Induction method for directionally differentiating hESC-melanocytes based on neural crest lineage

Abstract

The invention discloses an induction method for directionally differentiating hESC-melanocytes based on a neural crest lineage, and belongs to the technical field of cell engineering and cytobiology. According to the induction method, a complex differentiation process of embryonic stem cells-ectoderm-neural ectoderm-neural crest cells is abandoned, and the neural crest cells are directly obtained from embryonic stem cells, so that the differentiation process is simplified; the differentiation efficiency and controllability of the neural crest cells are remarkably improved, the obtained melanocytes have higher tyrosinase activity and melanin synthesis capacity, and a new way is provided for treating leucoderma by adopting a melanocyte transplantation method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell engineering and cell biology, and particularly relates to an induction method for hESC-melanocyte differentiation based on neural crest lineage orientation. BACKGROUND

[0002] Vitiligo is a common primary, localized or generalized skin depigmentation disease caused by the reduction or loss of functional melanocytes in the skin or hair follicle, which is an autoimmune disease. At present, the treatment means for vitiligo in clinic is very limited. For patients in the progression stage, immunomodulators combined with phototherapy and traditional Chinese medicine compound conditioning are usually used to delay the progression of vitiligo to a certain extent. For patients with large area in the stable stage, epidermal transplantation has obvious effect, but there are problems such as large wound surface caused by multiple skin grafting and patient pain. Therefore, it is a more ideal method to take melanocytes cultured in vitro for melanocyte transplantation. The repigmentation rate of melanocyte transplantation can reach 70%-90%, and the key of this method is to obtain enough melanocytes and ensure the survival rate of melanocyte transplantation.

[0003] The research for inducing differentiation of melanocytes focuses on adipose mesenchymal stem cells, dental pulp stem cells, induced pluripotent stem cells and human embryonic stem cells (hESC). Among them, hESC can differentiate into functional mature melanocytes in a relatively short time by simulating the differentiation strategy of neural crest development, which greatly shortens the differentiation period, and the low immunogenicity of hESC further improves the transplantation feasibility. This technology provides a more reliable source of transplanted cells for the treatment of patients with large-area stable vitiligo.

[0004] In the normal development process of an individual, the ectoderm of the gastrula embryo first differentiates into the neural ectoderm, and the differentiation lineage of the neural ectoderm includes the central nervous system, neuroendocrine cells, retina and neural crest cells, among which the neural crest cells are the precursor cells of melanocytes. Therefore, in the process of in vitro induction, obtaining a large number of neural crest cells is a key node to improve the efficiency of melanocyte differentiation. At present, the existing induction method is to first block the BMP and TGF-β signals by double Smad inhibition method to reduce the differentiation of hESCs to mesoderm and endoderm, and then to promote the differentiation of hESCs to ectoderm-neural ectoderm-neural crest cells by adding Wnt signal activator. Then, the neural crest cells are further induced to melanoblasts by melanocyte induction medium. Since the central nervous system cells are mainly differentiated in the neural ectoderm differentiation lineage, the proportion of neural crest cells is small, so it is difficult to obtain a sufficient number of neural crest cells, resulting in insufficient number of melanocytes obtained by final differentiation.

[0005] Recent literature research found that low concentration of BMP signal can directly drive the subpopulation of embryonic stem cells with no or low expression of transcription factor AP2a (TFN2A) to differentiate into neural crest cells, bypassing the complex sequence of embryonic stem cells-ectoderm-neural ectoderm-neural crest cells, and improving the efficiency and controllability of neural crest cell differentiation. However, in the conventional differentiation scheme, in addition to adding signal pathway inhibitors with clear composition, serum replacement KSR also needs to be added, which has complex composition and contains some animal-derived components (such as albumin), and has certain immunogenicity and infection risk, limiting its application in clinical practice, and high concentration of KSR may cause cell stress and easily induce the differentiation of non-target cells.

[0006] In order to overcome the above-mentioned defects, a new method for inducing hESC-melanocyte based on neural crest lineage directional differentiation is provided, which has great significance in the treatment of vitiligo. SUMMARY

[0007] Therefore, the purpose of the present application is to find a new method for inducing hESC-melanocyte based on neural crest lineage directional differentiation, which solves the problems of low differentiation efficiency and insufficient number of melanocytes in the current induction method, and possible immune risk.

[0008] The present application solves the above technical problems by the following technical means:

[0009] A method for inducing hESC-melanocyte based on neural crest lineage directional differentiation, the induction method comprising the following steps:

[0010] (1) H9 human embryonic stem cells are cultured, and when the cell confluence is ≥ 50%, H9 human embryonic stem cells are induced to differentiate into neural crest cells by using No. 1 Essential 6 culture medium and No. 2 Essential 6 culture medium in turn;

[0011] (2) The neural crest cells are induced to differentiate into melanoblasts by using a differentiation culture medium, and then melanocytes are obtained by culturing in a melanocyte selection culture medium.

[0012] The present application discards the complex differentiation process of embryonic stem cells-ectoderm-neural ectoderm-neural crest cells, and directly obtains neural crest cells from embryonic stem cells. In the disclosed differentiation melanocyte system, the serum-free culture medium Essential 6 without trophoblasts and with clear chemical composition is used to directly drive hESC to differentiate into neural crest lineage by precisely controlling the synergistic effect of BMP, WNT and TGF-β three signal pathways, reducing mesoderm or non-neural crest differentiation, and obtaining more neural crest cells, which can systematically improve the differentiation efficiency and quality of melanocytes.

[0013] In addition, the Essential 6 medium is free of complex additives such as serum, has a clear chemical composition, has stronger repeatability of experiments, and is free of immunogenic substances, thereby providing a basis for clinical application.

[0014] Further, the cell culture step of the H9 human embryonic stem cells in the induction method is as follows:

[0015] A. After coating the culture dish with DMEM / F12 medium containing Vitronectin, the H9 human embryonic stem cells are resuspended in mTeSR1 basic medium containing 1% penicillin-streptomycin for culture until the cell confluence rate reaches 80%, and then subcultured;

[0016] B. The H9 human embryonic stem cells are pre-seeded in a DMEM / F12 culture dish coated with Matrigel matrix glue, and cultured with mTeSR1 basic medium until the cell confluence is ≥50%.

[0017] Further, the specific operation process of A is as follows:

[0018] The culture dish is coated with DMEM / F12 medium containing 1 µg / cm 2 Vitronectin, and is placed at 37°C, 5% CO2 for at least 1 h, and after coating is completed, the liquid is aspirated and reserved for use;

[0019] The H9 human embryonic stem cells are resuspended in mTeSR1 basic medium added with 2.5 µM Blebbistatin and 1% penicillin-streptomycin, and then the cell suspension is transferred to the coated culture dish, and cultured at 37°C, 5% CO2, and after 24 h, the mTeSR1 basic medium (containing 1% penicillin-streptomycin) without Blebbistatin is replaced, and the mTeSR1 basic medium (containing 1% penicillin-streptomycin) is replaced every 24 h, and when the cell confluence rate reaches 80%, subculture is performed.

[0020] Further, the specific operation process of B is as follows:

[0021] The H9 human embryonic stem cells are pre-seeded in a DMEM / F12 culture dish coated with 10 mg / mL Matrigel matrix glue, and cultured with mTeSR1 basic medium (containing 1% penicillin-streptomycin) for 24 h; when the cell confluence reaches 50%-60%, subsequent differentiation culture is performed.

[0022] Further, the Essential 6 medium in (1) is: Essential 6 medium as the base, further added with 1 ng / mL BMP4, 10 µM SB431542 and 600 nM CHIR99021;

[0023] The second Essential 6 medium is: Essential 6 medium as the base, further added with 10 µM SB431542 and 1.5 µM CHIR99021.

[0024] Further, the differentiation culture of neural crest cells in (1) is cultured at 37°C, 5% CO2, and the specific operation is as follows:

[0025] Days 0-2: use the first Essential 6 medium to induce cell differentiation;

[0026] Days 3-5: use the second Essential 6 medium for culture to obtain neural crest cells.

[0027] Further, the differentiation medium in (2) includes:

[0028] The third differentiation medium: 50% Essential 6 differentiation medium and 50% N-2 differentiation medium are mixed as the base, and 3 µM CHIR99021, 25 ng / mL BMP4 and 100 nM EDN-3 are added;

[0029] The fourth differentiation medium: 25% Essential 6 differentiation medium and 75% N-2 differentiation medium are mixed as the base, and 3 µM CHIR99021, 25 ng / mL BMP4 and 100 nM EDN-3 are added;

[0030] The fifth differentiation medium: completely use N-2 differentiation medium as the base, and add 3 µM CHIR99021, 25 ng / mL BMP4 and 100 nM EDN-3;

[0031] The above Essential 6 differentiation medium is: Essential 6 medium as the base, added with 6 ng / mL FGF-2 and 0.1 mM β-mercaptoethanol;

[0032] The N-2 differentiation medium is: DMEM / F12 medium as the base, added with 1.55 g / L glucose and 1% N-2 supplement.

[0033] Further, the differentiation culture of melanoblasts is cultured at 37°C, 5% CO2, and the specific operation is as follows:

[0034] Day 6-7: culture with No. 3 differentiation medium;

[0035] Day 8-9: culture with No. 4 differentiation medium;

[0036] Day 10-11: culture with No. 5 differentiation medium, to obtain melanoblasts.

[0037] Further, the melanocyte selection medium in (2) is a DMEM / F12-based medium, with the addition of 6 ng / mL FGF-2, 500 µM dibutyryl cyclic AMP calcium, 0.8% insulin-transferrin-selenium, 3 µM CHIR99021 and 100 nM EDN-3.

[0038] Further, the differentiation culture of the melanocytes in (2) is carried out at 37°C, 5% CO2, and the specific operation is as follows:

[0039] Coat the well plate with 1 µg / mL laminin-1 and 10 mg / mL Materigel matrix gel, 4°C overnight, and wash with PBS before use;

[0040] Day 12-25: inoculate the cells in the well plate, add the melanocyte selection medium, and change the medium every other day.

[0041] Beneficial effects:

[0042] 1. The present application successfully establishes an efficient and stable preparation strategy for functional melanocytes derived from hESC based on the optimization of neural crest cell differentiation mechanism. The core innovation of the new scheme is to optimize the early differentiation path, use the serum-free medium Essential 6 without feeder layer and with clear chemical composition, and directly drive hESC to differentiate into neural crest lineage by precisely controlling the synergistic effect of BMP, WNT and TGF-β three signal pathways, thereby reducing mesoderm or non-neural crest differentiation, and significantly improving the differentiation efficiency and controllability of neural crest cells. This improvement not only significantly simplifies the differentiation process, but also greatly improves the differentiation efficiency and process controllability: the significant expression of neural crest fate markers can be detected at the early stage of differentiation (day 3), which is much earlier than the traditional scheme; by day 6, the expression levels of key transcription factors SOX9 and SOX10 are significantly up-regulated, indicating stronger melanocyte differentiation potential. The functional evaluation at the end of differentiation (day 25) shows that the new system is superior to the traditional scheme in terms of cell yield, melanocyte maturity (indicated by tyrosinase activity and melanin synthesis capacity) and related functional gene expression levels, and functional mature melanocytes are successfully obtained.

[0043] 2、The melanocytes obtained by the induction method of the present application not only express mature melanocyte markers (such as DCT, MITF, TYR and MLANA), but also have tyrosinase activity and melanin synthesis ability, which shows a clear treatment prospect in the treatment of vitiligo.

[0044] 3、In terms of treatment safety and effectiveness, in vivo experiments prove that the melanocytes obtained by differentiation of the system can be stably implanted in the host skin basement layer after transplantation, and long-term, self-reliant melanin synthesis function is achieved, and is not affected by the hair follicle cycle, and the formed pigment patches are stable and non-invasive, showing good biological safety and treatment potential. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 : hESCs were directionally induced to differentiate into neural crest cells for 6 days. (a) Day 0 (hESC stage) cells arranged closely, grew in aggregation, and were island-like or nest-like; (b) Day 6 (neural crest cell stage), the cell morphology was similar to that of fibroblasts; (c-d) On the third day of differentiation, the expression of the marker gene TFAP2A of the neural crest lineage and the neural ectoderm marker gene PAX6; (e-g) On the sixth day of differentiation, the expression of the neural crest cell marker genes SOX10, PAX3 and SOX9;

[0046] Figure 2 : Neural crest cells were directionally induced to differentiate into melanocytes; (a) On the 12th day of differentiation, melanoblast-like cells were obtained; (b) Day 12 (melanoblast stage) comparison of the number of cells in different differentiation schemes; (c-l) On the 12th day of differentiation, the relative expression of the melanoblast marker genes PMEL, TYR, MITF and DCT; (g) On the 25th day of differentiation, melanocyte-like cells were obtained; (h) Day 25 (melanocyte stage) comparison of the number of cells in different differentiation schemes; (i-l) On the 25th day of differentiation, the relative expression of the melanocyte marker genes MITF, TYR, Melan-A and DCT;

[0047] Figure 3 : Melanocyte function identification; (a) Dopachrome staining results; (b) Tyrosinase activity detection; (c-f) DCT, MITF, SOX10 and TYR immunofluorescence results;

[0048] Figure 4 : hESC-MC transplantation experiment; (a) hESC-melanocyte epidermis transplantation (the middle concentration group has more natural black patches); (b) HE staining result display; (c) DCT, Melan-A, SOX10 and TYR immunohistochemical result display;

[0049] Figure 5Safety analysis of hESC-MC transplantation; (a) On day 50 after transplantation, the skin pigmentation of the transplanted area was shown by epidermis transplantation method; (b) On day 50 after transplantation, the underlying muscle tissue of the transplanted area was shown; (c-e) The heart, spleen and kidney of the transplanted mouse were shown; (f) On day 12 after inoculation of low, medium and high concentrations of hESC-MC and B16F10 cells, the tumor formation of the nude mice by subcutaneous injection method. DETAILED DESCRIPTION

[0050] The present application will be described in detail below in conjunction with specific embodiments and drawings:

[0051] The experimental methods used in the following examples are conventional methods unless otherwise specified. All materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0052] The cells and reagents used in the present application are as follows:

[0053] The H9 human embryonic stem cells (H9 hESC) used in this experiment were donated by the Ecological Environment Research Center of the Chinese Academy of Sciences; Knockout DMEM medium (Gibco, 10829018, USA), DMEM / F12 medium (Savillex, China), mTeSR1 medium (Gibco, A1517001, USA), Essential 6 medium (Gibco, A15165-01, USA), 0.25% trypsin (Savillex, G4013, China), KSR serum substitute (Gibco, 10828028, USA), FGF-2 (R&D, 4114-TC-01M, USA), β-mercaptoethanol (Gibco, 21985-023, USA), glucose (Sigma, 49163-100M, Germany), 100x N-2 supplement (Gibco, 17502048, USA), dibutyryl cyclic adenosine monophosphate calcium (Sigma, D0260, Germany), insulin-transferrin-selenium (Gibco, 51500056, USA), CHIR99021 (STEMGENT, 04-0004, USA), endothelin-3 (MCE, HY-P024A, USA), LDN193189 (Selleck, S7507, USA), BMP4 (R&D, 314-BP-050, USA), laminin-1 (Sigma, L4544-100UL, Germany), Materigel (Corning, 356234, USA), Vitronectin (Zhongsheng Suyuan, RP01002, China), PBS buffer (Savillex, G0002, China), penicillin and streptomycin (abm, G255, Canada), L-DOPA (MCE, HY-N0304, USA).

[0054] Example 1: Induced differentiation method of hESC-melanocyte

[0055] (1) Culture dish pre-treatment: 60mm culture dish was coated with DMEM / F12 medium containing 1 μg / cm2vitronectin (VTN) and incubated at 37°C, 5% CO2 for at least 1 h, and the liquid was discarded after the coating was completed for standby use; 2 Vitronectin (VTN) was coated on a 60mm culture dish with DMEM / F12 medium, and incubated at 37°C, 5% CO2 for at least 1 h, and the liquid was discarded after the coating was completed for standby use;

[0056] Cell inoculation and initial culture: H9 hESC cells were resuspended in mTeSR1 basal medium supplemented with 2.5 μM Blebbistatin and 1% penicillin-streptomycin at a concentration of 5×10 5 The cell suspension was then transferred to the coated culture dish and incubated at 37°C, 5% CO2, and after 24 h, the medium was replaced with mTeSR1 basal medium without Blebbistatin (containing 1% penicillin-streptomycin), and the mTeSR1 basal medium (containing 1% penicillin-streptomycin) was replaced every 24 h, and the cells were passaged when the confluence reached 80%.

[0057] (2) Before cell induction and differentiation, H9 human embryonic stem cells (hESC) were pre-seeded in DMEM / F12 culture dishes coated with 10 mg / mL Matrigel matrix gel, and cultured with mTeSR1 basal medium for 24 h, and when the cell confluence reached 50%-60%, the subsequent differentiation culture was carried out.

[0058] (3) First stage (Day 0-5) H9 hESC cultured and differentiated into neural crest cells at 37°C, 5% CO2

[0059] On days 0-2, Essential 6 medium was used as the base and added with 1 ng / mL BMP4 (BMP pathway activator), 10 μM SB431542 (TGF-β pathway inhibitor), and 600 nM CHIR99021 (Wnt pathway inhibitor) to induce cell differentiation.

[0060] On days 3-5, Essential 6 medium was used as the base and added with 10 μM SB431542 (TGF-β pathway inhibitor) and 1.5 μM CHIR99021 (Wnt pathway inhibitor) to obtain neural crest cells.

[0061] (4) Second stage (Day 6-11) neural crest cells were cultured and differentiated into melanoblasts at 37°C, 5% CO2

[0062] Essential 6 differentiation medium is based on Essential 6 medium with the addition of 6 ng / mL FGF-2 and 0.1 mM beta-mercaptoethanol;

[0063] N-2 differentiation medium is based on DMEM / F12 medium with the addition of 1.55 g / L glucose and 1% N-2 supplement.

[0064] Day 6-7: 50% Essential 6 differentiation medium mixed with 50% N-2 differentiation medium with the addition of 3 mM CHIR99021 (Wnt pathway inhibitor), 25 ng / mL BMP4 (BMP pathway activator), and 100 nM EDN-3 (EDNRB pathway activator) to make a third differentiation medium;

[0065] Day 8-9: 25% Essential 6 differentiation medium mixed with 75% N-2 differentiation medium with the addition of 3 mM CHIR99021 (Wnt pathway inhibitor), 25 ng / mL BMP4 (BMP pathway activator), and 100 nM EDN-3 (EDNRB pathway activator) to make a fourth differentiation medium;

[0066] Day 10-11: Complete N-2 differentiation medium with the addition of 3 mM CHIR99021 (Wnt pathway inhibitor), 25 ng / mL BMP4 (BMP pathway activator), and 100 nM EDN-3 (EDNRB pathway activator) to make a fifth differentiation medium.

[0067] (4) Melanocyte induction from melanoblasts at 37°C, 5% CO2

[0068] Melanocyte selection medium: DMEM / F12 based medium with the addition of 6 ng / mL FGF-2, 500 mM dibutyryl cyclic AMP calcium, 0.8% insulin-transferrin-selenium, 3 mM CHIR99021, and 100 nM EDN-3.

[0069] 6-well plates were coated with 1 pg / mL Laminin-1 and 10 mg / mL Materigel Matrigel at 4°C overnight and washed three times with PBS before use;

[0070] Day 12-25: On day 12, 10 5The cells were seeded in 6-well plates at a density of 1 cell / well, and melanocyte selection medium was added, with medium changed every other day until the 25th day of differentiation, at which point differentiation was complete.

[0071] Comparative Example 1

[0072] After the same batch of H9 hESC cells were expanded in step (1) and step (2) of Example 1, the same batch of cells were subjected to differentiation culture under the same culture conditions as Comparative Example 1, and the specific operation process was as follows:

[0073] Two types of culture medium were used:

[0074] KSR differentiation medium: Knockout DMEM was used as the basic culture medium, and 10% KSR, 6 ng / mL FGF-2 and 0.1 mM β-mercaptoethanol were added.

[0075] N-2 differentiation medium: DMEM / F12 medium was used as the basic culture medium, and 1.55 g / L glucose and 1% N-2 supplement were added.

[0076] (1) First stage (Day 0-5) H9 hESC differentiation into neural crest cells

[0077] Days 0-1: Based on KSR differentiation medium, 100 nM LDN193189 (BMP pathway inhibitor) and 10 μM SB431542 (TGF-β pathway inhibitor) were added;

[0078] Day 2: Based on KSR differentiation medium, 100 nM LDN193189 (BMP pathway inhibitor) and 10 μM SB431542 (TGF-β pathway inhibitor), 3 μM CHIR99021 (Wnt pathway inhibitor) were added;

[0079] Day 3: Based on KSR differentiation medium, 10 μM SB431542 (TGF-β pathway inhibitor), 3 μM CHIR99021 (Wnt pathway inhibitor) were added;

[0080] Days 4-5: Based on a mixture of 75% KSR differentiation medium and 25% N-2 differentiation medium, then 3 μM CHIR99021 (Wnt pathway inhibitor) was added.

[0081] (2) Second stage (Day 6-11) neural crest cell differentiation into melanoblasts

[0082] Day 6-7: Based on 50% KSR differentiation medium and 50% N-2 differentiation medium, 3 μM CHIR99021 (Wnt pathway inhibitor), 25 ng / mL BMP4 (BMP pathway activator) and 100 nM EDN-3 (EDNRB pathway activator) were added.

[0083] Day 8-9: Based on 25% KSR differentiation medium and 75% N-2 differentiation medium, 3 μM CHIR99021 (Wnt pathway inhibitor), 25 ng / mL BMP4 (BMP pathway activator) and 100 nM EDN-3 (EDNRB pathway activator) were added.

[0084] Day 10-11: Based on N-2 differentiation medium, 3 μM CHIR99021 (Wnt pathway inhibitor), 25 ng / mL BMP4 (BMP pathway activator) and 100 nM EDN-3 (EDNRB pathway activator) were added.

[0085] The subsequent melanocyte induction and differentiation of melanoblasts were the same as the method of step (4) in Example 1.

[0086] Comparative Example 2

[0087] After the same batch of H9 hESCs cells were expanded and cultured according to the method of step (1) and step (2) in Example 1, the differentiation culture of Comparative Example 2 was carried out under the same culture conditions. The first stage was the same as the first stage differentiation of (3) in Example 1, and the second stage was the same as the second stage differentiation of Comparative Example 1. The specific differentiation operation process is as follows:

[0088] (1) First stage (Day 0-5) H9 hESC differentiation into neural crest cells

[0089] On day 0-2, No. 1 Essential 6 medium was used to induce cell differentiation, which was based on Essential 6 medium and added with 1 ng / mL BMP4 (BMP pathway activator), 10 μM SB431542 (TGF-β pathway inhibitor) and 600 nM CHIR99021 (Wnt pathway inhibitor).

[0090] On day 3-5, No. 2 Essential 6 medium was used for culture, which was based on Essential 6 medium and added with 10 μM SB431542 (TGF-β pathway inhibitor) and 1.5 μM CHIR99021 (Wnt pathway inhibitor), and neural crest cells were obtained.

[0091] (2) Second stage (Day 6-11) Neural crest cells differentiate into melanoblasts stage

[0092] Day 6-7: Based on 50% KSR differentiation medium and 50% N-2 differentiation medium, add 3 μM CHIR99021 (Wnt pathway inhibitor), 25 ng / mL BMP4 (BMP pathway activator) and 100 nM EDN-3 (EDNRB pathway activator);

[0093] Day 8-9: Based on 25% KSR differentiation medium and 75% N-2 differentiation medium, add 3 μM CHIR99021 (Wnt pathway inhibitor), 25 ng / mL BMP4 (BMP pathway activator) and 100 nM EDN-3 (EDNRB pathway activator);

[0094] Day 10-11: Based on complete use of N-2 differentiation medium, and add 3 μM CHIR99021 (Wnt pathway inhibitor), 25 ng / mL BMP4 (BMP pathway activator) and 100 nM EDN-3 (EDNRB pathway activator).

[0095] The subsequent melanoblast-induced differentiation of melanocytes is the same as the method of step (4) in Example 1.

[0096] The comparison table of the culture process of Example 1, Comparative Examples 1-2 is shown in Table 1:

[0097] Table 1

[0098]

[0099] Experiment 1, qRT-PCR molecular detection

[0100] The TRIzol method was used to extract the melanocyte RNA obtained from Example 1, Comparative Example 1 and Comparative Example 2, and a reverse transcription kit (Savil, G3337, China) was used to obtain cDNA. The reaction program was 95°C pre-denaturation for 30s, 95°C denaturation for 15s, 56°C annealing for 30s, 72°C extension for 30s, and repeated for 40 cycles. Human ACTB was used as a control to calculate the ΔCt value of each target gene, and the relative gene expression amount was calculated. The primers used are shown in Table 2.

[0101] Table 2 qRT-PCR primer sequences

[0102]

[0103] Experiment 2, dopa staining verification

[0104] According to 3×10 5The density of 1 cell / hole was inoculated in a 6-well plate, and the cells were cultured until the confluence reached 60%, and then subjected to dopa staining. After the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes, 0.1% L-DOPA staining solution was used for incubation at 37°C for 2 hours in the dark, and the color change was observed until black or gray-black precipitates were formed. The excess staining solution was washed off, and the cells were observed and photographed under a microscope.

[0105] Experiment 3, detection of tyrosinase activity

[0106] The tyrosinase activity was determined using a tyrosinase activity detection kit (Solabio, BC4055, China). The cells were collected in a centrifuge tube, and the extraction solution was added at a concentration of 10 6 cells / mL. The cells were broken by ultrasonic treatment in an ice bath (power 200W, ultrasonic treatment for 3s, interval 10s, repeated 30 times). The supernatant was obtained by centrifugation at 12000g and 4°C for 10 minutes, and the absorbance at 505nm was determined.

[0107] The tyrosinase activity in the lysate was calculated using the following formula: tyrosinase activity (U / 10 6 cells) = 52.5 ÷ N x ΔA measured x F.

[0108] N: total number of cells / million; ΔA = A measured - A blank; F: dilution factor. Three sets of replicates were set for each sample.

[0109] Experiment 4, immunohistochemistry

[0110] The fresh tissue was fixed in 4% paraformaldehyde overnight, and then dehydrated with gradient ethanol and transparentized with xylene, and then embedded in paraffin and sectioned. After the sections were deparaffinized with xylene and antigen repaired, 3% H2O2 was used for blocking for 10 minutes, and then 5% bovine serum albumin was used for blocking for 1 hour. The primary antibody was added and incubated at 4°C overnight. Then the secondary antibody was added and incubated at room temperature for 1 hour, followed by DAB color development, hematoxylin restaining, and hydrochloric acid-ethanol differentiation. Finally, the sections were dehydrated with gradient ethanol, transparentized with xylene, and then mounted with neutral resin.

[0111] Experiment 5, HE staining

[0112] The fresh tissue was fixed with 4% paraformaldehyde overnight, and then dehydrated with gradient ethanol and transparentized with xylene, and then embedded in paraffin and sectioned. The sections were baked at 60°C for 90 minutes. After deparaffinization with xylene and rehydration by gradient ethanol series, the sections were stained with hematoxylin solution for 2 minutes, rinsed with tap water to reverse blue, and then treated with 1% hydrochloric acid ethanol solution for about 3 seconds. Then, rinse with running water, and stain with eosin solution for 30 seconds, dehydrate with gradient ethanol series, transparentize with xylene, and mount with neutral resin. After air-drying at room temperature, observation was performed.

[0113] Experiment 6, Immunofluorescence detection

[0114] Cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS, permeabilized with 0.3% Triton X-100 for 10 minutes, and then blocked with 1% BSA solution for 1 hour at room temperature. Then, the first antibody specific to the molecule was added and incubated overnight at 4°C. The next day, after rewarming and washing with PBS, the second antibody labeled with fluorescein was added and incubated for 1 hour at room temperature in the dark. Finally, DAPI staining was performed for 10 minutes, and the mounting medium was used for mounting. The images were observed and collected under a confocal microscope.

[0115] Experiment 7, hESC-MC cell epidermal transplantation

[0116] A region of about 2 cm x 2 cm on the back of the mouse was selected, and the hair was removed to fully expose the skin on the back. The exposed skin was gently rubbed with sandpaper until slight bleeding appeared on the surface. The skin was washed with normal saline for 3 times to remove epidermal impurities and tissue debris. The differentiated melanocytes were evenly applied to the wound at low (2.5 x 10 6 cells / 150 μl), medium (2.5 x 10 6 cells / 150 μl), and high (5 x 10 6 cells / 150 μl) concentrations, and a medical bandage was used for fixation.

[0117] Experiment 8, Tumorigenicity test

[0118] Six-week-old BALB / c immunodeficient mice were selected, and the melanocytes induced in Example 1 were collected. The melanocytes were subcutaneously injected into the right axillary of the mice at different doses, and divided into three experimental groups: low concentration (2.5 x 10 6 cells per mouse), medium concentration (2.5 x 10 6 cells per mouse), and high concentration (5 x 10 6 cells per mouse). Normal saline was used as a negative control, and an equivalent amount of mouse-derived melanoma cell line B16F10 was used as a positive control. The tumorigenicity was observed.

[0119] The experimental data were analyzed using IBM SPSS software, t-test was used to analyze the differences between groups, and Graphpad 10.1.2 software was used for plotting, wherein * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. P < 0.05 means that the results have statistical significance.

[0120] The results of Experiments 1-8 are shown in Figures 1-5 , and the analysis results are as follows:

[0121] 1. A strategy based on neural crest lineage commitment promotes hESCs differentiation into neural crest cells

[0122] The same batch of hESCs cells were expanded and then induced to differentiate by three protocols: Example 1 (labeled as protocol two), Comparative Example 1 (labeled as protocol one), and Comparative Example 2 (labeled as protocol three). As shown in Figure 1 , on the sixth day of differentiation, hESCs were observed to differentiate into neural crest-like cells with morphology of polygonal or fusiform shape, with highly dynamic cytoplasmic processes or pseudopods in all three protocols. However, in protocols two and three, the cell state was significantly better than that in protocol one (see Figure 1 a). The number of cells was counted, and the results showed that the number of cells on the sixth day of differentiation in all three protocols increased significantly, indicating that all three culture media were suitable for inducing hESCs to differentiate into neural crest cells at this stage, and there was no significant difference in cell proliferation (see Figure 1 b).

[0123] Gene expression detection on the third day of differentiation found that in protocol one, the neural ectoderm marker gene PAX6 was highly expressed, while in protocols two and three, PAX6 was low or not expressed (see Figure 1 c). In addition, the neural crest lineage development marker gene-TFAP2A was significantly highly expressed in protocols two and three (see Figure 1 d), which was consistent with the differences in the reported differentiation mechanisms, suggesting that protocols two and three had already deviated towards neural crest fate at this stage. On the sixth day of differentiation, neural crest marker gene PAX3 was highly expressed in all three protocols (see Figure 1 e), indicating that all three protocols could successfully induce hESCs to differentiate into neural crest cells. In addition, the expression levels of SOX10 and SOX9 were significantly increased in protocols two and three (see Figure 1 f and Figure 1 g), and the synergistic effect of these two molecules could further activate the downstream melanocyte expression-related target genes of MITF transcription, laying a molecular foundation for subsequent differentiation of the melanocyte lineage.

[0124] 2. A new differentiation strategy can promote neural crest cells to differentiate into melanocytes

[0125] At the key stage of differentiation of neural crest cells into melanoblasts, the three differentiation protocols used different culture strategies: protocols one and three continued to use the traditional method of continuous induction based on KSR differentiation medium, while protocol two innovatively used Essential 6 medium for differentiation throughout the process. On the twelfth day of differentiation, melanoblast-like cells were successfully obtained in all three protocols (see Figure 2 a). However, in terms of cell number, protocol two was significantly higher than protocols one and three (seeFigure 2 (b) suggests that the Essential 6 culture medium system is more conducive to the expansion of melanocytes.

[0126] Further analysis at the molecular level, including temporal expression assays, revealed that the expression levels of melanocyte marker molecules in Scheme 2, including PMEL (premelanosome matrix protein), TYR (tyrosinase), MITF (microphthalmia-associated transcription factor), and DCT (dopachrome isomerase), were significantly higher than those in Schemes 1 and 3. Figure 2 (cf) This indicates that the differentiation system constructed based on Essential 6 medium is significantly superior to conventional methods (Schemes 1 and 3) in inducing melanocytes. By day 25 of differentiation, melanocytes further mature into melanocytes with typical morphological characteristics, exhibiting polygonal or irregular shapes, and extending multiple slender dendritic processes to transfer melanin. Figure 2 g). At the terminal stage of differentiation, scheme two still maintains an advantage in cell number ( Figure 2 h). Molecular-level detection results also showed that in melanocytes obtained through scheme two, the expression levels of marker genes (MITF, TYR, DCT, Melan-A (melanocyte lineage-specific antigen A)) were significantly better than in other schemes. Figure 2 (il). Of particular note is that MITF, as a core regulator of melanocyte lineage differentiation, shows a positive correlation between its high expression level and the degree of terminal differentiation of melanocytes. This suggests that scheme two not only leads in differentiation efficiency but also performs best in promoting cells to a functionally mature state.

[0127] 3. The new differentiation strategy results in a stronger melanin synthesis capacity.

[0128] Dopa staining analysis confirmed that all three differentiation protocols could successfully obtain melanocytes with melanin-producing function. Figure 3 a). Further quantitative detection of intracellular tyrosinase (TYR) activity showed that the tyrosinase activity of melanocytes differentiated from scheme 2 was significantly higher than that of the control group and other scheme groups. Figure 3 (b) indicates that this optimized system has a significant advantage in promoting melanocyte pigment synthesis. Immunofluorescence experiments showed that melanocytes induced by scheme two expressed key melanocyte marker proteins such as DCT, MITF, SOX10, and TYR, and their expression patterns were highly consistent with those of normal human epidermal melanocytes (NHEM). Figure 3 c) indicates that the differentiated cells are extremely similar to natural melanocytes in molecular phenotype. In summary, Scheme 2 not only efficiently drives hESCs to differentiate into melanocytes, but also shows significant advantages in functional maturity and pigment synthesis capacity.

[0129] 4. hESC-MC cell transplantation

[0130] To evaluate the transplantation potential of hESC-MC, the melanocytes obtained in protocol two were transplanted into the epidermis of the back of mice at different concentrations (low, medium, and high). On the 12th day after transplantation, obvious pigmentation was observed in the transplanted areas at all three concentrations, indicating that the melanocytes had successfully colonized and begun to function to synthesize melanin. The pigmented area and the degree of pigmentation no longer expanded after the 30th day after transplantation, and by the 50th day, the pigmentation of the epidermis in the transplanted area was more uniform, exhibiting controllable, non-invasive growth characteristics. Figure 4 a). In addition, the pigmented area did not disappear with the periodic changes in the mouse hair follicle cycle (anagen, catagen, telogen), indicating that the transplanted human melanocytes had successfully and stably integrated into the microenvironment of the skin, and their survival and function were independent of the periodic activation of the mouse hair follicle cycle, demonstrating long-term stability of the transplantation.

[0131] The safety and functionality were further verified by histological analysis: HE staining showed that there were a large number of pigment-rich melanocytes in the subcutaneous tissue of the transplanted area, with normal cell morphology and orderly arrangement, and no signs of abnormal proliferation. Figure 4 b). Immunohistochemical experiments showed that the expression levels of the melanocyte marker molecules DOPA chromotropic enzyme (DCT), Melan-A, SOX10, and tyrosinase (TYR) in the skin of the transplanted area were significantly higher than those in the control group. Figure 4 c). This confirmed the functional activity of the transplanted cells at the molecular level. The above results indicate that when hESCs are induced to differentiate into mature melanocytes and transplanted into the skin, they can achieve safe, stable, and long-term functional regeneration.

[0132] 5. Analysis of the safety of hESC-MC transplantation

[0133] To verify the safety of hESC-MC transplantation, we transplanted different concentrations of hESC-MC into the skin of mice in different ways. The low, medium, and high concentrations of hESC-MC were transplanted into the skin of mice in the form of cell epidermis, and no tumors or any abnormal morphology were observed in the epidermis up to the 50th day after transplantation. Figure 5 a). No black patches or tumor tissue were found in the subcutaneous muscle tissue of the transplanted area. Figure 5 b). Meanwhile, histological examination of the heart, spleen, and kidney of the experimental animals showed no abnormal lesions in their morphology and structure. Figure 5c-e). These results preliminarily confirmed that the effect of hESC-MC in the dorsal skin graft model was highly localized and tissue-specific, and its biological behavior was effectively limited to the epidermis, without adverse effects on deep tissues or distant organs, thus preliminarily verifying its safety under this specific transplantation condition. In addition, in order to further clarify the safety of hESC-MC, hESC-MC was transplanted into the axillary region of immunodeficient mice by subcutaneous injection, and the results showed that on the 12th day of transplantation, tumor tissues appeared in mice inoculated with three concentrations of hESC-MC cells Figure 5 f). The above results suggest that the use of hESC-MC for epidermal transplantation has good biological safety, but attention should be paid to the inoculation method during transplantation. If subcutaneous injection is performed, it has tumorigenicity.

[0134] In summary, the present application provides a completely new differentiation melanocyte induction system. Compared with the traditional melanocyte differentiation system, the core difference lies in the optimization of the hESC-neural crest cell-melanoblast stage. The new scheme discards the complex differentiation process of embryonic stem cells-ectoderm-neural ectoderm-neural crest cells, and instead directly obtains neural crest cells from embryonic stem cells. This improvement significantly improves the differentiation efficiency and controllability of neural crest cells. In addition, at the 3rd day of differentiation, neural crest fate markers show significant expression, while the traditional differentiation scheme is still in the neural ectoderm stage, and the cells have not yet obviously biased towards the neural crest fate. By the 6th day of differentiation, neural crest marker genes are expressed in all differentiation schemes, and the new system can significantly activate SOX10 and SOX9, which are downstream target genes of MITF transcription related to melanocyte expression, and their expression levels are much higher than those of the traditional scheme. When the differentiation enters the melanoblast stage, the new differentiation system shows significant advantages: the expression levels of genes related to melanocyte maturation are all higher than those of other differentiation groups. By the 25th day of differentiation, melanocytes are successfully obtained in three schemes, and the new induction system, i.e. scheme two, shows stronger expression in cell number, expression of related genes and melanocyte function, such as tyrosinase activity and melanin synthesis ability.

[0135] In terms of therapeutic potential, the present study confirmed that the melanocytes obtained by induced differentiation of hESC-MC have significant clinical application value. These functional cells can successfully colonize the host skin tissue and stably perform the physiological function of synthesizing melanin for a long time. More importantly, their survival and function maintenance do not depend on the periodic changes of the host's own hair follicles, showing good autonomy and stability, which provides a solid foundation for realizing long-term functional regeneration. The transplanted cells exhibit controllable and non-invasive growth characteristics, and the pigmentation range of the transplanted area is stable without abnormal expansion or invasion, indicating that they have good biological safety and will not cause adverse effects on the surrounding normal tissues and other distant organ functions.

[0136] The above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application, and all should be covered in the scope of the claims of the present application. The technical, shape and structure parts not described in detail in the present application are well-known technologies.

Claims

1. A method for inducing hESC-melanocytes based on the directed differentiation of neural crest lineage, characterized in that, The induction method includes the following steps: (1) H9 human embryonic stem cells were cultured. When the cell confluence was ≥50%, H9 human embryonic stem cells were induced to differentiate into neural crest cells by sequentially using Essential 6 medium No. 1 and Essential 6 medium No.

2. (2) Neural crest cells were induced to differentiate into melanocytes using differentiation culture medium, and then melanocytes were cultured using melanocyte selection culture medium.

2. The method according to claim 1, characterized in that, The cell culture steps for H9 human embryonic stem cells in the induction method are as follows: A. After coating the culture dish with DMEM / F12 medium containing Vitronectin, H9 human embryonic stem cells were resuspended in mTeSR1 basal medium containing 1% penicillin-streptomycin and cultured until the cell confluence reached 80% and then passaged. B. H9 human embryonic stem cells were pre-seeded in DMEM / F12 culture dishes coated with Matrigel and cultured in mTeSR1 basal medium until the cell confluence was ≥50%.

3. The method according to claim 2, characterized in that, The specific operation process of A is as follows: Use containing 1µg / cm 2 Vitronectin was coated in DMEM / F12 medium and incubated at 37°C and 5% CO2 for at least 1 hour. After coating, the liquid was aspirated and the medium was used for later use. H9 human embryonic stem cells were resuspended in mTeSR1 basal medium supplemented with 2.5 µM Blebbistatin and 1% penicillin-streptomycin. The cell suspension was then transferred to coated culture dishes and cultured at 37 °C and 5% CO2. After 24 h, the medium was replaced with mTeSR1 basal medium (containing 1% penicillin-streptomycin) without Blebbistatin. The mTeSR1 basal medium (containing 1% penicillin-streptomycin) was replaced every 24 hours. When the cell confluence reached 80%, the cells were passaged.

4. The method according to claim 2, characterized in that, The specific operation process of B is as follows: H9 human embryonic stem cells were pre-seeded in DMEM / F12 culture dishes coated with 10 mg / mL Matrigel and cultured in mTeSR1 basal medium (containing 1% penicillin-streptomycin) for 24 hours; when the cell confluence reached 50%-60%, subsequent differentiation culture was carried out.

5. The method according to any one of claims 1-4, characterized in that, The Essential 6 medium in (1) is: based on Essential 6 medium, with the addition of 1 ng / mL BMP4, 10µMSB431542 and 600nM CHIR99021; The second Essential 6 medium is based on Essential 6 medium, with the addition of 10 µM SB431542 and 1.5 µM CHIR99021.

6. The method according to claim 5, characterized in that, The specific procedures for differentiating and culturing neural crest cells in (1) are as follows: Days 0-2: Cell differentiation was induced using Essential 6 medium (No. 1); Days 3-5: The cells were cultured in Essential 6 medium (No. 2) to obtain neural crest cells.

7. The method according to any one of claims 1-4, characterized in that, The differentiation culture medium in (2) includes: Differentiation medium No. 3: Based on a mixture of 50% Essential 6 differentiation medium and 50% N-2 differentiation medium, with the addition of 3 μM CHIR99021, 25 ng / mL BMP4 and 100 nM EDN-3; Differentiation medium No. 4: Based on a mixture of 25% Essential 6 differentiation medium and 75% N-2 differentiation medium, with the addition of 3 μM CHIR99021, 25 ng / mL BMP4 and 100 nM EDN-3; Differentiation medium No. 5: Based entirely on N-2 differentiation medium, with the addition of 3µM CHIR99021, 25ng / mL BMP4 and 100nM EDN-3; The Essential 6 differentiation medium mentioned above is based on Essential 6 medium, with the addition of 6 ng / mLFGF-2 and 0.1 mM β-mercaptoethanol; The N-2 differentiation medium is based on DMEM / F12 medium with the addition of 1.55 g / L glucose and 1% N-2 supplement.

8. The method according to claim 7, characterized in that, The specific procedures for the differentiation and culture of melanocytes are as follows: Days 6-7: Cultured using differentiation medium No. 3; Days 8-9: Cultured using differentiation medium No. 4; Days 10-11: Melanocytes were obtained by culturing in differentiation medium No.

5.

9. The method according to any one of claims 1-4, characterized in that, The melanocyte selective medium in (2) is based on DMEM / F12, with the addition of 6 ng / mL FGF-2, 500µM dibutyryl cyclic adenosine monophosphate calcium, 0.8% insulin-transferrin-selenium, 3µM CHIR99021 and 100nM EDN-3.

10. The method according to claim 9, characterized in that, The specific procedures for the differentiation and culture of melanocytes in (2) are as follows: The well plates were coated with 1 µg / mL laminin-1 and 10 mg / mL Materigel matrix gel overnight and washed with PBS before use. Days 12-25: Seed cells in well plates, add melanocyte selection medium, and change the medium every other day.

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