基于单细胞转录组测序结果探索人胚胎干细胞向黑素细胞定向分化中关键因子的研究

组织工程表皮移植是申请人主持开发的白癜风治疗新技术，临床效果显著。黑素细胞来源不足限制其进一步发展。我们前期从人胚胎干细胞（hESC）成功分化出了黑素细胞，但仍存在分化效率低、方案不明确等问题。黑素细胞分化关键通路的发现有助于开发新的方案。我们采用单细胞转录组测序技术，发现随着黑素细胞的生成，EDNRB与MITF基因的表达逐渐升高，EDNRB相关通路的活化程度远高于其他通路，因此推测EDN3-EDNRB-MITF通路可能是hESC向黑素细胞分化的关键通路。本项目将进一步验证EDN3-EDNRB-MITF通路在黑素细胞分化过程中的作用和机制；验证EDN3、骨化三醇、罗格列酮等可激活EDN3-EDNRB-MITF通路的物质成分对hESC黑素细胞分化的作用；找到EDN3、骨化三醇、罗格列酮的最佳添加剂量与时间，形成优化的 hESC黑素细胞分化方案，提高分化效率，为未来实现临床应用奠定基础。

Tissue Engineering Stratified Epithelial Sheets technology was developed by the applicant as the principle investigator, and it already showed significant clinical effects. However, lack of source of melanocytes restricts its further development. We successfully differentiated melanocyte from human embryonic stem cells（hESCs）earlier, but there are still problems such as differentiation is not efficient enough and the method need to be improved. The discovery of the key pathway of melanocyte differentiation helps the development of new method. We adopted 10x single-cell transcriptome technology, and found that as melanocytes generated, expressions of both EDNRB and MITF increased gradually and up-expression level of EDNRB related pathways was much higher than that of other pathways, therefore we infer that EDN3-EDNRB-MITF pathway could be the key pathway of hESCs differentiates to melanocyte. The project will further verify the role and mechanism of EDN3-EDNRB-MITF pathway in the process of melanocyte differentiation. The project will also verify the role of the chemical compounds that activates EDN3-EDNRB-MITF pathway such as EDN3, 1,25-dihydroxyvitamin D3 and rosiglitazone in melanocyte differentiation. The project will find the optimal dosage and timing of EDN3, 1,25-dihydroxyvitamin D3, and rosiglitazone to improve the melanocyte differentiation method, increase differentiation efficiency, and layout the foundation of future clinical application.