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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i PS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i PSCs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i PS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 i PSCs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。
Objective To explore the interaction between these two in vitro differentiated cells using inner ear hair cells and supporting cells that induced the differentiation of pluripotent stem cells. Methods Firstly, three iPS cells (wild-type strain, MYO7A-deficient strain and MYO7A-corrected strain) were induced to differentiate into inner earlobe progenitor cells and inner ear hair cells by two-step cell wall adherent method. Secondly, the interaction between inner ear hair cells and supporting cells differentiated in vitro was explored by the method of cellular immunochemistry. Finally, the epithelial-like inner ear progenitor cells expressing green fluorescent protein (EGFP) The method of round window membrane puncture was transplanted into the inner ear of Baihua Rongchang pig to observe and analyze the migration, differentiation and connection of transplanted cells in vivo. Results Differentiation of three iPS cells into hair cells of the inner ear resulted in the differentiation of some cells into supporting cells. The immunofluorescence results of E-cadherin, N-cadherin and ZO-1 in differentiated cells showed that E-cadherin, The expression of N-cadherin and ZO-1 in the sertoli-supporting cells and the supporting cells-hair cell junctions were also observed. Immunostaining of the cochlea after 4 weeks of transplantation showed that a few cells were successfully transplanted in the three different transplanted cells Migrated to the damaged hair follicles - Keratei, and express the hair cell marker protein MYO7A. There was E-cadherin, N-cadherin and ZO-1 expression between the transplanted cells and between the transplanted cells and the host cells. Conclusion Intracellular and extracellular hair cells and supporting cells induced by PSCs can form Ca2 + -adhesive and tight junctions. These findings have certain scientific significance for the perfection and development of the detoxification strategy of hair cell replacement therapy.