摘要目的:探究体外将人诱导多能干细胞(hiPSCs)定向分化成视网膜类器官(ROs)的进程并进行小鼠在体细胞移植。方法:使用定向分化培养基使BC1-eGFP hiPSCs悬浮培养得到神经球，培养第7天时将神经球贴壁培养诱导出神经视网膜上皮结构，然后人工分离类视网膜结构悬浮培养，进一步定向诱导分化得到成熟的ROs。采用实时荧光定量PCR检测培养第0、7、15、21和30周标志基因的表达水平变化，采用免疫荧光染色法检测培养第8天、第15天、第15周、第21周和第30周标志基因的蛋白水平变化来鉴定诱导分化进程及效果。在体移植ROs实验中，首先破坏外界膜，然后将经消化的RO细胞悬液从角巩膜缘注射到 Gnat1-/-小鼠视网膜下腔，细胞移植后5个月进行视网膜切片的免疫荧光染色检测植入细胞的存活情况、与宿主视网膜的整合以及进一步的成熟分化情况。 结果:形态学和免疫荧光染色结果显示，体外诱导hiPSCs早期形成的眼区细胞高表达神经视网膜上皮特异性标志物PAX6和SOX1，随后细胞可以表达视网膜祖细胞特异性标志物LHX2，集落外圈逐渐形成圆形透明马蹄状的类视网膜结构。机械分离并悬浮培养得到的ROs直径约为1 mm，类视网膜组织逐渐增厚并出现类视网膜色素上皮细胞。实时荧光定量PCR结果显示视网膜祖细胞标志基因 VSX2表达在第7周即达峰值并持续高表达( F＝168.30， P<0.01)，视网膜前体细胞标志基因 RCVRN在第7周也开始出现并逐渐高表达( F＝271.60， P<0.01)，感光蛋白基因 RHO在第15周开始表达( F＝95.02， P<0.01)，而成熟感光细胞标志 OPN1LW/ MW的表达在第21周明显升高( F＝40.57， P<0.01)。免疫荧光染色进一步检测到感光蛋白RHO在第30周达高表达状态。RO细胞移植后3周，自身带有绿色荧光的细胞成功在宿主小鼠的视网膜外核层中长期存活，移植后5个月植入细胞表达功能性光信号转导蛋白GNAT1。 结论:体外3D培养诱导hiPSCs生成ROs可成功地模拟人体内视网膜的发育过程，移植的RO细胞能在受体小鼠眼内长期存活，进入外核层并进一步发育成熟为类感光细胞。

abstractsObjective:To investigate the differentiation process of human-induced pluripotent stem cells (hiPSCs) into retinal organoids (ROs) in vitro and its potential application in cell transplantation, and to provide a fundamental study for stem-cell therapy. Methods:BC1-eGFP hiPSCs were differentiated into neurospheres in directional differentiation medium via suspension culture.On day 7, hiPSCs-induced-neurospheres were seeded onto Geltrex-coated dishes to induce neural retinal (NR) domains.On day 28, the NR domains were manually detached and collected.These NRs were cultured until the maturation of ROs.The quantitative real-time PCR (at week 0, week 7, week 15, week 21 and week 30 group) and immunohistochemistry (at day 8, day 15, week 15, week 21 and week 30 group) were used to characterize the differentiation process of hiPSCs.For ROs transplantation, the ROs were digested, and the cell suspension was injected into the subretinal cavity of Gnat1-/- mice with the outer limiting membrane damaged in retina.Immunohistochemistry was also used to detect the survival and integration ability of the implanted cells 5 months after transplantation. Results:The morphology and immunofluorescence staining illustrated that the cells induced from hiPSCs highly expressed the neural-retinal-epithelial specific markers PAX6 and SOX1 in the early stage, then the cells expressed the retinal-progenitor-cell specific marker LHX2 and a transparent and horseshoe-shaped NR domain was formed at the outer region of the colony.ROs was obtained by manual isolation and suspension culture.The diameter of ROs was about 1 millimeter.The retinal-like tissue gradually became thicker, even formed retinal pigment epithelial cells.Quantitative real-time PCR results showed that the expression of retinal-progenitor marker VSX2 peaked at week 7 and maintained its high expression thereafter ( F=168.30, P<0.01); the expression of retinal-precursor marker RCVRN also appeared at week 7 and increased thereafter ( F=271.60, P<0.01); the expressions of RHO was detected at week 15 ( F=95.02, P<0.01), and the expression of OPN1LW/MW was detected at week 21 ( F=40.57, P<0.01). Moreover, the expression of photoreceptor protein RHO maintained in a relatively high expression state at week 30.Three weeks after the transplantation of RO cells, cells with green fluorescence were successfully moved into the outer nuclear layer of the host retina.Four to six months after transplantation, the implanted cells expressed the functional light signal transduction protein GNAT1. Conclusions:Transplantation of retinal organoids in vivo can recapitulate the development of human retina.Transplanted RO cells can effectively move into the outer nuclear layer, differentiate into photoreceptors and survive in the recipient mice's retina over several months.