Tanaka Taku, Yokoi Tadashi, Tamalu Fuminobu, Watanabe Shu-Ichi, Nishina Sachiko, Azuma Noriyuki
Department of Ophthalmology and Laboratory for Visual Science National Center for Child Health and Development, Tokyo, Japan.
Department of Physiology, Faculty of Medicine, Saitama Medical University, Saitama, Japan.
Invest Ophthalmol Vis Sci. 2016 Jun 1;57(7):3348-59. doi: 10.1167/iovs.16-19166.
We previously generated self-induced retinal ganglion cells (RGCs) with functional axons from human induced pluripotent stem cells (hiPSCs). We investigated whether self-induced RGCs from mouse embryonic stem cells (mESCs) and induced pluripotent stem cells (miPSCs) are realized by the similar induction protocol.
Retinal ganglion cells were induced using a protocol in which floating embryoid bodies (EBs) were differentiated into a retinal cell lineage in three-dimensional culture and subsequently attached to two-dimensional culture dishes with brain-derived neurotrophic factor (BDNF) supplementation.
Retinal ganglion cells developed in an attached clump of cells originating from the optic vesicle, and most axons grew from RGC cell bodies at the margins of the clump. The differentiation of RGCs was confirmed by the expression of specific markers, including Brn3a and Math5. The axons contained neurofilament subtypes and tau, and manifested axonal transport and sodium-dependent action potentials. The RGCs derived from mESCs and miPSCs generally showed similar profiles, including RNA and protein expression levels and function.
Retinal ganglion cells generated from mESCs and miPSCs, especially the latter, may contribute to research associated with RGCs and to in vitro analyses of genetically modified mice.
我们之前已从人诱导多能干细胞（hiPSC）中生成了具有功能性轴突的自诱导视网膜神经节细胞（RGC）。我们研究了通过类似的诱导方案是否能从小鼠胚胎干细胞（mESC）和诱导多能干细胞（miPSC）中实现自诱导RGC。
使用一种方案诱导视网膜神经节细胞，即悬浮的胚状体（EB）在三维培养中分化为视网膜细胞谱系，随后附着于补充有脑源性神经营养因子（BDNF）的二维培养皿上。
视网膜神经节细胞在源自视泡的附着细胞团中发育，大多数轴突从细胞团边缘的RGC细胞体长出。通过包括Brn3a和Math5在内的特异性标志物的表达证实了RGC的分化。轴突含有神经丝亚型和tau，并表现出轴突运输和钠依赖性动作电位。源自mESC和miPSC的RGC通常表现出相似的特征，包括RNA和蛋白质表达水平及功能。
从mESC和miPSC生成的视网膜神经节细胞，尤其是后者，可能有助于与RGC相关的研究以及对基因修饰小鼠的体外分析。
