CN115418341A - Method for transdifferentiation of fibroblasts into hair papilla cells and application thereof - Google Patents
Method for transdifferentiation of fibroblasts into hair papilla cells and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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Abstract
The invention provides a method for transdifferentiation of fibroblasts to hair papilla cells and application thereof, and relates to the technical field of biotechnology. Wherein the small molecule drug refers to at least one of Ribociclib hydrochloride, selumetinib and Tucidinostat. The method can obtain a large amount of hair papilla cells in vitro, and solves the problem of the prior art that functional hair papilla cells with hair induction capability are obtained by taking non-hair source cells as initial cells.
Description
Technical Field
The invention relates to the technical field of biotechnology, in particular to a method for transdifferentiation of fibroblasts into papilla pili cells and application thereof.
Background
Alopecia is a common disease caused by various factors such as illness, age, and mental stress. Is a sub-health problem affecting billions of men and women worldwide. Hair transplantation, which is the most effective means for treating alopecia at present, is to repair hair follicles in unaffected parts of the scalp by extraction and re-transplantation into an area where hair is lost. However, the cost and labor required to extract and implant a unit of hair follicles, and the limitation in the number of hair follicles available for transplantation, have prompted the search for new alternative therapies.
With advances in tissue engineering and regenerative medicine, the harvesting of regenerative hair follicles via cell-based methods provides an alternative treatment for hair loss. The hair follicle is a major cutaneous appendage derived from the ectoderm, and morphogenesis and regeneration of the hair follicle has been shown to depend on close cooperation of the epithelium (epidermal stem cells) and the hair-induced mesenchymal components (hair papilla cells). The hair papilla, which is the main mesenchymal dermal component in the hair follicle, is an instructive niche for embryonic development and periodic cycle of the hair follicle and plays an indispensable role in the reconstruction of the hair follicle. However, the specific and necessary micro-environment of hair follicles, the lack of efficient isolation methods and the loss of in vitro intrinsic properties make it a huge challenge to obtain sufficient human hair papilla cells with hair-inducing ability by in vitro expansion. Therefore, in the case of donor hair shortage, it is of great significance to select non-hair-derived cells to obtain functional hair papilla cells having hair-inducing ability.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the present invention is to provide a method for transdifferentiation of fibroblasts into hair papilla cells, which alleviates the problem of the prior art that a functional hair papilla cell having hair-inducing ability is not obtained by using a non-hair-derived cell as an initial cell.
The second object of the present invention is to provide a medium capable of inducing the transdifferentiation of fibroblasts into papilla cells.
The third object of the present invention is to provide the method for transdifferentiation of fibroblasts into papilla cells, or the use of the induction medium.
In order to solve the technical problems, the invention adopts the following technical scheme:
according to one aspect of the present invention, there is provided a method for transdifferentiation of fibroblasts into papilla pili cells, comprising inducing the fibroblasts using a small molecule drug to transdifferentiate the fibroblasts into papilla pili cells;
the small molecule drug is selected from at least one of Ribociclib hydrochloride, selumetinib and Tucidinostat.
Preferably, the method comprises culturing fibroblasts in an induction medium containing at least one of Ribociclib hydrochloride, selumitinib and Tucidinostat;
preferably, the concentration of Ribociclib hydrochloride in the induction medium is 2-10 μ M, preferably 10 μ M;
preferably, the concentration of Selumetinib in the induction medium is 2-10. Mu.M, preferably 10. Mu.M;
preferably, the concentration of Tucidinostat in the induction medium is 2-10 μ M, preferably 10 μ M;
preferably, the induction medium is a basal medium containing the small molecule drug, and the basal medium is high-glucose DMEM containing serum.
Preferably, the fibroblast is of human origin;
preferably, the fibroblasts are derived from skin tissue;
preferably, the fibroblasts are derived from scalp tissue or foreskin tissue.
Preferably, the fibroblasts are primary fibroblasts;
preferably, the culture medium of the primary fibroblasts also contains antibiotics;
preferably, the antibiotic comprises streptomycin and/or penicillin.
Preferably, the method for isolating primary fibroblasts comprises: the washed skin tissue was digested with dispase and then the epidermis was separated, and the epidermis was digested with collagenase and then fibroblasts were separated.
Preferably, the method further comprises: and (3) adopting alkaline phosphatase staining and detecting the expression condition of the related papilla specific marker gene to judge whether the fibroblast is successfully transdifferentiated into papilla cells.
According to another aspect of the present invention, the present invention also provides an induction medium for inducing the transdifferentiation of fibroblasts into hair papilla cells, the induction medium containing a small molecule drug selected from at least one of Ribociclib hydrochloride, selumetinib, and Tucidinostat.
Preferably, the induction medium contains Ribociclib hydrochloride with a working concentration of 2-10. Mu.M; the working concentration of Ribociclib hydrochloride is preferably 10 μ M;
preferably, the induction medium contains Selumetinib at a working concentration of 2-10. Mu.M; the working concentration of Selumetinib is preferably 10. Mu.M;
preferably, the induction medium contains Tucidinostat at a working concentration of 2-10 μ M; the working concentration of Tucidinostat is preferably 10 μ M;
preferably, the induction medium is a basal medium containing the small molecule drug, and the basal medium is high-glucose DMEM containing serum.
According to another aspect of the invention, the invention also provides a method for transdifferentiation of the above-described fibroblasts into hair papilla cells, or use of the above-described induction medium for hair follicle regeneration for non-diagnostic and therapeutic purposes.
According to another aspect of the present invention, the present invention also provides a method for transdifferentiation of the above fibroblast into hair papilla cells, or use of the above induction medium for preparing a product for hair follicle regeneration.
According to another aspect of the present invention, there is also provided a product for hair follicle regeneration, which comprises hair papilla cells prepared by the above-described method for transdifferentiation of fibroblasts into hair papilla cells, or the above-described induction medium.
Compared with the prior art, the invention has the following beneficial effects:
the method for transdifferentiation of fibroblasts into hair papilla cells comprises inducing fibroblasts using at least one of Ribociclib hydrochloride, selumetinib, and Tucidinostat, and transdifferentiating the fibroblasts into hair papilla cells.
The method provided by the invention has the advantages that the transdifferentiated primary cells are fibroblasts, the fibroblasts have high availability and can be easily separated from skin tissues, and in addition, the culture, propagation and cryopreservation characteristics of the fibroblasts also have culture advantages in terms of nutrient requirements and culture viability.
The effective component for inducing the fibroblast to transdifferentiate into hair papilla cells adopts at least one of small molecular drugs Ribociclib hydrochloride, selumetinib and Tucidinostat. The small molecules have the advantages of clear chemical components, high purity and small batch-to-batch difference, and also have the characteristics of rapid action and dose-dependent biological activity. The method for inducing the fate transition differentiation of the fibroblast based on the micromolecule can obtain a large number of hair papilla cells in vitro, is a method for obtaining functional hair papilla cells with hair induction capability by using non-hair source cells, and has important application value for replacing a hair follicle regeneration method based on natural hair papilla cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1A is a diagram of the morphology of primary human foreskin fibroblasts extracted in example 1;
FIG. 1B is a graph of the fluorescent staining identification result of primary human foreskin fibroblasts extracted in example 1;
FIG. 2 is a graph showing the results of morphological changes of cells induced by the small molecule drug Ribociclib hydrochloride in example 2;
FIG. 3 is a graph showing the results of morphological changes in cells induced by the small molecule drug Selumetinib in example 2;
FIG. 4 is a graph showing the results of morphological changes in cells induced by small molecule drug Tucidinostat in example 2;
FIG. 5A shows ALP staining results of small molecule drug Ribociclib hydrochloride-induced cells in effect example 1;
FIG. 5B shows ALP staining results of cells induced by small molecule drug Selumetinib in effect example 1;
FIG. 5C shows ALP staining results of small molecule drug Tucidinostat-induced cells in Effect example 1;
FIGS. 6A to 6E show the expression of marker genes ALPL, VCAN, BMP4, LEF1 and THY1 in cell hair cream induced by small molecule drug Ribociclib hydrochloride in effect example 2;
FIGS. 7A to 7F show the expression of marker genes ALPL, SOX2, VCAN, BMP4, LEF1 and WIF1 in hair cream cells induced by small molecule Selumetinib in effect example 3;
FIGS. 8A to 8G are graphs showing the expression of marker genes ALPL, SOX2, VCAN, NOG, BMP4, LEF1 and WIF1 in cell hair cream induced by small molecule drugs Tucidinostat in Effect example 4.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to one aspect of the present invention, there is provided a method for transdifferentiation of fibroblasts into hair papilla cells, comprising inducing the fibroblasts using a small molecule drug, and transdifferentiating the fibroblasts into hair papilla cells; the small molecule drug is selected from at least one of Ribociclib hydrochloride, selumetinib and Tucidinostat. The method for transdifferentiation of fibroblasts into hair papilla cells according to the present invention is not intended for diagnosis or treatment.
The invention takes fibroblasts as the initial cells for transdifferentiation. Fibroblasts are the main cells constituting connective tissues and are present in most tissues and organs of the human body. In vivo, the ability of fibroblasts to produce and secrete ECM and to communicate with other cells, one of the most abundant cell types in connective tissue, makes them a central component of tissue biology and responsible for tissue homeostasis under normal physiological conditions. When tissue is injured, fibroblasts play an important role in wound healing and inflammation by interacting with immunocompetent cells (e.g., T cells) and regulating neuropeptides. In vitro, human fibroblast cultures play an important role in drug testing or toxicology screening, providing a favorable model for inflammation and disease research; and even to support the growth of human ESCs and other cell types. In recent years, one of the main reasons for the rapid development of regenerative medicine is the progress of cell retrodifferentiation and transdifferentiation, and fibroblasts with various biological functions, as the most commonly used seed cells, show wide application potential in regenerative medicine. The high availability of fibroblasts, ease of isolation from skin tissue, and the culturing, propagation and cryopreservation characteristics of fibroblasts also provide culture advantages in terms of nutritional requirements and culture viability.
The substance for transdifferentiating fibroblast into hair papilla cell is at least one of small molecule drugs Ribociclib hydrochloride, selumetinib and Tucidinostat.
The micromolecule has the characteristics of clear chemical components, high purity, small difference among batches, rapidness and dosage-dependent biological activity; the appropriately targeted delivery and controlled release of small molecules can modulate their in vivo effects spatially and temporally. Therefore, the small molecules have wide application in stem cell biology with unique advantages and have great application potential in regenerative medicine. Chemical methods using small molecules have achieved exciting results in the induction and differentiation of pluripotent stem cells, and in lineage reprogramming of somatic cells. The induction of transdifferentiation of human fibroblasts into neurons and cardiomyocytes has been successfully achieved by small molecule compounds.
The screening of the invention discovers that Ribociclib hydrochloride, selumetinib and Tucidinostat can induce the fibroblast to transdifferentiate to hair papilla cells. Ribociclib hydrochloride (Ribociclib hydrochloride, LEE011 hydrochloride, CAS No.: 1211443-80-9) is a highly specific CDK4/6 inhibitor; selumetinib (Semetinib, AZD6244, CAS No: 606143-52-6) is an inhibitor of mitogen-activated protein kinases 1 and 2 (MEK 1/2), and is suitable for patients 2 years old and older with neurofibromatosis type 1 (NF 1) and inoperable pelvic neurofibromas; tucidinostat (Sida benamine, HBI-8000, CAS No.: 1616493-44-7) is a potent, orally available inhibitor of HDAC class I HDAC1/2/3 and IIb HDAC 10.
Fibroblast cells are induced by Ribociclib hydrochloride, and the trans-differentiated cells express hair papilla marker genes ALPL, VCAN, BMP4, LEF1 and THY1.
Fibroblasts were induced by Selumetinib, and the trans-differentiated cells expressed the hair papilla marker genes ALPL, SOX2, VCAN, BMP4, LEF1 and WIF1.
Fibroblasts were induced by Tucidinostat, and the trans-differentiated cells expressed the hair papilla marker genes ALPL, SOX2, VCAN, NOG, BMP4, LEF1 and WIF1.
The fibroblasts used as the starting material for transdifferentiation are preferably primary fibroblasts, which are isolated directly from human specimens, retain the morphological and functional characteristics of the tissue from which they originate, and do not undergo any genetic manipulation, which helps to solve this limitation. Fibroblasts can be isolated from various tissues, and the invention does not limit the tissue from which the fibroblasts are derived, but human skin tissue is more accessible, less invasive and less ethical in comparison with other sources.
Fibroblasts isolated from skin tissue show a wide range of potential applications, providing abundant materials for different studies. Circumcision is one of the most surgical procedures performed worldwide, and most of the removed foreskin is usually discarded as biological waste. Foreskin contains many cells, but fibroblasts are the most abundant cells in its dermis. Therefore, foreskin is an important source for isolating human primary fibroblasts, and it is preferable to prepare hair papilla cells using foreskin fibroblasts as the starting cells for transdifferentiation.
The isolation method of primary fibroblasts preferably comprises: the cleaned skin tissue is digested with dispase to separate the true epidermis, and the true epidermis is digested with collagenase to separate the fibroblasts. Please particularly preferably wash the skin tissue with a buffer containing antibiotics; isolating fibroblasts preferably comprises collecting cells by filtration using a 70 μm cell sieve.
Preferably 0.2 to 0.5% w/v of Dispase II, preferably digestion conditions of Dispase of from 2 to 6 ℃ for from 12 to 24h; the working concentration of the dispase may be, for example, but not limited to, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5% w/v; the digestion temperature may be, for example, but not limited to, 2, 3, 4, 5, or 6 ℃, and the digestion time may be, for example, but not limited to, 16, 17, or 18 hours. Preferably the dispersive enzymatic digestion conditions are: 0.25% w/v Dispase II was digested in a refrigerator at 4 ℃ overnight for 16-18 h.
Collagenase is preferably 0.2 to 0.5% w/v collagenase type I, and the digestion conditions are preferably 35 to 38 ℃ for 2 to 4 hours. The working collagenase concentration may be, for example, but not limited to, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5% w/v; digestion temperatures may be, for example, but are not limited to, 35, 36, 37, or 38 ℃; the digestion time may be, for example, but not limited to, 2, 3, or 4 hours. Preferably the collagenase digestion conditions are: 0.25% w/v collagenase type I digested at 37 ℃ for 2-4 h.
The medium used for culturing the fibroblasts is optionally a common cell culture medium acceptable in the art, and the present invention is not limited thereto, and in some optional embodiments, serum-containing high-glucose DMEM is used, for example, 10% by volume of fetal bovine serum-containing high-glucose DMEM. When fibroblasts are derived from human waste skin tissue, belonging to primary cells, antibiotics acceptable in the art, such as streptomycin and/or penicillin, may be added before the culture.
In the step of inducing the transdifferentiation of fibroblasts into hair papilla cells using the small molecule drug, it is preferable to culture the fibroblasts in an induction medium containing the small molecule drug to transdifferentiate the fibroblasts into hair papilla cells.
The concentration of Ribociclib hydrochloride in the induction medium is preferably 2 to 10. Mu.M, and may be, for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Mu.M, preferably 10. Mu.M.
The concentration of Selumetinib in the induction medium is preferably 2 to 10. Mu.M, and may be, for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Mu.M, preferably 10. Mu.M.
The concentration of Tucidinostat in the induction medium is preferably 2 to 10. Mu.M, and may be, for example but not limited to, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Mu.M, preferably 10. Mu.M.
The concentration of the small molecular drug is verified by experiments, the transdifferentiation effect is poor due to too low concentration, the severe cytotoxicity is caused due to too high concentration, and the experiment shows that the induced transdifferentiation effect is optimal when the concentration is 10 mu M.
In addition to the small molecule drug, the induction medium may also contain conventional basal medium for culturing cells, which is acceptable in the art, but the invention is not limited thereto, and in some alternative embodiments, serum-containing high-glucose DMEM is used, for example, 10% by volume fetal bovine serum-containing high-glucose DMEM. The preferred mode of induction culture is as follows:
(a) Inoculating fibroblast cells onto a cell culture dish, culturing in a basal medium, incubating the cells at 37 deg.C with 5% CO 2 Culturing in an incubator;
(b) And after 24h, abandoning the basic culture medium, replacing the induction culture medium, continuously carrying out induction culture for 6-10 days, preferably 8 days, replacing a new induction culture medium every two days, and obtaining the papilla cells which are transformed and differentiated from the fibroblasts.
In some preferred embodiments, the preparation method further comprises using alkaline phosphatase staining and detecting the expression of the related papilla-specific marker gene to determine whether the fibroblast cells are successfully transdifferentiated into papilla cells.
The use of alkaline phosphatase staining is due to the fact that hair follicles are highly regenerative and known to express ALP, and previous reports indicate that hair papillae exhibit a high level of ALP activity throughout the hair follicle cycle, and the high expression of ALP in hair papilla cells has been used as a simple and reliable method of distinguishing hair papillae from other hair follicle structures throughout the hair follicle cycle. McElwee et al found that low-generation hair papilla cells highly expressing ALP activity induced hair follicle formation when transplanted into recipient ear skin wounds, while high-generation hair papilla cells losing ALP activity failed to produce hair follicles. It can be seen that ALP activity is not only a specific hair papilla cell marker in hair follicles, but also a key marker reflecting their hair-inducing ability. The basic characteristics of cells obtained after transdifferentiation can be identified by detecting the expression condition of the related papilla specific marker genes, and the qRT-PCR detection can be carried out by adopting a mode of extracting total RNA.
According to another aspect of the present invention, there is also provided an induction medium for inducing differentiation of fibroblasts into hair papilla cells, the induction medium containing a small molecule drug selected from at least one of Ribociclib hydrochloride, selumetinib and Tucidinostat.
In the induction medium, the working concentration of Ribociclib hydrochloride is preferably 2 to 10 μ M, and may be, for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μ M, and preferably 10 μ M.
In the induction medium, the working concentration of Selumetinib is preferably 2 to 10. Mu.M, and may be, for example, but not limited to, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Mu.M, and is preferably 10. Mu.M.
The working concentration of Tucidinostat in the induction medium is preferably 2-10. Mu.M, and may be, for example but not limited to, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Mu.M, preferably 10. Mu.M.
Besides the small molecule drugs, the induction culture medium also contains substances which are accepted by the field and are used for culturing cells in a conventional way, such as basic substances for supplying nutrition to the cells and promoting the reproduction and proliferation of the cells and substances for maintaining the growth environment of the cells; specific examples may be, for example, but are not limited to, one or more of sugars, amino acids, buffer substances, antibiotics, growth factors, vitamins, inorganic ions, and serum. The induction culture medium provided by the invention can induce the transdifferentiation of fibroblasts into hair papilla cells.
Based on the beneficial effects of the method for transdifferentiating fibroblasts into hair papilla cells provided by the invention, the hair papilla cells and the induction medium obtained based on the inventive concept, the invention also provides the method for transdifferentiating the fibroblasts into the hair papilla cells, or the application of the induction medium in hair follicle regeneration for non-diagnosis and treatment purposes, or the application in preparing products for hair follicle regeneration. The hair papilla cells obtained by the in vitro transdifferentiation of the invention can obtain a large amount of cells for hair follicle regeneration, and provide a large amount of raw materials for the research of hair follicle regeneration; the cost of preparing the product for hair follicle regeneration is reduced.
According to another aspect of the present invention, there is also provided a product for hair follicle regeneration, which comprises hair papilla cells prepared by the above-described method for transdifferentiation of fibroblasts into hair papilla cells, or the above-described induction medium.
The technical solutions and advantageous effects of the present invention will be further described below with reference to preferred embodiments.
In the following examples, the basal medium used was high-glucose DMEM containing 10% fetal bovine serum, unless otherwise specified; the induction culture medium is prepared by adding 10 μ M small molecule drug Ribociclib hydrochloride, selumetinib or Tucidinostat on the basis of basal culture medium.
Example 1
Extracting and identifying human foreskin fibroblasts:
fresh human foreskin tissue is taken from a patient who performs a conventional circumcision operation, and the foreskin tissue cut off in the operation is put into a centrifuge tube containing normal saline or PBSPlaced in ice and brought back to the laboratory and immediately started processing. After the foreskin tissue is repeatedly washed for 3-5 times by sterile PBS containing 2% double antibodies, redundant adipose tissues are removed as much as possible under a body type dissecting mirror. Cutting foreskin tissue into small pieces with sterile scissors, adding 0.25% of Dispase II (Dispase II) to digest in a refrigerator at 4 ℃ overnight for 16-18 h, separating true epidermis, cutting dermis part into small pieces with sterile scissors, and digesting with 0.25% of I-type collagenase at 37 ℃ for 2-4 h. The cell-containing digest was filtered through a 70 μm cell sieve, centrifuged, washed 2-3 times with PBS, then the cells were resuspended using complete medium (DMEM +10% FBS +1% double antibody), the cells were transferred to a petri dish, placed at 37 ℃ 5% CO 2 Culturing in an incubator, and changing the culture medium every 3 days until the cells grow full for passage. And (3) inoculating the primary human foreskin fibroblasts separated and extracted into a cell slide, and identifying the cells by immunofluorescence staining.
The results are shown in FIG. 1A, and the isolated cells have a typical fibroblast morphology: the cells are slender, spindle-shaped and have obvious cell boundaries; growth was continued until complete confluence, with cells in the confluence region growing close to each other in a parallel arrangement. Fibroblasts synthesize extracellular matrix and Collagen, with type I and type III Collagen (Collagen I and Collagen III) highly expressed in skin fibroblasts. In addition, fibroblast specific protein 1 (FSP-1) is a fibroblast marker, also known as S100A4, a member of the S100 superfamily of intracellular proteins. Immunofluorescent staining (fig. 1B) showed that almost all cells expressed S100A4, collagen I and Collagen III at high levels, further verifying that the cells cultured separately from foreskin were fibroblasts.
Example 2
The primary fibroblasts isolated and extracted in example 1 were inoculated into a culture dish, and the induction medium containing the small molecule drug Ribociclib hydrochloride was replaced for 24h to culture for 8 days, and the induction medium was replaced every two days. The cell morphology after induction is changed to some extent, and the cell morphology after induction is shown in FIG. 2. In this example, the induction medium was a basal medium containing 10. Mu.M Ribociclib hydrochloride, and the basal medium was high-glucose DMEM containing 10% fetal bovine serum.
Example 3
The primary fibroblasts isolated and extracted in example 1 were inoculated in a culture dish, and cultured for 24h while replacing the induction medium containing the small molecule drug Selumetinib for 8 days, and the induction medium was replaced every two days. After induction, the cell morphology has changed to some extent, and the cell morphology after induction is shown in FIG. 3. In this example, the induction medium was a basal medium containing 10. Mu.M Selumetinib, and the basal medium was high-glucose DMEM containing 10% fetal bovine serum.
Example 4
The primary fibroblasts separated and extracted in example 1 were inoculated into a culture dish, and the induction medium containing the small molecule drug Tucidinostat was replaced for 24h for 8 days, and the induction medium was replaced every two days. The cell morphology after induction is changed to some extent, and the cell morphology after induction is shown in FIG. 4. In this example, the induction medium was a basal medium containing 10. Mu.M of Tucidinostat, and the basal medium was high-glucose DMEM containing 10% fetal bovine serum.
Effect example 1
ALP staining was performed on cells cultured for 8 days in the induction medium containing the small molecule drugs Ribociclib hydrochloride, selumetinib and Tucidinostat in examples 2 to 4, respectively, and as a result, the cells induced with the small molecule drugs Ribociclib hydrochloride, selumetinib and Tucidinostat showed strong ALP activity, as shown in FIGS. 5A to 5C.
Effect example 2
The expression of hair papilla marker genes ALPL, VCAN, BMP4, LEF1, and THY1 was detected by qRT-PCR extraction of RNA of the cells induced by Ribociclib hydrochloride in example 2, and as a result, the expression of the specific marker genes of hair papilla cells was significantly improved as shown in fig. 6A to 6E. It can be seen that the cells after induction have the molecular characteristics of dermal papilla cells.
Effect example 3
Extraction example 3 qRT-PCR of RNA from cells induced by Selumetinib was performed to detect the expression of the papilla marker genes ALPL, SOX2, VCAN, BMP4, LEF1 and WIF1, and as a result, the expression of the papilla marker genes specific to the papilla cells was significantly improved as shown in FIGS. 7A to 7F. It can be seen that the cells after induction have the molecular characteristics of dermal papilla cells.
Effect example 4
Extraction example 4 qRT-PCR of RNA from cells induced by Tucidinostat was used to detect the expression of the hair papilla marker genes ALPL, SOX2, VCAN, NOG, BMP4, LEF1 and WIF1, and the results are shown in FIGS. 8A to 8G, indicating that the expression of the specific marker gene of hair papilla cells was significantly improved. It can be seen that the cells after induction have the molecular characteristics of dermal papilla cells.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for transdifferentiation of fibroblasts into hair papilla cells, which is characterized by comprising inducing the fibroblasts by using a small molecule drug to transdifferentiate the fibroblasts into the hair papilla cells;
the small molecule drug is selected from at least one of Ribociclib hydrochloride, selumetinib and Tucidinostat.
2. The method according to claim 1, comprising culturing fibroblasts in an induction medium comprising at least one of Ribociclib hydrochloride, selumetinib and Tucidinostat;
preferably, the concentration of Ribociclib hydrochloride in the induction medium is 2-10 μ M, preferably 10 μ M;
preferably, the concentration of Selumetinib in the induction medium is 2-10. Mu.M, preferably 10. Mu.M;
preferably, the concentration of Tucidinostat in the induction medium is 2-10 μ M, preferably 10 μ M;
preferably, the induction medium is a basic medium containing the small molecule drug, and the basic medium is high-glucose DMEM containing serum.
3. The method according to claim 2, wherein the fibroblast cells are cultured using the induction medium for 6 to 10 days;
preferably, the fibroblasts are cultured using the induction medium for 8 days;
preferably, the medium is renewed every second day;
preferably, the induction medium is replaced 24h after fibroblast seeding.
4. The method of claim 1, wherein the fibroblast cells are of human origin;
preferably, the fibroblasts are derived from skin tissue;
preferably, the fibroblasts are derived from scalp tissue or foreskin tissue;
preferably, the fibroblasts are primary fibroblasts;
preferably, the culture medium of the primary fibroblasts is the basal medium described in claim 2;
preferably, the culture medium of the primary fibroblasts also contains antibiotics;
preferably, the antibiotic comprises streptomycin and/or penicillin.
5. The method of claim 4, wherein the primary fibroblast cell isolation method comprises: digesting the cleaned skin tissue by using dispase, separating true epidermis, digesting the true epidermis by using collagenase, and separating fibroblast;
preferably, the W/v Dispase II is digested at 2-6 ℃ for 16-18 h using 0.2-0.5% w/v;
preferably, the collagenase type I is digested at 35-38 ℃ for 2-4 hours at a rate of 0.2-0.5% w/v;
preferably, the isolating fibroblasts comprises collecting the cells using a 70 μm cell sieve filtration;
preferably, the skin tissue is washed with a buffer containing an antibiotic.
6. The method according to any one of claims 1-5, further comprising: and (3) adopting alkaline phosphatase staining and detecting the expression condition of the related papilla specific marker gene to judge whether the fibroblast is successfully transdifferentiated into papilla cells.
7. An induction medium for inducing the transdifferentiation of fibroblasts into hair papilla cells, wherein the induction medium contains a small molecule drug selected from at least one of Ribociclib hydrochloride, selumetinib, and Tucidinostat;
preferably, the induction medium contains Ribociclib hydrochloride with a working concentration of 2-10 μ M; the working concentration of Ribociclib hydrochloride is preferably 10 μ M;
preferably, the induction medium contains Selumetinib at a working concentration of 2-10. Mu.M; the working concentration of Selumetinib is preferably 10. Mu.M;
preferably, the induction medium contains Tucidinostat at a working concentration of 2-10 μ M; the working concentration of Tucidinostat is preferably 10 μ M;
preferably, the induction medium is a basal medium containing the small molecule drug, and the basal medium is high-glucose DMEM containing serum.
8. A method for transdifferentiation of fibroblasts into hair papilla cells according to any one of claims 1 to 6, or use of the induction medium according to claim 7 for hair follicle regeneration for non-diagnostic and therapeutic purposes.
9. Use of the method for transdifferentiation of fibroblasts into hair papilla cells according to any one of claims 1 to 6 or the induction medium according to claim 7 for the preparation of a product for hair follicle regeneration.
10. A product for hair follicle regeneration, which comprises hair papilla cells prepared by the method for transdifferentiation of fibroblasts into hair papilla cells according to any one of claims 1 to 6, or the induction medium according to claim 7.
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| KR20190009716A (en) * | 2017-07-19 | 2019-01-29 | 서울대학교산학협력단 | Method for differentiation of dermal papilla precursor cells from human induced pluripotent stem cells and uses thereof |
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| KR20190009716A (en) * | 2017-07-19 | 2019-01-29 | 서울대학교산학협력단 | Method for differentiation of dermal papilla precursor cells from human induced pluripotent stem cells and uses thereof |
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