Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.
Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.
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DOI：
10.1038/srep16595
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发表时间：
2015-11-13
影响因子：
4.6
通讯作者：
Zack DJ
中科院分区：
文献类型：
--
作者：
Sluch VM;Davis CH;Ranganathan V;Kerr JM;Krick K;Martin R;Berlinicke CA;Marsh-Armstrong N;Diamond JS;Mao HQ;Zack DJ
青光眼和其他形式的视神经疾病引起的视网膜神经节细胞(RGC)损伤和细胞死亡是导致不可逆性视力丧失和失明的主要原因。人类多能干细胞(HPSC)来源的RGCs可以为开发新的治疗分子以及潜在的基于细胞的治疗提供细胞来源。此外，这样的细胞可以为人类RGC的发育、基因调控和神经元生物学提供见解。在这里，我们报告了一种简单、贴壁的细胞培养方案，用于使用CRISPR工程的RGC荧光报告干细胞系将hPSCs分化为RGC。对分化的培养物进行荧光激活的细胞分选产生高度纯化的细胞群，这些细胞群表达一系列RGC丰富的标志物，并表现出典型的RGC的形态和生理特性。此外，我们证明定向的纳米纤维基质可以用于指导hPSC来源的视网膜节细胞的轴突生长，用于体外视神经样建模。最后，利用这一方法，我们发现Forskolin是RGC分化的一个强有力的启动子。
Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation.