fibroblasts were mixed with newborn mouse keratinocytes. In the negative control group, only keratinocytes were used for all hair follicle regeneration experiments. Dimethyl sulfoxide solution was used as the DMSO treatment group.

[0087] like Figure 5 shown.

[0088] Figure 5 Conclusion: If Figure 5 As shown, the blank control group treated with DMSO and the experimental group (Peficitinib and VitaminD2) after combined induction could not observe obvious hair growth under the naked eye. Further observation through a dissecting microscope and an optical microscope showed that no hair was generated in the control group, while obvious hair growth was observed in the experimental group after induction. These formed hairs could not be observed by the naked eye under the skin of the nude mouse back because the number of hairs was small and could not stand out in the skin of the nude mouse. The results showed that the human foreskin fibroblasts induced by the experimental group had the ability to induce hair follicle formation.

[0089] 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 reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells, characterized in that: The following steps are involved: Human primary foreskin fibroblasts were extracted, expanded and cultured. When the cell density of human primary foreskin fibroblasts was 80% to 90%, culture medium containing 1-15 μM peficitinib and 0.1-1 μM Vitamin D2 was added to induce the conversion of human primary foreskin fibroblasts into human hair follicle dermal papilla-like cells.

2. The method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to claim 1, characterized in that: When the effective concentration of peficitinib is 1-5 μM and the effective concentration of Vitamin D2 is 0.1-0.5 μM, the induction time is 8 days, and the culture medium is replaced every 4 days of induction.

3. The method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to claim 1, characterized in that: When the effective concentration of peficitinib is 5-10 μM and the effective concentration of vitamin D2 is 0.5-0.75 μM, the induction time is 6 days, and the culture medium is replaced every 3 days of induction.

4. The method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to claim 1, characterized in that: When the effective concentration of peficitinib is 10-15 μM, when the effective concentration of vitamin D2 is 0.75-1 μM, the induction time is 4 days, and the culture medium is replaced every 2 days of induction.

5. The method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to claim 1, characterized in that: The primary human foreskin fibroblasts are human foreskin fibroblasts extracted from discarded human foreskin tissues.

6. The method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to claim 1, characterized in that: The culture medium is a DMEM culture medium containing FBS with a mass concentration of 10% to 20%.

7. The method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to claim 1, characterized in that: The peficitinib and Vitamin D2 are dissolved in dimethyl sulfoxide solution.

8. The method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to claim 1, characterized in that: The steps of extracting, amplifying and culturing primary human foreskin fibroblasts include: 1) Rinse the foreskin tissue with sterile phosphate buffer, then cut the foreskin tissue into pieces and digest it in a neutral protease II solution to separate the dermis tissue; 2) The dermal tissue was digested with type I collagenase, filtered, and the obtained cells were suspended in a DMEM culture medium containing fetal bovine serum and penicillin / streptomycin, and then cultured at 37° C. and 5% CO 2 .

9. Use of the method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to any one of claims 1 to 8 in preparing hair follicle regeneration products.

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