三维培养hESCs来源视杯样组织中c-kit和SSEA-4的时空表达特点研究

Spatiotemporal expression profiles of c-kit and SSEA-4 in optic cup-like tissues derived from human embryonic stem cells by three-dimensional culture

目的研究人胚胎干细胞(human embryonic stem cells,h ESCs)三维培养后形成的视杯样组织中c-kit和SSEA-4表达的时空特性。方法 H1人胚胎干细胞行三维培养和神经诱导分化,在分化7、14、21、30、49、70、90 d固定组织并行免疫荧光鉴定检测c-kit和SSEA-4的表达,同时在相应时间点将分化组织消化成单细胞行流式细胞仪检测验证。结果三维培养第14天可见Rax+视泡(optic vesicle,OV)样结构,第30天形成具有内层Pax6+、外层Mitf+的双层视杯样(optic cup,OC)结构,第90天可见部分Crx+细胞。c-kit主要表达于h ESCs来源OV状及OC状的神经上皮样结构中,SSEA-4早期均匀表达于整个组织,晚期则主要集中于周边部。c-kit+细胞比例呈先升高(P<0.01)后下降(P<0.01)趋势,并在分化14 d达40%的峰值。SSEA-4+细胞比例从分化0 d至70 d持续下降(P<0.01),直到90 d一直维持于该低水平。结论在三维培养h ESCs来源OV或OC状组织中,c-kit基因随诱导分化而激活,且主要表达于神经上皮层,呈先增高后下降趋势。SSEA-4早期高表达于整个组织,之后逐渐减少至周边部并维持于低水平状态。

Objective To investigate the spatiotemporal expression profiles of c-kit and stage-specific embryonic antigen-4（ SSEA-4） in optic cup（ OC）-like tissues self-organized from human embryonic stem cells（ h ESCs） by three-dimensional culture. Methods The expression of c-kit and SSEA-4 in optic vesiclelike or optic cup-like tissues derived from the h ESCs were identified by flow cytometry and immunofluorescence assay on the 7th,14 th,21st,30 th,49th,70 th,and 90 th days. Results On the 14 th day,Rax-positive optic vesicle（ OV）-like structures were observed,and OC-like structures with Pax6-positive inner layer and Mitf-positive outer layer were observed on the 30 th day. Furthermore,a few cells expressing Crx were found on the 90 th day. C-kit was mostly expressed in the OV-like and OC-like neuroretina. The percentage of cells with c-kit expression was increased（ P 〈 0. 01）,and then reached the peak of 40% in 14 d,and then decreased gradually（ P 〈 0. 01）. However,the amount of cells positive to SSEA-4 was decreased homogeneously in the whole tissue at early stage,and continued to decline to a low level till the 90 th day. Conclusion In the OV-like or OC-like tissues derived from h ESCs,c-kit is activated in the neuroretina,and its expression level shows a descending trend after the first rise. However,SSEA-4 is highly expressed in the whole tissue at early stage and then gradually decreased to maintain at a low level,and its expression is restricted to the peripheral region.