Method for inducing differentiation of uterine blood mesenchymal stem cells into hair follicle-derived melanocytes

Abstract

The invention discloses a method for inducing differentiation of uterine blood mesenchymal stem cells into hair follicle-derived melanocytes, which is characterized in that directional differentiation is realized by simulating hair follicle microenvironment conditions and adopting a two-stage induction strategy: in the first stage, the uterine blood mesenchymal stem cells are induced to be differentiated into hair follicle melanocytes; in the second stage, the hair follicle melanin stem cells are further induced to be differentiated into hair follicle-derived melanocytes. The hair follicle-derived melanocyte has dual application values: melanin can be synthesized, hair coloring can be realized after transplantation, and the hair follicle-derived melanocyte can be homed to skin for treating pigment disorder diseases. Researches prove that the T cell immune response of a vitiligo patient has no aggressiveness to hair follicle-derived melanocytes, so that the T cell immune response can become an ideal cell for transplantation treatment of vitiligo compared with skin-derived melanocytes. According to the method, the uterine blood mesenchymal stem cells are induced and differentiated into the hair follicle-derived melanocytes, and a new thought is provided for obtaining the hair follicle-derived melanocytes.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for inducing uterine mesenchymal stem cells to differentiate into hair follicle-derived melanocytes. BACKGROUND

[0002] Vitiligo is a common acquired depigmenting disease caused by a reduction or complete loss of functional melanocytes in the skin and / or hair follicles. It is clinically characterized by depigmented macules and / or patches without subjective symptoms. To date, the etiology and pathogenesis of vitiligo have not been elucidated. In recent years, two new types of vitiligo treatment technologies, tissue-engineered epidermis transplantation and cell transplantation, have achieved good results. Both treatment methods involve expanding melanocytes in vitro and allowing them to migrate to the white spot through or without a carrier for growth and pigmentation, and maintaining pigmentation to prevent disease recurrence. Melanocytes are the key to treating vitiligo. However, the current approach to obtaining patients' own melanocytes is to extract them from healthy skin through one or more incisions. This process not only increases the patients' treatment pain but also limits the number of melanocytes extracted from the skin. Therefore, there is an urgent need to develop a safe and efficient method to obtain patients' autologous melanocytes to reduce the treatment pain of vitiligo patients.

[0003] In human skin, melanocytes are mainly distributed in the basal layer of the epidermis and the hair follicle. They are responsible for synthesizing melanin, which affects the color of the skin and hair. There have been many studies on epidermal melanocytes, but epidermal-derived melanocytes cannot color hair. Hair follicle-derived melanocytes not only color hair but also home to the skin to color the white spots. Melanocytes in the hair follicle mainly include hair follicle-derived melanocytes located in the hair bulb and melanocyte stem cells located in the hair follicle bulge. The former is responsible for hair pigmentation, and the latter acts as a reserve source to undertake the functions of self-renewal and differentiation of melanocytes. Hair follicles have periodic growth characteristics and cycle in sequence according to the growth phase, degeneration phase, and resting phase. During the growth phase, melanocyte stem cells in the hair follicle migrate downward to the hair bulb and differentiate into hair follicle-derived melanocytes to color hair. When the hair follicle enters the degeneration and resting phases, hair follicle-derived melanocytes can de-differentiate and return to the bulge area. The hair follicle bulge is a relatively immune-exempt area. When vitiligo occurs, the melanocyte stem cells in the hair follicle bulge of the affected area are not only immune from autoimmune attacks but also can migrate to the vitiligo lesion to continuously repigment it.

[0004] Mesenchymal stem cells are adult stem cells with multi-directional differentiation potential and immune regulation, which can maintain long-term differentiation and self-renewal ability. When mesenchymal stem cells are stimulated by different stimuli, they can be induced to differentiate into different cells or tissues. Uterine blood mesenchymal stem cells are a new type of stem cells derived from female menstrual blood, which have multi-directional differentiation potential and immune regulation functions, and are increasingly used in disease treatment. It is derived from female autologous, has all the commonness of mesenchymal stem cells, is easy to obtain and has no ethical and traumatic problems, and because it has multi-directional differentiation potential, it provides a new idea for the acquisition of hair follicle-derived melanocyte cells. SUMMARY

[0005] The present application coats the culture environment with mussel byssus protein, collagen and keratin, and then inoculates HaCaT cells as feeder layer to construct the hair follicle microenvironment. Then the HaCaT cells are treated with mitomycin C to stop at a non-proliferative but metabolically active state. Then the uterine blood mesenchymal stem cells are co-cultured with the HaCaT cells, and then the first stage induction medium is used to differentiate the uterine blood mesenchymal stem cells into non-activated melanocyte stem cells in the hair follicle; then the second stage induction medium is used in combination with ultraviolet irradiation culture, and the dopa rich in mussel byssus protein is used to induce the melanocyte stem cells to migrate downward in the hair follicle microenvironment and differentiate into hair follicle-derived melanocyte cells capable of producing melanin granules.

[0006] To solve the above problems, the technical scheme adopted by the present application is: A method for differentiating uterine blood mesenchymal stem cells into hair follicle-derived melanocyte cells, the specific steps are as follows: (1) Extraction of uterine blood mesenchymal stem cells: uterine blood mesenchymal stem cells are extracted from menstrual blood samples of female volunteers during menstruation.

[0007] (2) Pretreatment of hair follicle microenvironment: the culture environment is coated with mussel byssus protein with a final concentration of 0.3-1 mg / mL, collagen with a final concentration of 6-10 μg / mL, and keratin with a final concentration of 0.5-1 mg / mL in sequence; wherein the mussel byssus protein is wrapped with collagen to form a fibrous structure, and then the fibrous structure of the mussel byssus protein is wrapped with keratin to create a structure similar to the hair root in the hair follicle. HaCaT cells are used as feeder layer cells, and the HaCaT cells are resuscitated and inoculated into the coated environment to construct the hair follicle microenvironment attached to the skin hair follicle. When the confluence of HaCaT cells reaches 100%, mitomycin C is used for treatment to stop the HaCaT cells at a non-proliferative but metabolically active state, providing a nutritional basis for the subsequent culture and differentiation of uterine blood mesenchymal stem cells.

[0008] (3) Co-culture of kerotinocytes and uteroplacental mesenchymal stem cells: Kerotinocytes are the main body of the skin, and each melanocyte in the skin is closely associated with about 36 kerotinocytes adjacent thereto to form an interactive unit. The kerotinocytes secrete granulocyte macrophage colony stimulating factor and exosomes during growth to participate in the formation and growth of melanocytes. At the same time, the melanocytes generate melanin under the action of external factors such as ultraviolet light, and the melanin particles are transported to the surrounding epidermis and growing hair matrix kerotinocytes by the dendrites of the melanocytes into the kerotinocytes. Through co-culture, the kerotinocytes provide a basis for the uteroplacental mesenchymal stem cells to differentiate into hair follicle-derived melanocytes.

[0009] (4) Induced differentiation of uteroplacental mesenchymal stem cells: The first stage of induced differentiation makes the uteroplacental mesenchymal stem cells differentiate into non-activated melanin stem cells in the hair follicle: the culture medium of the co-culture of kerotinocytes and uteroplacental mesenchymal stem cells is discarded, and the first stage of induced culture medium containing 10-15 μg / mL MITF, 10-15 ng / mL PAX3, 20-25 ng / mL BFGF, 1-5 μmol / L α-MSH, and 10-15 mmol / L L-lactic acid is added, and the continuous induced culture is performed for 8 days.

[0010] The second stage of induced differentiation makes the melanin stem cells migrate downward in the microenvironment of the hair follicle and differentiate into hair follicle-derived melanocytes capable of generating melanin particles: the first stage of induced culture medium is completely discarded, and the second stage of induced culture medium containing 1-3 μmol / L DES, 1 mg / mL tyrosine, and 200-300 nmol / L 5-HT is added, and 30-50 μm / cm 2 of ultraviolet (365 nm) irradiation is used for 30 min, and the continuous induced culture is performed for 3 days.

[0011] (5) Identification: dopa staining is used to identify the effect of the uteroplacental mesenchymal stem cells on the differentiation into hair follicle-derived melanocytes.

[0012] The advantages of the present application are: A hair root-like structure was constructed using mussel byssal proteins, collagen, and keratin. Keratinocytes were then seeded onto this structure to form skin hair follicle appendages. Simultaneously, the soluble factors and exosomes secreted by these keratinocytes, acting as a feeder layer, continuously provided a nutritional basis for the growth and proliferation of follicle-derived melanocytes differentiated from mesenchymal stem cells co-cultured with them. The mesenchymal stem cells were then induced to gradually proliferate and differentiate into unactivated, achromatic follicular melanocyte stem cells using the first inducing factors MITF, PAX3, BFGF, α-MSH, and L-lactic acid. The achromatic melanocyte stem cells were then stimulated by the second inducing factors DES, tyrosine, 5-HT, and ultraviolet irradiation to differentiate into follicle-derived melanocytes and generate melanin granules. These melanin granules were then transferred to keratinocytes via dendrites to resist UV damage to keratinocyte DNA. Simultaneously, during melanin production, the dopamine-rich components in mussel byssal proteins regulated the downward migration of melanocytes within the constructed hair follicle microstructure, forming melanocytes localized at the hair follicle. Follicular melanocytes can synthesize melanin and, after transplantation, can color hair and home to the skin for the treatment of skin pigmentation disorders. Furthermore, studies have shown that the T-cell immune response triggered by vitiligo is not aggressive towards follicular melanocytes, making them a more ideal cell type for vitiligo transplantation than skin-derived melanocytes. This method, by inducing autologous uterine blood mesenchymal stem cells to differentiate into follicular melanocytes, effectively reduces the recurrence rate in vitiligo transplantation treatment and alleviates the treatment suffering of vitiligo patients. Attached Figure Description

[0013] Figure 1 This is a flow cytometry result of P1 generation mesenchymal stem cells from uterine blood.

[0014] Figure 2 This is a diagram of the structure formed by the combination of mussel byssal proteins and collagen.

[0015] Figure 3 This is a comparison image of immunofluorescence detection of DCT in the first stage of induced differentiation of cells.

[0016] Figure 4 This is a comparison chart of the results of dopa staining identification of cells in the first stage of induced differentiation.

[0017] Figure 5 This is a comparison chart of cell growth and migration during the second stage of induced differentiation.

[0018] Figure 6 This is a comparison chart of the results of dopa identification in the second stage of induced differentiation of cells.

[0019] Figure 7 This is a comparison chart showing the effects of ultraviolet irradiation on cell differentiation induction in the second stage. Detailed Implementation

[0020] The application will be described in detail below with reference to specific embodiments, but not as a limitation to the application.

[0021] The reagents and raw materials used in the embodiments of the application can be purchased from the market. Among them, the α-MEM culture medium is purchased from Gibco, item number C12571500CP; the lymph separation liquid is purchased from Haoyang, item number P24000052413; the mesenchymal stem cell culture medium is purchased from Youkang, item number NC0103; the mussel byssal protein is purchased from Merck, item number 767719-500UL; the collagen is purchased from Merck, item number C5533-5MG; the keratin is purchased from Merck, item number K0253-5MG; the DMEM culture medium is purchased from Gibco, item number C11995500BT; the F12 culture medium is purchased from Gibco, item number 11765-054; the FBS is purchased from Gibco, item number 10100147; the mitomycin C is purchased from Merck, item number M5353-0.2ML; the MITF is purchased from Huamei Biology, item number CSB-EP304681NGSa3; the PAX3 is purchased from Huamei Biology, item number CSB-YP017489HU; the BFGF is purchased from Tongli Haiyuan, item number GMP-TL401; the L-lactic acid is purchased from Merck, item number L6402-1G; the α-MSH is purchased from Merck, item number M4135; the DES is purchased from Merck, item number D4628-1G; the tyrosine is purchased from Merck, item number T2900000; the 5-HT is purchased from Merck, item number H9523-25MG; the Cnt-40 culture medium is purchased from CELLNTEC, item number 2007225; the primary antibody Anti-DCT antibody produced in rabbit is purchased from Merck, item number HPA010742-100UL; the fluorescent secondary antibody Goat Anti-Rabbit IgG (H+L) Antibody, FITC conjugated is purchased from Huamei Biology, item number CSB-PA198023. Unless otherwise specified, the methods in the embodiments are conventional methods and will not be described in detail.

[0022] Embodiment 1: S1: Extraction of uterine blood mesenchymal stem cells: S1-1: During the menstrual period of a female volunteer, use a sterile catheter tampon to collect menstrual blood; place the collected tampon containing menstrual blood in 50mL of α-MEM culture medium, and repeatedly squeeze the tampon with sterile forceps within 6h to squeeze out all the menstrual blood and mix it well with the culture medium, a total of 71mL of mixed liquid is collected, and then the blood sample mixture is supplemented to 90mL with α-MEM culture medium.

[0023] S1-2: Take 3 50 mL centrifuge tubes, add 15 mL lymph separation liquid to each, then add 30 mL blood sample mixture to each, centrifuge at 800 g for 30 min.

[0024] S1-3: After centrifugation, the white membrane layer in each tube is taken and added to a new 50 mL centrifuge tube, 50 mL of normal saline is added to each tube, mixed and centrifuged at 800 g for 10 min.

[0025] S1-4: After centrifugation, the supernatant is discarded, and 50 mL of normal saline is used to resuspend the cell precipitate in the three tubes and combine them into the same 50 mL centrifuge tube, and centrifuged at 300 g for 10 min.

[0026] S1-5: After centrifugation, the cell precipitate is resuspended with 10 mL of mesenchymal stem cell culture medium, and all of it is inoculated into a T75 culture bottle, which is placed in a 37°C, 5% CO2 incubator for culture; during the culture process, fresh mesenchymal stem cell culture medium is replaced every 3 days; when the cell confluence reaches 80%, 4.1 x 10 6 cells of P1 generation are harvested.

[0027] 2.1 x 10 6 cells of P1 generation are reserved for later use, and the remaining 2 x 10 6 cells of P1 generation are used for flow cytometry detection, and the flow cytometry detection results are: the total expression rate of cell surface markers CD90 + 98.6%, CD73 + 100.0%, CD105 + 99.0%, and CD45 + / CD34 + / CD11b + / CD19 + / HLA-DR + is 2.2%( Figure 1 ). The results confirm that the obtained cells meet the phenotypic characteristics of uterine blood mesenchymal stem cells, and the purity of the extracted uterine blood mesenchymal stem cells is high.

[0028] S2: Pretreatment of hair follicle microenvironment: S2-1: Prepare a 0.5 mg / mL solution of mussel byssus protein, sterilize the solution, and then take 500 μL of the solution and add it to each of the A, B, C, and D wells of a 6-well plate, ensuring that the solution completely covers the bottom of the wells; the four wells are placed at room temperature for 2 hours of coating, then the remaining protein solution is discarded, and the wells are rinsed twice with PBS; 500 μL of 6 μg / mL collagen protein solution is added to each well, and the solution is placed at 37°C for 30 minutes to allow the mussel byssus protein to bind to the collagen protein and solidify; then the remaining protein solution in each well is discarded, the wells are rinsed twice with PBS, and the remaining PBS is discarded, and the wells are dried at room temperature.

[0029] S2-2: Under ice bath condition, 1 mL of pre-cooled NBT / Glycinate staining solution (2 M glycinate potassium solution containing 0.24 mM NBT, pH = 10) was added into the D well (identification well); after the addition was completed, it was placed in a 25°C water bath and kept in the dark for 1 h; after the reaction was completed, the results were observed under a 400-fold optical microscope. As shown in FIG. 2B, after NBT / Glycinate staining, the blue-purple mussel byssal protein was observed; the black arrow indicates that the region can be seen to be solidified into a fibrous structure after the combination of mussel byssal protein and collagen. Figure 2

[0030] S2-3: 500 μL of 0.6 mg / mL keratin solution was added into the A, B, and C wells, respectively, and coated at 37°C for 24 h, ready for use.

[0031] S2-4: Feeding layer cell culture: S2-4-1: Preparation of D / F12 complete medium: S2-4-2: A tube of keratinocytes (1 × 10 6 P1 generation) was taken out from the liquid nitrogen tank, thawed to 80% at 37°C, and then transferred to a 50 mL centrifuge tube containing 30 mL of D / F12 complete medium, washed by blowing, and centrifuged at 500 g for 5 min.

[0032] S2-4-3: After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in 20 mL of D / F12 complete medium. After mixing, 2 mL of cell suspension was taken and inoculated into the A, B, C, E, F, and G wells, which were coated or not coated, respectively, and cultured in a 37°C, 5% CO2 incubator.

[0033] S2-4-4: After three days of culture, the cell confluence reached 100%, the old culture medium was discarded, 1 mL of mitomycin C solution with a final concentration of 4 μg / mL was added into the A, B, C, E, F, and G wells, respectively, and placed at 37°C for 90 min, then the liquid was removed; washed twice with α-MEM medium, then added 2 mL of D / F12 complete medium, and placed in the incubator for 8 h before use.

[0034] S3: Feeding layer cell and uterine mesenchymal stem cell co-culture: S3-1: The D / F12 complete medium in the A, B, C, E, F, and G wells was discarded.

[0035] S3-2: 2.1 × 10 6 ​P1 generation of uterine blood mesenchymal stem cells, adjust the cell density to 5 x 10 4 2 mL of each of A, B, C, E, F, and G, and place in a 37°C, 5% CO2 incubator for co-culture.

[0036] S4: First stage induction and differentiation of uterine blood mesenchymal stem cells S4-1: After 24 hours of co-culture, when the uterine blood mesenchymal stem cells adhere, remove the old mesenchymal stem cell culture medium in A, B, C, E, F, and G, and add 2 mL of first stage induction medium (containing mesenchymal stem cell culture medium with a final concentration of 10 μg / mL MITF, a final concentration of 10 ng / mL PAX3, a final concentration of 20 ng / mL BFGF, a final concentration of 10 mmol / L L-lactic acid, and a final concentration of 1 μmol / L α-MSH) to each well, and place in a 37°C, 5% CO2 incubator for culture; thereafter, replace the fresh first stage induction medium every 24 hours, and continuously induce for 8 days.

[0037] S4-2: Immunofluorescence identification of first stage induction and differentiation S4-2-1: Remove the old culture medium in C and G, and immerse each well with 2 mL of PBS for 3 times, 3 min each time.

[0038] S4-2-2: Add 2 mL of 4% paraformaldehyde to each well for fixation for 15 min, and then immerse each well with 2 mL of PBS for 3 times, 3 min each time.

[0039] S4-2-3: Absorb the PBS in the well with absorbent paper, and add 5% goat serum to each well to ensure that the cells are not covered by the bottom of the well, and seal at room temperature for 30 min.

[0040] S4-2-4: Primary antibody incubation: absorb the sealing liquid in each well with absorbent paper, and add 500 μL of primary antibody (diluted 1:100 with PBS) to each well, and incubate at 4°C overnight.

[0041] S4-2-5: Green fluorescent secondary antibody incubation: immerse each of the two wells with PBS for 3 times, 3 min each time; after absorbing the excess liquid in the well with absorbent paper, add 500 μL of fluorescent secondary antibody (diluted 1:100 with PBS) to each well, and incubate at 37°C for 1 h; then immerse each well with PBS for 3 times, 3 min each time. Then observe under a fluorescence microscope and collect images. The results are shown in Figure 3 T1 shows that DCT in the C well is expressed in the cytoplasm, showing green fluorescence, indicating that the target cell is a DCT positive cell, and the cell is oval, has a large nucleus-cytoplasm ratio, and has no protrusions; T2 shows that no obvious fluorescence signal is observed in the G well, indicating that the target cell is a DCT negative cell.

[0042] S4-3: Dopachrome staining identification of the first stage induced differentiation S4-3-1: Preparation of 10x PB buffer: weigh 143.5 g of disodium hydrogen phosphate and 16.5 g of sodium dihydrogen phosphate, dissolve in 500 mL of deionized water; dilute the 10x PB buffer with deionized water to 1x PB buffer.

[0043] S4-3-2: Remove the old culture medium in B and F wells respectively, rinse each well with 2 mL of 1x PB buffer for 2 times, 3 min each time; then add 2 mL of 4% paraformaldehyde for 10 min, and then rinse with 2 mL of 1x PB buffer for 2 times, 3 min each time; dry the well plate naturally at room temperature for 4 hours.

[0044] S4-3-3: Take a 50 mL centrifuge tube, weigh 15 mg of levodopa, add 15 mL of 1x PB buffer, and after complete dissolution, take 2 mL and add to B and F wells respectively; stain at 37°C for 6 h in the dark, and then observe under a microscope and collect images. The results are shown in Figure 4 T1 shows that no melanin granules appear in the B well, and combined with the immunofluorescence identification results, it can be known that the cells differentiated from the uterine blood mesenchymal stem cells after the first stage induction express DCT molecular markers, the cells are oval, have a large nucleus-cytoplasm ratio, have no processes, and do not express melanin granules, and the above characteristics are consistent with the phenotype of hair follicle melanin stem cells; T2 shows that no melanin granules appear in the F well, and combined with the immunofluorescence identification results, it can be known that the uterine blood mesenchymal stem cells do not effectively differentiate.

[0045] S5: Second stage induction of uterine blood mesenchymal stem cells: S5-1: After 8 days of first stage induction and culture of A and E wells, discard the first stage induction medium, add 2 mL of second stage induction medium (Cnt-40 medium containing a final concentration of 1 μmol / L DES, a final concentration of 1 mg / mL tyrosine, and a final concentration of 200 nmol / L 5-HT) to each well, and then irradiate the A and E wells with an ultraviolet lamp tube (wavelength 365 nm) with an irradiation intensity of 30 μw / cm 2 2 for 30 min at a distance of 15 cm from the well plate, and after irradiation, place in a 37°C, 5% CO2 incubator; thereafter, replace the fresh second stage induction medium in each well every 24 h, and at the same time, perform 30 min of ultraviolet irradiation, and after 3 days of continuous induction, observe the cell state under a microscope. Figure 5T1a and T1b show the migration of the cells in the A hole. In the T1a picture (200x), the melanocyte can be observed at the black arrow, but no melanocyte can be observed. In the T1b picture (50x), the melanocyte can be observed at the black arrow, and the cell at the red arrow shows the typical multi-level dendritic morphology of the melanocyte. No cell with dendritic morphology can be observed in the layer where the melanocyte is located. The melanocyte with multi-level dendritic morphology can be observed in the layer below the melanocyte layer. T2 shows the migration of the cells in the E hole. In the picture, the melanocyte can be observed at the black arrow, and the cell at the red arrow shows the typical bipolar dendritic structure of the epidermal melanocyte.

[0046] S5-2: Dopachrome staining of the second stage induced differentiation cells: Dopachrome staining of the cells in the A and E holes was performed according to the method in S4, and the staining was observed. Figure 6 Figure 6 T1a is a 50x picture of the A hole. The picture shows that the cytoplasm of the dendritic cell is gray-black, and the dopachrome staining is positive. The cells grow in multiple places. T1b is a 200x picture of the A hole. The picture shows that the dendritic cells grow in the protrusions at the bottom of the hole, which confirms that the melanocyte stem cells can migrate downward and grow in the hair follicle microenvironment in the protrusions of the hair follicle-like roots under the regulation of the dopa in the second stage induction and the mussel byssal protein. The melanocyte stem cells can successfully differentiate into hair follicle-derived melanocytes that can generate melanin granules. T2 is a 200x picture of the E hole. The picture shows that the cytoplasm of the dendritic cell is gray-black, and the dopachrome staining is positive. The cells grow in the upper layer of the melanocyte and are uniformly distributed without migrating to the lower layer of the melanocyte. Compared with the hair follicle-derived melanocytes in the A hole, which show a three-level to multi-polar dendritic structure, the epidermal melanocytes in the E hole show a bipolar dendritic structure.

[0047] Example 2: The A hole experimental hole and the B hole control hole were set on the six-hole plate. The A and B holes were operated synchronously according to the steps in Example 1 to induce and differentiate until the first stage. After 8 days of induction, the second stage of induction and differentiation was performed. In the second stage of induction and differentiation, 2 mL of the second stage of induction medium (formula same as Example 1) was added to the A hole, and then the irradiation intensity was 30 μw / cm 2 ​The cells in the two wells were irradiated with the UV lamp (wavelength 365 nm) at a distance of 15 cm for 30 min, and then cultured in a 37℃, 5% CO2 incubator. After that, the cells in each well were replaced with fresh second-stage induction medium every 24 h, and irradiated with the UV lamp for 30 min. The induction was continued for 3 days. During the second-stage induction differentiation, 2 mL of second-stage induction medium (formula same as in Example 1) was added to the B well, and the cells were cultured in a 37℃, 5% CO2 incubator. After that, the cells in each well were replaced with fresh second-stage induction medium every 24 h, and the induction was continued for 3 days. The cells in the A and B wells were subjected to dopa staining identification, and the effect of UV irradiation on the second-stage differentiation of the cells was compared.

[0048] The results are shown in Figure 7 T1 is the case of the cells in the A well, and T2 is the case of the cells in the B well. As can be seen by comparing the two, the melanocytes growing in the aggregation in T1 are significantly more than the melanocytes in T2, and the dopa staining color of the melanocytes in T1 is deeper than that of the melanocytes in T2. This proves that UV irradiation can increase the differentiation efficiency and the expression ability of melanin granules of the hair follicle-derived melanocytes during the second-stage induction differentiation.

[0049] The above description is only the preferred embodiment of the present application, and any equivalent changes and modifications made within the scope of the present application should be included in the scope of the present application.

Claims

1. A method of inducing differentiation of uterine mesenchymal stem cells into hair follicle-derived melanocytes, characterized by: The method comprises the following steps:

2. The method of claim 1, wherein: S1: Pretreatment of hair follicle microenvironment: coating a 6-well plate with Mytilus galloprovincialis byssus protein, collagen and keratin, and then inoculating feeder layer cells in the well plate after coating is completed; S2: Co-culture of uteroplacental mesenchymal stem cells and feeder layer cells: inoculating uteroplacental mesenchymal stem cells into the 6-well plate treated in S1 and co-culturing with the feeder layer cells; S3: First-stage induction and differentiation: after the uteroplacental mesenchymal stem cells are adhered in the co-culture of S2, continuously inducing and culturing for 8 days with a first-stage induction medium; S3: Second-stage induction and differentiation: after the induction and culture in S3 is completed, continuously inducing and culturing for 3 days with a second-stage induction medium while being treated with ultraviolet irradiation. The 6-well plate is coated in the order of the first Mytilus galloprovincialis byssus protein, the second collagen and the third keratin; the coating concentration of the Mytilus galloprovincialis byssus protein is 0.3-1 mg / mL, the coating concentration of the collagen is 6-10 μg / mL, and the coating concentration of the keratin is 0.5-1 mg / mL.

3. The method of claim 2, wherein: The feeder layer cells are keratinocytes; the feeder layer cells are treated with mitomycin C before being co-cultured with the uteroplacental mesenchymal stem cells.

4. The method of claim 2, wherein: The first-stage induction medium contains the following additives with the following final concentrations: 10-15 μg / mL MITF, 10-15 ng / mL PAX3, 20-25 ng / mL BFGF, 1-5 μmol / L α-MSH and 10-15 mmol / L L-lactic acid.

5. The method of claim 2, wherein: The concentration of the uterine mesenchymal stem cells for inoculation is 5-8×10 4 / mL.

6. The method of claim 2, wherein: The second-stage induction medium contains the following additives with the following final concentrations: 1-3 μmol / L DES, 1 mg / mL tyrosine and 200-300 nmol / L 5-HT.

7. The method of claim 2, wherein:

9. The use of the method of claim 1 in the preparation of hair follicle melanin stem cells and hair follicle-derived melanocytes.

8. The method of claim 2, wherein: The condition of the ultraviolet irradiation treatment is: irradiation for 30 min every 24 h, irradiation intensity 30-50 μw / cm 2 , and irradiation wavelength 365 nm.

10. The use of the hair follicle melanin stem cells and hair follicle-derived melanocytes obtained by the method of claim 1 in the preparation of products for treating pigmentary disorders. ​

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