iPSCs定向分化的内耳毛细胞与支持细胞间相互作用的研究

摘要

目的 利用诱导多能性干细胞定向分化的内耳毛细胞和支持细胞探究这两种体外诱导分化细胞之间的相互作用.方法 首先,利用细胞单层贴壁两步诱导法将三株iPS细胞(野生株、MYO7A缺陷株、MYO7A校正株)向内耳祖细胞及内耳毛细胞诱导分化,探究iPSCs定向分化内耳毛细胞的过程中是否有支持细胞的产生;其次,通过细胞免疫化学的方法探究体外分化的内耳毛细胞和支持细胞间的相互作用;最后,将表达绿色荧光蛋白(EGFP)的上皮样内耳祖细胞以圆窗膜穿刺的方法移植到白化荣昌猪的内耳中观察分析移植细胞在体内的迁移、分化以及在体内形成的联系.结果 三株iPS细胞诱导分化为内耳毛细胞的过程中均有一部分细胞分化为支持细胞;对分化细胞进行E-cadherin、N-cadherin和ZO-1的免疫荧光检测结果显示,E-cadherin、N-cadherin和ZO-1在支持细胞-支持细胞连接和支持细胞-毛细胞连接间都有表达;移植4周后,耳蜗免疫组织化学结果显示,三株不同来源的移植细胞均有少量细胞成功迁移到了毛细胞受损部位—柯底氏器,并表达毛细胞标志性蛋白MYO7A.移植细胞之间以及移植细胞与宿主细胞之间有E-cadherin、N-cadherin和ZO-1的表达.结论 iPSCs诱导分化的内耳毛细胞和支持细胞在体内、外均能形成钙粘连接和紧密连接.这些研究结果对毛细胞取代法治疗耳聋策略的完善与发展有一定的科学意义.%Obiective To examine the relationships between inner ear hair cells and supporting cells from induced pluripotent stem cells (iPSCs). Methods Normal iPSCs derived from a healthy donor (C-iPS), iPSCs with myosin7A mutation derived from a deaf patient (P-iPS) and iPSCs with myosin 7A corrected genetically (CP-iPS) were induced to differentiate into otic progenitors, and then OEPs were isolated from otic progenitors and induced to differentiate into in-ner ear hair cell-like cells and supporting cells. Immunofluorescence was used to examine in vitro formation of intercel-lular junctions between the induced hair cell-like cells and supporting cells. Furthermore, EGFP-OEPs derived from three iPSCs were transplanted into the inner ear of albino Rong-Chang swine via round widow to examine the migra-tion, differentiation and intercellular junctions between these cells. Results OEPs derived from iPSCs could be induced into hair cell-like cells and supporting cells simultaneously, and intercellular junctions such as adhering junctions and tight junctions were formed between hair cell-like cells and supporting cells derived from OEPs. After transplantation, the grafted cells could migrate from the scala tympani to the scala media. However, only a limited number of cells were integrated into the native auditory epithelial tissue, and some EGFP-cells integrated into the organ of Corti and ex-pressed the hair cell marker MYO7A, indicating that these exogenous cells had differentiated into hair cell-like cells. Further immunolabeling assays showed production of adhering junction proteins (E-cadherin and N-cadherin) and a tight junction protein (ZO-1) between these differentiated cells and other cells, similar to what was observed in vitro. Conclusions Cell-cell interactions such as adhering junctions and tight junctions between hair cell-like cells and support-ing cells derived from iPSCs can be formed not only in vitro but also in vivo. These results may facilitate the develop-ment of hair cells replacement-based strategy for deafness.