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

Abstract

The invention relates to the technical field of biology, in particular to a method for inversely differentiating into dermal papilla-like cells and application, and the method comprises the following steps: extracting, amplifying and culturing human primary foreskin fibroblasts, adding a culture medium containing Selumetinib with the action concentration of 1-10 [mu] M for induction, and culturing the human primary foreskin fibroblasts to obtain the dermal papilla-like cells. The human primary foreskin fibroblasts are inversely differentiated into dermal papilla-like cells. According to the application, the human primary foreskin fibroblasts are inversely differentiated into the dermal papilla-like cells by adding Selumetinib with the action concentration of 1-10 mu M for induction, and the obtained dermal papilla-like cells have marker alkaline phosphatase expression and symbolic gene expression, have high biomolecular expression similarity with primary human scalp hair follicle dermal papilla cells, and can be used for preparing the dermal papilla-like cells. The hair follicle regeneration is facilitated.

Description

Technical Field

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

[0002] As an appendage of the skin, hair plays an important role in controlling body temperature and protecting the skin. Diseases and drug treatments can cause hair loss. Although hair loss does not directly threaten human life, it can cause certain distress to the patient's psychology and quality of life.

[0003] 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.

[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.

[0005] Therefore, obtaining a large number of dermal papilla-like cells with hair-inducing ability is crucial for efficient hair follicle regeneration. 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 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 reverse differentiation into dermal papillary cells, comprising the following steps:

[0009] Human primary foreskin fibroblasts were extracted, expanded and cultured, and induced by adding culture medium containing selumetinib at an effective concentration of 1 to 10 μM. Human primary foreskin fibroblasts were reversely differentiated into dermal papillary cells (which have extremely high molecular expression similarity with dermal papilla cells).

[0010] In the technical scheme of the present application, the present application induces the reverse differentiation of human primary foreskin fibroblasts into dermal papilla-like cells by adding Selumetinib at an effective concentration of 1 to 10 μM. The obtained dermal papilla-like cells have a high similarity in biomolecular expression with primary human scalp hair follicle dermal papilla cells, which is beneficial to hair follicle regeneration.

[0011] As a preferred embodiment of the method for reverse differentiation into dermal papillary cells described in the present application, the effective concentration of Selumetinib is 5 to 10 μM.

[0012] As a preferred embodiment of the method for reverse differentiation into dermal papillary cells described in the present application, the effective concentration of Selumetinib is 10 μM.

[0013] The use of Selumetinib at the above-mentioned preferred concentration can better reversely differentiate human primary foreskin fibroblasts into dermal papilla-like cells, which is beneficial to hair follicle regeneration and is suitable for people with hair loss symptoms.

[0014] As a preferred embodiment of the method for reverse differentiation into dermal papillary cells described in the present application, the Selumetinib is dissolved in a dimethyl sulfoxide solution.

[0015] As a preferred embodiment of the method for reverse differentiation into dermal papillary-like cells described in the present application, the culture medium is RPMI 1640 culture medium containing FBS at a mass concentration of 5-15%.

[0016] Preferably, the culture medium is RPMI 1640 culture medium containing FBS with a mass concentration of 10%.

[0017] As a preferred embodiment of the method for reverse differentiation into dermal papillary-like cells described in the present application, the induction time is 6 to 8 days (preferably 8 days), and the culture medium is replaced every 2 days of induction.

[0018] As a preferred embodiment of the method for reverse differentiation into dermal papillary cells described in the present application, the steps of extracting, amplifying and culturing primary human foreskin fibroblasts include:

[0019] 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;

[0020] 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 .

[0021] As a preferred embodiment of the method for reverse differentiation into dermal papillary cells described in the present application, the primary human foreskin fibroblasts are human foreskin fibroblasts extracted from discarded human foreskin tissue.

[0022] As a preferred embodiment of the method for reverse differentiation into dermal papillary-like cells described in the present application, the dermal papillary-like cells have expression of the marker alkaline phosphatase and the marker gene expression.

[0023] As a preferred embodiment of the method for reverse differentiation into dermal papillary cells described in the present application, the marker genes include at least one of ALPL, BMP4, LEF1, SOX2, THY1, VCAN and WIF1.

[0024] The dermal papilla-like cells obtained by the method of the present application have the expression of the marker alkaline phosphatase and the expression of the signature genes (ALPL, BMP4, LEF1, SOX2, THY1, VCAN and WIF1), have a high similarity in biomolecular expression with the primary human scalp hair follicle dermal papilla cells, and have the effect of hair follicle regeneration.

[0025] As a preferred embodiment of the method for reverse differentiation into dermal papilla-like cells described in the present application, the dermal papilla-like cells are human hair follicle dermal papilla-like cells.

[0026] By adopting the method of the present application, human primary foreskin fibroblasts are reversely differentiated into human hair follicle dermal papilla-like cells, which can achieve hair follicle regeneration and is suitable for people with hair loss symptoms.

[0027] The present application provides the use of the above-mentioned method of reverse differentiation into dermal papilla-like cells in the preparation of hair follicle regeneration products.

[0028] 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.

[0029] Compared with the prior art, this application has the following beneficial effects:

[0030] The present application provides a method and application of reverse differentiation into dermal papilla-like cells. The present application induces reverse differentiation of human primary foreskin fibroblasts into dermal papilla-like cells by adding Selumetinib at an effective concentration of 1 to 10 μM. The obtained dermal papilla-like cells have the expression of marker alkaline phosphatase and marker genes (ALPL, BMP4, LEF1, SOX2, THY1, VCAN and WIF1), have a high degree of biomolecular expression similarity with primary human scalp hair follicle dermal papilla cells, and are beneficial to hair follicle regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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;

[0032] Figure 2 The human hair follicle dermal papilla cells extraction process and adhesion schematic diagram and ALP staining diagram obtained in Example 2;

[0033] Figure 3 This is a diagram showing the morphological changes and ALP staining of primary human foreskin fibroblasts induced by Selumetinib in Example 3;

[0034] Figure 4 This is a graph showing the expression of marker genes in human hair follicle dermal papilla-like cells obtained by the method of Example 3. DETAILED DESCRIPTION

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] DMEM culture medium, penicillin / streptomycin (P / S) and fetal bovine serum (FBS) were purchased from Gibco, USA, with catalog numbers C11995500BT, 15140122 and 10270106, respectively.

[0040] Type I collagenase was purchased from Sigma, USA, with the catalog number C2674; commercial culture medium for human dermal papilla cells was purchased from Promocell, Germany, with the catalog number C26501; Selumetinib was purchased from MCE, with the catalog number HY-50706;

[0041] QuickBlockTM 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.

[0042] Example 1: Extraction, amplification and culture of primary human foreskin fibroblasts and immunofluorescence staining verification

[0043] This example provides specific steps for extracting, amplifying and culturing primary human foreskin fibroblasts, including:

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 5) The culture medium was changed every 3 days until the cells reached confluence for passaging.

[0049] 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.

[0050] 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.

[0051] 8) Nuclear DNA was counterstained blue with DAPI.

[0052] 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.

[0053] Figure 1 Conclusion: Firstly, primary human foreskin fibroblasts were isolated and amplified from foreskin tissue, and then identified by combining morphology and immunofluorescence staining. 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.

[0054] Immunofluorescence staining ( Figure 1 Middle 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.

[0055] Example 2: Extraction, amplification and culture of human hair follicle dermal papilla cells and ALP staining verification This example provides specific steps for extracting, amplifying and culturing human hair follicle dermal papilla cells, including:

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 5) In approximately one week, cells will migrate from the dermal papillae and adhere to the surface of the culture plate.

[0061] 6) Until the cells reach confluence for passaging.

[0062] 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.

[0063] 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.

[0064] Figure 2 in conclusion:

[0065] 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.

[0066] Example 3: A method for reverse differentiation into dermal papillary cells

[0067] This embodiment provides a method for reverse differentiation into dermal papilla-like cells (specifically human hair follicle dermal papilla-like cells), comprising the following steps:

[0068] 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%.

[0069] 2) On the second day (day 0), the original culture medium was replaced with a complete culture medium supplemented with 10 μM Selumetinib (dissolved in dimethyl sulfoxide solution), wherein the complete culture medium was RPMI 1640 culture medium supplemented with 10% FBS.

[0070] 3) Replace the complete culture medium containing Selumetinib every 2 days.

[0071] After an 8-day incubation period, human primary foreskin fibroblasts were reversely differentiated 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, and the DMSO-treated group was used as a control.

[0072] Morphological changes of primary human foreskin fibroblasts induced by Selumetinib Figure 3 As shown on the left, ALP staining of human primary foreskin fibroblasts induced by Selumetinib Figure 3 Shown on the right.

[0073] Figure 3 in conclusion: Figure 3 The results showed that Selumetinib induced changes in the cell morphology of human foreskin fibroblasts, with the cells becoming larger and rounder and showing obvious ALP-positive results.

[0074] Example 4: Detection of the expression of marker genes in human hair follicle dermal papilla-like cells obtained by the method of Example 3

[0075] 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:

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] Table 1

[0081]

[0082] like Figure 4 As shown, human hair follicle dermal papilla-like cells obtained from human primary foreskin fibroblasts induced by Selumetinib expressed the marker alkaline phosphatase and signature genes (ALPL, BMP4, LEF1, SOX2, THY1, VCAN and WIF1), and had a high degree of biomolecular expression similarity with primary human scalp hair follicle dermal papilla cells, which is beneficial to hair follicle regeneration.

[0083] The present application induces the reverse differentiation of human primary foreskin fibroblasts into dermal papilla-like cells by adding Selumetinib at an effective concentration of 1 to 10 μM. The obtained dermal papilla-like cells have the expression of the marker alkaline phosphatase and the expression of signature genes (ALPL, BMP4, LEF1, SOX2, THY1, VCAN and WIF1), have a high similarity in biomolecular expression with primary human scalp hair follicle dermal papilla cells, have hair induction ability, and are beneficial to hair follicle regeneration.

[0084] 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 into dermal papillary cells, characterized in that: The following steps are involved: Primary human foreskin fibroblasts were extracted, expanded and cultured, and induced to reversely differentiate into dermal papillary cells by adding culture medium containing selumetinib at an effective concentration of 1 to 10 μM.

2. The method for reverse differentiation into dermal papillary cells according to claim 1, characterized in that: The effective concentration of Selumetinib is 5 to 10 μM.

3. The method for reverse differentiation into dermal papillary cells according to claim 2, characterized in that: The effective concentration of Selumetinib is 10 μM.

4. The method for reverse differentiation into dermal papillary cells according to claim 1, characterized in that: The Selumetinib is dissolved in dimethyl sulfoxide solution.

5. The method for reverse differentiation into dermal papillary cells according to claim 1, characterized in that: The culture medium is RPMI 1640 culture medium containing FBS with a mass concentration of 5-15%.

6. The method for reverse differentiation into dermal papillary cells according to claim 1, characterized in that: The induction time is 6 to 8 days, and the culture medium is replaced every 2 days of induction.

7. The method for reverse differentiation into dermal papillary cells according to claim 1, characterized in that: The dermal papillary cells express marker alkaline phosphatase and characteristic genes.

8. The method for reverse differentiation into dermal papillary cells according to claim 7, characterized in that: The marker genes include at least one of ALPL, BMP4, LEF1, SOX2, THY1, VCAN and WIF1.

9. The method for reverse differentiation into dermal papillary cells according to claim 1, characterized in that: The dermal papilla-like cells are human hair follicle dermal papilla-like cells.

10. Use of the method for reverse differentiation into dermal papilla-like cells according to any one of claims 1 to 9 in the preparation of hair follicle regeneration products.

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