CN119506195A

CN119506195A - Method and application of reverse differentiation of human foreskin fibroblasts into human hair follicle dermal papilla-like cells

Info

Publication number: CN119506195A
Application number: CN202411641152.XA
Authority: CN
Inventors: 曾清岚; 王旭升
Original assignee: Sun Yat Sen University; Sun Yat Sen University Shenzhen Campus
Current assignee: Sun Yat Sen University; Sun Yat Sen University Shenzhen Campus
Priority date: 2024-11-18
Filing date: 2024-11-18
Publication date: 2025-02-25

Abstract

The present application relates to the field of biotechnology, and specifically discloses 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 joint induction. The expression of alkaline phosphatase, a marker of human hair follicle dermal papilla-like cells, and the expression of marker genes (SOX2, NOG, VCAN, LEF1, and α-SMA) of the present application human hair follicle dermal papilla-like cells are highly similar to those of primary human hair follicle dermal papilla cells, and can induce hair follicle regeneration in vivo, and is suitable for people with hair loss symptoms.

Description

Method for retrodifferentiating human foreskin fibroblast into human hair follicle dermal papilla-like cell and application

Technical Field

The application relates to the technical field of biology, in particular to a method for inversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells and application thereof.

Background

Alopecia is a common skin disorder associated with aging, illness, mental stress, medication and severe wound damage. Current treatments include drug therapy and autologous hair transplantation, but both are limited by adverse reactions or insufficient hair follicles.

With the development of regenerative medicine, cell-based hair follicle regeneration provides an alternative therapy. Human dermal papilla cells are a specialized population of dermal fibroblasts that originate at the bottom of the hair follicle. They can restart the epithelial-mesenchymal interactions with functional keratinocytes, essential for hair follicle morphogenesis, regulation of hair cycle and maintenance of follicular homeostasis. Thus, human hair follicle regeneration is largely dependent on culturing the expanded dermal papilla cells.

However, conventional techniques for in vitro culture of dermal papilla cells present significant challenges, including limited proliferation of dermal papilla cells and gradual decrease in hair induction potential of dermal papilla cells over a longer period of time. Thus, obtaining a large number of dermal papilla cells with hair induction capability is critical for efficient hair follicle regeneration.

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

However, in the field of hair follicle regeneration, there is still little research on the use of single small molecules to convert human fibroblasts into dermal papilla-like cells capable of inducing hair growth.

Disclosure of Invention

The application aims to overcome the defects of the prior art and provide a method for retrodifferentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the application is as follows:

The application provides a method for inversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells, which comprises the following steps:

Extracting, amplifying and culturing human primary prepuce fibroblasts, and adding a culture medium with the combined action concentration of peficitinib to 15 mu M and 0.1 to 1 mu MVITAMIN D2 for induction when the cell density of the human primary prepuce fibroblasts is 80 to 90 percent, wherein the human primary prepuce fibroblasts are converted into human hair follicle dermis papilla-like cells.

The human hair follicle dermal papilla-like cells have high similarity to the primary human hair follicle dermal papilla cells in terms of expression of biomolecules by adding peficitinib and Vitamin D2 for combined induction, the human hair follicle dermal papilla-like cells can be directly transformed into the human hair follicle dermal papilla-like cells, the marker alkaline phosphatase expression and the marker gene (SOX 2, NOG, VCAN, LEF1 and alpha-SMA) expression of the human hair follicle dermal papilla-like cells have high similarity to the primary human hair follicle dermal papilla-like cells, and hair follicle regeneration can be induced in vivo.

As a preferred embodiment of the method for inversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells, the culture medium is changed every 4 days when the acting concentration of peficitinib is 1-5 mu M and the acting concentration of Vitamin D2 is 0.1-0.5 mu M.

As a preferred embodiment of the method for inversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells, the culture medium is changed every 3 days when the acting concentration of Peficitinib is 5-10 mu M and the acting concentration of Vitamin D2 is 0.5-0.75 mu M.

As a preferred implementation mode of the method for inversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells, when the action concentration of Peficitinib is 10-15 mu M and the action concentration of Vitamin D2 is 0.75-1 mu M, the induction time is 4 days, and the culture medium is changed every 2 days of induction.

Preferably, peficitinib acts at a concentration of 10. Mu.M and Vitamin D2 acts at a concentration of 0.75. Mu.M.

By adopting Peficitinib and Vitamin D2 with the concentration, the application can convert human foreskin fibroblasts into human hair follicle dermal papilla-like cells, thereby realizing hair follicle regeneration and being suitable for people with alopecia symptoms.

As a preferred embodiment of the method for inversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to the application, the human primary foreskin fibroblasts are human foreskin fibroblasts extracted from foreskin tissues of abandoned humans.

In a preferred embodiment of the method for inversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to the present application, the medium is DMEM medium containing 10% -20% FBS by mass, preferably, the medium is DMEM medium containing 10% FBS by mass.

As a preferred embodiment of the method for retrodifferentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to the application, the peficitinib and Vitamin D2 are dissolved in a dimethyl sulfoxide solution.

As a preferred embodiment of the method for retrodifferentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to the application, the steps of extracting, expanding and culturing human primary foreskin fibroblasts include:

1) Flushing foreskin tissue with sterile phosphate buffer solution, shearing foreskin tissue, and digesting in neutral proteinase II solution to separate dermis tissue;

2) The dermal tissue was digested with collagenase type I, filtered, and the resulting cells were suspended in DMEM medium containing fetal bovine serum and penicillin/streptomycin and then incubated at 37 ℃ in 5% co ₂.

The application also provides application of the method for preparing the hair follicle regeneration product by the retrodifferentiated human foreskin fibroblasts and human hair follicle dermal papilla-like cells.

Follicular regeneration refers to a minimally invasive method of in vivo follicular regeneration based on cell transplantation, which involves the transplantation of stem cells or a mixture of epidermis and dermis components, and the injection of a mixture containing epidermal stem cells and dermis papilla-like cells into nude mice can induce new hair follicles having the correct tissue structure.

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

The application provides a method for inversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla cells and application thereof, wherein the human primary foreskin fibroblasts can be directly converted into human hair follicle dermal papilla cells by adding peficitinib and Vitamin D2 for induction, and the expression of the marker alkaline phosphatase and the expression of the marker genes (SOX 2, NOG, VCAN, LEF1 and alpha-SMA) of the human hair follicle dermal papilla cells have high similarity with the expression of the primary human hair follicle dermal papilla cells, can induce hair follicle regeneration in vivo, and are suitable for people with alopecia symptoms.

Drawings

FIG. 1 is a state diagram of the first day, third day, and immunofluorescence staining diagrams of human primary prepuce fibroblasts extracted from human primary prepuce fibroblasts obtained in example 1;

FIG. 2 is a schematic drawing of the extraction process and the adherence of dermal papilla cells of human hair follicle obtained in example 2 and an ALP staining chart;

FIG. 3 is a graph showing cell morphology change and ALP staining after the combined induction of peficitinib and Vitamin D2 in example 3;

FIG. 4 is a graph showing the results of detecting the expression of a marker gene in dermal papilla-like cells of human hair follicle obtained by the method of example 3;

FIG. 5 is a graph showing experimental results of human primary foreskin fibroblasts induced hair follicle regeneration after combined induction with peficitinib and Vitamin D2.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to the accompanying drawings and specific embodiments.

In the following examples and comparative examples, the experimental methods used were conventional methods unless otherwise specified, the materials, reagents and the like used were commercially available, and the component materials used in each parallel experiment were the same.

The experimental animals in the examples below were immunodeficient mice (BALB/C-nu/nu) and mice (C57 BL/6), purchased from the medical experimental animal center in Guangdong province.

Neonatal mouse fibroblasts and epidermal cells were isolated using 1-3 day old C57BL/6 mice. Human foreskin tissue samples were collected from outpatients receiving conventional circumcision, and scalp hair follicles were collected from the occipital area of patients receiving hair transplantation, with patient consent.

EDTA-free trypsin solution, DMEM medium, penicillin/streptomycin (P/S) and Fetal Bovine Serum (FBS) were purchased from Gibco, U.S.A., under the designations 25200072, C11995500BT, 15140122 and 10270106, respectively.

Type I collagenase and type II neutral protease were purchased from Sigma Co., USA under the product numbers C2674 and D4693, respectively, human dermal papilla cell commercial medium was purchased from Promocell Co., germany under the product number C26501, peficitinib was purchased from MCE Co., USA under the product number HY-L022M, vitamin D2 was purchased from MCE Co., USA under the product number HY-76542, quickBlock ^TM immunostaining blocking buffer and BCIP/NBT alkaline phosphatase chromogenic kit were purchased from Biyun Co., under the product numbers P0260 and C3206, respectively, reverse transcription kit and TBPremix Ex Taq ^TM II was purchased from Takara corporation of Japan under the accession numbers RR047A and RR820A, respectively, and matrigel was purchased from Corning corporation of America under the accession number 354234.

Example 1 extraction, expansion and culture of human Primary foreskin fibroblasts and immunofluorescent staining validation

The embodiment provides specific steps of extracting, amplifying and culturing human primary foreskin fibroblasts, comprising:

1) Human foreskin tissue samples were thoroughly rinsed 3-5 times with sterile Phosphate Buffered Saline (PBS). After irrigation, the foreskin tissue should be carefully cut into small pieces using sterile scissors. The pieces were then immersed in a solution containing 0.25% dispase II and stored overnight in a refrigerator at 4 ℃.

2) After one night of digestion, the dermis and epidermis were carefully separated from the human foreskin tissue sample using sterile forceps, ensuring that the dermis portion was preserved. The dermis section was then washed three times with PBS to remove any residual debris.

3) Dermal tissue was further minced using sterile scissors and treated with 0.25% collagenase type I and digested in an incubator at 37 ℃ until completed.

4) After digestion, the cell suspension was filtered through a cell filter and washed with PBS. The cells were then suspended in DMEM medium and 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (P/S) were added. The suspended cells were transferred to a petri dish and incubated in a 37 ℃ 5% CO ₂ incubator.

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

6) Cells were fixed in 4% paraformaldehyde for 20 min and then blocked with QuickBlock ^TM immunostaining blocking solution for 30 min. Subsequently, cells were incubated with primary antibodies overnight at 4 ℃.

7) Following incubation of the primary antibody, cells were allowed to interact with goat anti-mouse or anti-rabbit IgG H & L pre-adsorbed secondary antibodies for 1 hour at room temperature.

8) Nuclear DNA was counterstained blue with DAPI.

9) Finally, image analysis was performed using a confocal microscope, as shown in FIG. 1 (the left side of FIG. 1 shows a state diagram of the first and third day of extraction; and the right side of FIG. 1 shows immunofluorescence staining of cells).

FIG. 1 concludes that human primary foreskin fibroblasts are first isolated and expanded from foreskin tissue and identified in combination with morphological and immunofluorescent staining.

As shown in FIG. 1A, the isolated cells have a typical fibroblast morphology, the cells are elongated, spindle-shaped, have distinct cell boundaries, and continue to grow until the cells at the fully confluent, confluent region grow in parallel alignment adjacent to each other. Fibroblasts synthesize extracellular matrix and Collagen, with type I and type III Collagen (Collagen I and Collagen III) being highly expressed in skin fibroblasts. Furthermore, fibroblast-specific protein 1 (FSP-1) is a fibroblast cytoplasmic marker, also known as S100A4, a member of the intracellular protein S100 superfamily.

Immunofluorescent staining (B in FIG. 1) showed that almost all cells expressed S100A4, collagen I and Collagen III at higher levels, further verifying that the isolated cultured cells from foreskin according to the application were fibroblasts, which were used in subsequent experiments.

Example 2 extraction, expansion and culture of human follicular dermal papilla cells and ALP staining verification

The embodiment provides specific steps of extracting, amplifying and culturing human hair follicle dermal papilla cells, comprising:

1) First, the hair follicle is subjected to a stringent washing procedure, which involves three consecutive washes with PBS buffer containing penicillin/streptomycin (P/S) at a concentration of 2%. Subsequently, surrounding skin and adipose tissue surrounding the hair follicle are carefully removed using a dissecting microscope.

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

3) Immediately after complete digestion of the tissue surrounding the dermal papilla and exposure of the dermal papilla itself, DMEM complete medium supplemented with 10% FBS and 1% penicillin/streptomycin (P/S) was added to stop the enzyme activity. Care was taken to collect the completely detached dermal papilla.

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

5) Within about one week, cells will migrate from the dermal papilla and adhere to the surface of the culture plate.

6) Until the cells reach confluence for passaging.

7) Cells were fixed with 4% paraformaldehyde at room temperature for 20min and washed three more times with PBS. Subsequently, the immobilized cells were stained using the BCIP/NBT alkaline phosphatase chromogenic kit according to the manufacturer's instructions.

The results are shown in FIG. 2, wherein the left side of FIG. 2 is a schematic diagram showing the extraction process and the adherence of human hair follicle dermal papilla cells, and FIG. 2 is a diagram showing the results of alkaline phosphatase staining of human hair follicle dermal papilla cells.

Fig. 2 conclusion:

The hair follicle papilla DP was isolated using enzymatic digestion combined with microdissection. As shown in FIG. 2, the treated single hair follicle ends carry intact flame-like DP structures, which are only encapsulated by the basement membrane, and collagenase can be used to digest collagen fibers, adipose tissue, etc. surrounding the DP, thereby freeing the DP. The free DP was excised and completely isolated, and the hair shaft and large undigested tissue were picked under a microscope. At this time, a large amount of digested collagen components and cells are mixed around the digested DP, the DP is precipitated by low-speed centrifugation by utilizing the characteristic that the DP is in a lump, and other fat, collagen and cell components are suspended in the supernatant to be discarded, so that purer DP can be obtained after repeated times. On the 3 rd day of culture, DP adheres to the wall, cells in polygon or short fusiform form begin to migrate around, on the 6 th day of culture, cells grow radially around DP, and on the 18 th day, DP cells grow in multiple layers after fusion. ALP staining was performed on the extracted DP cells. As a result, as shown in FIG. 2, the extracted DP cell center showed strong ALP activity, while the peripheral migratory cells gradually decreased.

Example 3A method of retrodifferentiating human foreskin fibroblasts into human dermal papilla-like cells of hair follicles

The embodiment provides a method for inversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells, which comprises the following steps:

1) The human primary foreskin fibroblasts obtained in example 1 were seeded into 10cm cell culture dishes at 80% density.

2) The next day (day 0), the original medium was replaced with complete medium supplemented with 10 μ M peficitinib and 0.75 μ MVITAMIN D2 (dissolved in dimethyl sulfoxide solution), and the complete medium was DMEM medium supplemented with 10% FBS.

3) The medium containing the small molecules was changed every 4 days.

After an incubation period of 8 days, human primary prepuce fibroblasts were transformed into human hair follicle dermal papilla-like cells, the fibroblasts were fixed with 4% paraformaldehyde at room temperature for 20 minutes, and washed three times with PBS. Subsequently, the immobilized cells were stained using the BCIP/NBT alkaline phosphatase chromogenic kit according to the manufacturer's instructions.

The morphological changes of human primary foreskin fibroblasts induced by peficitinib and Vitamin D2 are shown on the left side of FIG. 3, and the ALP staining pattern of human primary foreskin fibroblasts induced by peficitinib is shown on the right side of FIG. 3.

FIG. 3 shows that FIG. 3 shows peficitinib and Vitamin D2 in combination induce changes in the cell morphology of human foreskin fibroblasts, cells become enlarged and rounded, and show significant ALP positive results.

Example 4 detection of the expression of the marker Gene of human dermal papilla-like cells of hair follicle obtained by the method of example 3

An experiment for detecting the expression of a marker gene of human hair follicle dermal papilla-like cells obtained by the method of example 3, specifically comprising the steps of:

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

2) Subsequently, 1. Mu.g of RNA was reverse transcribed into cDNA using PRIMESCRIPT ^TM RT kit with GDNA ERASER. The reverse transcription reaction was performed according to the manufacturer's protocol.

3) Then use TBThe obtained cDNA was subjected to qRT-PCR according to the manufacturer's instructions by Premix Ex Taq ^TM II, and the primer sequences are shown in Table 1 below. The relative fold change is calculated by adopting a2 (-delta CT) method, and each experiment is carried out three times, so that three biological repetitions are realized.

Statistical tests were performed using GRAPHPAD PRISM software (GraphPad 9.0), unpaired t-test was performed for differences between the two groups, all data were expressed as mean ± SD of multiple samples (n.gtoreq.3), and significance level P <0.05 was statistically significant.

TABLE 1

As shown in FIG. 4, the expression of the marker gene of human hair follicle dermal papilla-like cells obtained by the combined induction of peficitinib and Vitamin D2 with human primary foreskin fibroblasts was highly consistent with that of the human hair follicle dermal papilla cells obtained in example 2.

Example 5 experiment to induce follicular regeneration with human Primary foreskin fibroblasts induced by a combination of peficitinib and Vitamin D2

An experiment for inducing hair follicle regeneration by human primary prepuce fibroblasts in the method of example 3 (after combined induction with peficitinib and Vitamin D2) specifically comprises the following steps:

1. in vivo hair follicle reconstruction was performed using 5-6 week old BALB/c-nu/nu nude mice.

2. A symmetrical full-thickness skin wound was made in the back area using a skin biopsy punch with a diameter of 4 mm. 100 ten thousand human primary foreskin fibroblasts induced by the combination of peficitinib and Vitamin D2 were mixed with 100 ten thousand neonatal mouse keratinocytes and added to 30 μl matrigel to obtain a mixture.

3. The mixture was then incubated in a 37 ℃ incubator for 30 minutes until the matrigel solidified. Subsequently, the coagulated cell-matrigel mixture was transplanted into a skin incision and the wound was covered with a 3M transparent dressing and a self-adhesive elastic bandage to prevent infection.

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

As shown in fig. 5.

FIG. 5 shows that neither the DMSO-treated blank nor the experimental group (Peficitinib and Vitamin D2) induced in combination was able to observe significant hair growth with the naked eye, as shown in FIG. 5. Further, by observation with a dissecting scope and an optical microscope, it was found that the control group had no hair formation and that the experimental group induced significant hair growth was observed after the experimental group, and these formed hairs could not be visually observed under the skin of the back of the nude mice, because the small number of hairs could not be highlighted in the skin of the nude mice. The results indicate that human foreskin fibroblasts induced by this experimental group have the ability to induce hair follicle formation.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted equally without departing from the spirit 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, comprising the following steps:

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 as claimed in claim 1, characterized in that when the effective concentration of peficitinib is 1 to 5 μM and the effective concentration of Vitamin D2 is 0.1 to 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 tissue.

6 . The method for reversely differentiating human foreskin fibroblasts into human hair follicle dermal papilla-like cells according to claim 1 , wherein the culture medium is a DMEM culture medium containing FBS at 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 a 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 comprise:

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.