Establishment of retinal progenitor cell clones by transfection with Pax6 gene of mouse induced pluripotent stem (iPS) cells
Establishment of retinal progenitor cell clones by transfection with Pax6 gene of mouse induced pluripotent stem (iPS) cells
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DOI：
10.1016/j.neulet.2011.12.055
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发表时间：
2012-02-16
影响因子：
2.5
通讯作者：
Tadokoro, Mamoru
中科院分区：
文献类型：
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作者：
Suzuki, Noboru;Shimizu, Jun;Tadokoro, Mamoru
我们以前曾报道，将调控眼发育早期事件的Pax6基因导入小鼠ES细胞，可使其分化为视网膜前体细胞。在这里，我们试图通过将Pax6基因导入小鼠诱导的多能干细胞(IPS)来建立具有进一步分化为光感受器样细胞能力的克隆视网膜前体细胞。未分化的iPS细胞转染Pax6基因后，用G418进行筛选。经过有限稀释培养，我们选择了同时表达Nestin、Musashi1、Six3和Chx10 mRNAs的克隆Pax6细胞进行进一步的鉴定。共获得8个克隆扩增的Pax6转基因细胞。开始表达Brn3b的mRNAs。视紫红质和视紫红质；流式细胞仪分析显示，几乎一半的细胞是光感受器前体的标志CD73+。Western blotting证实视紫红质在细胞质中有蛋白表达。钙显像显示，高钾刺激使细胞内游离钙内流增加。转Pax6基因的iPS细胞经有限稀释法培养后，成为视网膜祖细胞，包括光感受器样细胞。克隆的细胞系可用于分析视网膜祖细胞的分化需求。(C)2012爱思唯尔爱尔兰有限公司。保留所有权利。
We previously reported that transfection of Pax6 gene which regulated early events in eye development into mouse ES cells brought about their differentiation into retinal progenitors. Here, we attempted to establish cloned retinal progenitors which had ability to further differentiate into photoreceptor like cells by transfecting mouse induced pluripotent stem (iPS) cells with Pax6 gene. Undifferentiated iPS cells were transfected with Pax6 cDNA, followed by selection with G418. After limiting dilution culture, we selected cloned Pax6-transfected cells, which simultaneously expressed mRNAs of Nestin, Musashi1, Six3 and Chx10 for further characterization. We obtained totally 8 clonally expanding Pax6-transfected cells. They started to express mRNAs of Brn3b. Cone-rod homeobox (Crx), pkc, CD73, rhodopsin and the gamma-subunit of rod cGMP phosphodiesterase (PDE gamma). Flow cytometric analysis revealed that almost half of the cells were CD73+, a marker of photoreceptor precursors. Western blotting confirmed cytoplasmic protein expression of rhodopsin. High KCl stimulation increased free Ca influx into the cells on Ca2+ imaging. iPS cells transfected with Pax6 gene, followed by subsequent limiting dilution culture became retinal progenitors including photoreceptor like cells. The cloned cell lines may be useful for analyzing differentiation requirement of retinal progenitors. (C) 2012 Elsevier Ireland Ltd. All rights reserved.