论文部分内容阅读
目的:从人诱导多能干细胞(hiPSC)分化而来的视网膜色素上皮(RPE)细胞在治疗视网膜退行性疾病方面有很大的前景.本研究将探讨非集落样、分散的hiPSC分化为功能性RPE细胞(hiPSC-RPE细胞)的可能性,并提供一种基于细胞球的可选移植方法.方法:通过RT-PCR、免疫荧光和流式细胞术分析鉴定hiPSC-RPE.通过荧光素渗漏实验、跨上皮电阻测定、原子力显微镜观察、光感受器外节段吞噬实验、冰冻切片、死/活细胞染色、衰老相关β-半乳糖苷酶(SA-β-Gal)染色和免疫组化实验评估hiPSC-RPE的体内和体外功能.结果:hiPSC-RPE细胞阳性表达RPE细胞的生物标志物但不表达iPSC的生物标志物,例如CRALBP(97.4％)、EMMPRIN(93.8％)、Oct4(2.1％)和Sox2(2.0％).hiPSC-RPE细胞表现出与RPE细胞一样的特性,包括屏障功能、吞噬活性和极性膜.hiPSC-RPE成球处理后细胞阳性表达nestin,并且SA-β-Gal染色减少(P<0.01).hiPSC-RPE细胞球在脱细胞基质角膜上培养能形成单层.在碘酸钠诱导的RPE退化的青紫蓝兔视网膜下腔移植1个月,hiPSC-RPE细胞球能存活下来,并维持部分片状结构生长.结论:非集落样、分散的hiPSC能有效分化为功能性RPE细胞,而且移植后的hiPSC-RPE细胞球能在RPE退化的青紫蓝兔视网膜下腔维持部分片状结构生长.本研究可能为未来治疗RPE退行性疾病提供一种可选的细胞球移植方法.“,”AIM:To explore the feasibility of non-colony dissociated human induced pluripotent stem cells (hiPSCs)to differentiate into functional retinal pigment epithelial(RPE)cells(hiPSC-RPE cells),and offer an alterna-tive transplantation method based on cell spheroids. METHODS:The hiPSC-RPE cells were identified by RT-PCR,im-munofluorescence assay,and flow cytometry. The in vitro and in vivo functions of hiPSC-RPE were assessed by fluorescein leakage test,transepithelial electrical resistance assay,atomic force microscopic observation,photoreceptor outer segment (POS)phagocytosis assay,frozen tissue sections,live/dead cell staining assay,senescence-associated β-galactosidase (SA-β-Gal)staining,and immunocytochemistry. RESULTS:The hiPSC-RPE cells positively expressed biomarkers of RPE cells but not iPSCs,such as CRALBP(97. 4％),EMMPRIN(93. 8％),Oct4(2. 1％),and Sox2(2. 0％). These cells displayed RPE cell-like characteristics including barrier function,phagocytic activity,and polarized membrane. The cells derived from hiPSC-RPE cell spheroids positively expressed nestin and exhibited reduced SA-β-Gal staining(P<0. 01). The hiPSC-RPE cell spheroids were able to form monolayer on decellularized corneal matrices(DCM). After 1 month of subretinal transplantation,the hiPSC-RPE cell spheroids survived and maintained segmental sheet growth in Chinchilla rabbits with sodium iodate-induced RPE degeneration. CONCLUSION:Non-colony dissociated hiPSCs can effectively differentiate into functional RPE cells,and hiPSC-RPE spheroids maintain segmental sheet growth in the sub-retinal space of RPE-degenerative Chinchilla rabbits in vivo,which may offer cell spheroid transplantation as an alternative method for the treatment of RPE degenerative diseases in the future.