miR-138调控iPSC源性神经嵴细胞向雪旺细胞定向分化

Schwann cells (SC) transplant can promote the repair of nerve injuries, however, it is always faced with the problems of difficult preparation and limited sources. The induced Pluripotent Stem Cells（iPSC）which can be induced to differentiate into SC are promising to solve these difficulties. However, the differentiation process experiences the middle stage of neual crest cells (NCC). iPSCs can be induced to differentiate into NCC quickly and efficiently, however, the differentiation of NCC into SC is long time-consuming with a low differentiation efficiency. Previous study showed that transcription factors and cyclins play an important role in the process of NCC differentiation into SC. According to literature reports, miRNA expression spectrum and functional analysis in the process of SC development, bioinformatics predictions, we infer that miR-138 may regulate their expression levels and then regulate the differentiation process of NCC into SC. In this study, we plan to use our previously established iPSC cell line and experimental platform of inducing iPSC quick, efficient and steady differentiation into NCC. We examine the expression level of miR-138 during the differentiation process of NCC into SC, detect the expression levels of its potential target genes by altering the cellular level of miR-138. Luciferase assays are also employed to verify the regulation targets of miR-138. This study is aimed to establish the efficiently differentiation platform of iPSC derived NCC into SC, elucidate the regulatory effect and mechanism of miR-138 in this process. The anticipated results are helpful to elucidate the mechanism of stem cell directional differentiation, and provide new strategies for nerve injury repair.

雪旺细胞（SC）移植能够促进神经损伤的修复，但常面临制备困难、来源受限的难题。利用诱导多能干细胞（iPSC）向SC分化，有望解决此困境。该分化过程经历神经嵴细胞（NCC）阶段，iPSC能够快速高效向NCC分化，但NCC向SC分化效率不高，耗时很长。转录因子和细胞周期蛋白在NCC向SC分化中发挥关键作用，根据SC发育过程miRNA表达谱与功能分析和生物信息预测，我们推断miR-138可能调控它们的表达进而调控分化进程。本课题使用已经建立的iPSC细胞系和iPSC向NCC快速高效稳定分化的实验平台，检测miR-138在NCC向SC分化过程的表达，改变其胞内水平促进分化，检测其潜在靶基因表达，采用荧光素酶检测验证其调控靶点。本研究旨在构建iPSC源性NCC向SC高效分化的实验平台，探讨miR-138在此过程中的调控作用及机理，预期成果将有助于阐明干细胞定向分化机理，并为神经损伤的修复提供新策略。

雪旺细胞（Schwann cell, SC）移植能够促进神经损伤的修复，但常面临制备困难、来源受限的难题。本课题利用诱导多能干细胞（induced pluripotent stem cell, iPSC）来源的神经嵴细胞(neural crest cell, NCC)向SC分化，采用诱导剂诱导和miR-138调控的方法，调控NCC向SC分化，以期解决神经修复过程中SC来源受限的困境。本课题通过诱导剂诱导的方法由iPSC制备NCC。诱导14天左右出现NCC标记物HNK1和p75呈阳性。经过20天的时间，iPSC诱导分化成NCC的分化率达55.8±12.3%，经过传代培养，得到纯度达90%以上的NCC。.为检测miR-138在NCC向SC分化过程中的作用和机理，设定三个实验组：诱导剂诱导分化组（A）、单独miR-138转染组（B），miR-138转染+诱导剂分化组（C），于3d, 7d和14d转染。研究发现，A组于21.2±1.4天时出现SC形态；S100阳性率达80.2±6.9%。检测A组NCC向SC分化过程中的miR-138表达水平，SC分化相关转录因子和Ccnd1 mRNA表达水平,其结果显示，miR-138和Egr2表达水平上升；Ccnd1、Sox2和Sox5 mRNA表达水平下降。B组未显示出向SC定向分化的趋势。C组于平均17.6±1.9天出现SC形态，S100阳性率为81.7±13.2%。.Real-time PCR结果显示，miR-138分别与Ccnd1、Sox2和Sox5的表达呈现负相关，根据生物信息学预测结果显示，它们可能是miR-138调控的靶基因。通过双荧光素酶报告基因检测技术验证miR-138 调控的靶位点，证实了Ccnd1和Sox2是miR-138 调控NCC向SC分化的靶基因。.本研究构建了NCC向 SC 高效分化的实验平台，由iPSC作为起始细胞，制备了高纯度的雪旺细胞，为神经损伤后获取大量神经修复细胞提供了可能性。转染miR-138，对分化率的影响不大，却可以缩短NCC向SC诱导分化的时间，为损伤修复缩短时间。探讨了miR-138 在此过程中的调控作用及机理，丰富了干细胞定向分化的机理。