非集落样、分散的人诱导多能干细胞可分化为球型可移植的功能性视网膜色素上皮细胞

Spheroid transplantable and functional retinal pigment epithelium derived from non-colony dissociated human induced pluripotent stem cells

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-associatedβ-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.