Sun Xuerong, Jiang Ruzhang, Zhang Yuehong, Chen Mengfei, Xiang Peng, Qi Ying, Gao Qianying, Huang Bing, Ge Jian
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Mol Vis. 2009 Dec 2;15:2503-14.
Retinal progenitor cells (RPCs) are the most valuable seed cells in replacement therapy for neural retinal diseases. The competence of RPCs changes with retinal development. Gene expression plays a fundamental role in determining the competence. To improve the selection of the right-timing RPCs for replacement therapy, we compared the gene expression between embryonic day (E) 13.5 and E17.5 RPCs and further explored their gene expression and differentiation capacity in vitro.
Timed-pregnant E13.5 and E17.5 RPCs were freshly harvested and cultured in proliferation conditions for 4 days and then in differentiation conditions for 8 days. At different time points, the expression of key genes involved in retinal development was investigated by quantitative reverse transcription-PCR or immunofluorescence.
The expression of 14 key genes involved in retinal development was investigated in freshly harvested E13.5 and E17.5 RPCs. The freshly harvested E13.5 RPCs showed a high expression of retinal ganglion cell (RGC)-related genes, including Math5, Brn3b, Islet1, and Nfl, while the freshly harvested E17.5 RPCs displayed a high expression for Nrl, GFAP, and Thy1, the key genes involved in rod photoreceptor development, glial cell development, and synaptogenesis, respectively. During proliferation culture in vitro, the gene expression changed dramatically in both RPCs. After the 4 days of proliferation culture, the expression levels of most genes (11 of the 14 genes) in E13.5 RPCs came close to those in the freshly harvested E17.5 RPCs. Differentiation of RPCs in vitro was verified by the significant decrease in Nestin expression and BruU incorporation efficiency. After the 8 days of differentiation in vitro, the expression level of RGC-related genes (Math5, Brn3b, and Islet1) was still significantly higher in E13.5 RPCs than in E17.5 RPCs. In contrast, the expression level of Nrl and GFAP was significantly higher in E17.5 RPCs than in E13.5 RPCs. In morphology, the differentiated E13.5 RPCs displayed more robust process outgrowth than did the differentiated E17.5 RPCs. Immunofluorescence showed that, after the 8 days of differentiation, E13.5 RPCs contained more Brn3b- and Map2-positive cells, while E17.5 RPCs contained more GFAP-, GS-, and Rhodopsin-positive cells.
The results implied that E13.5 RPCs might be a better choice for RGC replacement therapy, while E17.5 RPCs might be better for photoreceptor replacement therapy. The duration of in vitro culture should be timed, since the expression of key genes kept changing in the proliferating RPCs.
视网膜祖细胞（RPCs）是神经视网膜疾病替代治疗中最有价值的种子细胞。RPCs的能力随视网膜发育而变化。基因表达在决定这种能力方面起着基本作用。为了改善用于替代治疗的适时RPCs的选择，我们比较了胚胎第（E）13.5天和E17.5天RPCs之间的基因表达，并进一步探讨了它们在体外的基因表达和分化能力。
对处于特定孕期的E13.5天和E17.5天的RPCs进行新鲜采集，并在增殖条件下培养4天，然后在分化条件下培养8天。在不同时间点，通过定量逆转录PCR或免疫荧光研究参与视网膜发育的关键基因的表达。
在新鲜采集的E13.5天和E17.5天的RPCs中研究了14个参与视网膜发育的关键基因的表达。新鲜采集的E13.5天的RPCs显示出视网膜神经节细胞（RGC）相关基因的高表达，包括Math5、Brn3b、Islet1和Nfl，而新鲜采集的E17.5天的RPCs分别显示出Nrl、GFAP和Thy1的高表达，这些分别是参与视杆光感受器发育、神经胶质细胞发育和突触形成的关键基因。在体外增殖培养期间，两种RPCs中的基因表达都发生了显著变化。增殖培养4天后，E13.5天的RPCs中大多数基因（14个基因中的11个）的表达水平接近新鲜采集的E17.5天的RPCs中的表达水平。通过Nestin表达和BrdU掺入效率的显著降低验证了RPCs的体外分化。体外分化8天后，E13.5天的RPCs中RGC相关基因（Math5、Brn3b和Islet1）的表达水平仍显著高于E17.5天的RPCs。相反，E17.5天的RPCs中Nrl和GFAP的表达水平显著高于E13.5天的RPCs。在形态上，分化后的E13.5天的RPCs比分化后的E17.5天的RPCs显示出更强壮的突起生长。免疫荧光显示，分化8天后，E13.5天的RPCs含有更多的Brn3b和Map2阳性细胞，而E17.5天的RPCs含有更多的GFAP、GS和视紫红质阳性细胞。
结果表明，E13.5天的RPCs可能是RGC替代治疗的更好选择，而E17.5天的RPCs可能更适合光感受器替代治疗。体外培养的持续时间应加以控制，因为在增殖的RPCs中关键基因的表达一直在变化。
