Three-dimensional retinal organoids from mouse pluripotent stem cells mimic in vivo development with enhanced stratification and rod photoreceptor differentiation
Three-dimensional retinal organoids from mouse pluripotent stem cells mimic in vivo development with enhanced stratification and rod photoreceptor differentiation
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发表时间：
2016-09
期刊：
影响因子：
2.2
通讯作者：
H. Chen;Koray D. Kaya;Lijin Dong;A. Swaroop
中科院分区：
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作者：
H. Chen;Koray D. Kaya;Lijin Dong;A. Swaroop
目的利用多能干细胞构建具有视杯状结构的三维有机体，为研究哺乳动物视网膜的体外发育创造了条件。然而，培养中的视网膜有机体并不能完全反映杆状优势的小鼠视网膜的发育状态和体内结构。这项研究的目的是开发一种从干细胞产生视网膜类器官的有效方案，并在体外检测视杆的形态发生。方法利用由亮氨酸拉链转录因子NRL启动子调控的视杆特异表达绿色荧光蛋白(GFP)的NRL绿色荧光蛋白(GFP)小鼠，评价视杆感受器在视网膜类器官中的分化。利用来自NRL-GFP小鼠的胚胎干细胞和诱导多能干细胞(分别为ESCs和IPSCs)，我们成功地使用改进的培养条件建立了长期的视网膜器官培养(称为高效低氧诱导视网膜有机体中光感受器的生成，简称HiPRO)。结果我们证实了多能干细胞向视网膜结构的有效分化。7d时，70%以上的胚状体形成视泡；10d时，50%的胚状体形成视杯，大多数胚状体至少存活到35d。HiPro有机体包括具有一定层状结构的独特的视网膜内神经元和横跨整个视网膜的成熟Müler胶质细胞。视网膜类器官中近70%的细胞是杆状感光细胞，呈现细长的纤毛。从D25-35的有机物中纯化的GFP+视杆感受器的转录组图谱显示，与从P2到P6的小鼠视网膜提纯视杆的基因图谱高度相关，表明它们处于早期分化状态。结论用HiPro方法生成的3D视网膜有机体能较好地模拟体内视网膜的发生，为研究光感受器发育和模拟疾病病理提供了有效的体外模型。
Purpose The generation of three-dimensional (3D) organoids with optic cup–like structures from pluripotent stem cells has created opportunities for investigating mammalian retinal development in vitro. However, retinal organoids in culture do not completely reflect the developmental state and in vivo architecture of the rod-dominant mouse retina. The goals of this study were to develop an efficient protocol for generating retinal organoids from stem cells and examine the morphogenesis of rods in vitro. Methods To assess rod photoreceptor differentiation in retinal organoids, we took advantage of Nrl-green fluorescent protein (GFP) mice that show rod-specific expression of GFP directed by the promoter of leucine zipper transcription factor NRL. Using embryonic and induced pluripotent stem cells (ESCs and iPSCs, respectively) derived from the Nrl-GFP mouse, we were successful in establishing long-term retinal organoid cultures using modified culture conditions (called High Efficiency Hypoxia Induced Generation of Photoreceptors in Retinal Organoids, or HIPRO). Results We demonstrated efficient differentiation of pluripotent stem cells to retinal structures. More than 70% of embryoid bodies formed optic vesicles at day (D) 7, >50% produced optic cups by D10, and most of them survived until at least D35. The HIPRO organoids included distinct inner retina neurons in a somewhat stratified architecture and mature Müller glia spanning the entire retina. Almost 70% of the cells in the retinal organoids were rod photoreceptors that exhibited elongated cilia. Transcriptome profiles of GFP+ rod photoreceptors, purified from organoids at D25–35, demonstrated a high correlation with the gene profiles of purified rods from the mouse retina at P2 to P6, indicating their early state of differentiation. Conclusions The 3D retinal organoids, generated by HIPRO method, closely mimic in vivo retinogenesis and provide an efficient in vitro model to investigate photoreceptor development and modeling disease pathology.