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Establishment of efficient differentiation method of retinal ganglion cells from human iPS cells.

人iPS细胞高效分化视网膜神经节细胞方法的建立

基本信息

批准号：
22791689
负责人：
YUKI Kenya
金额：
$ 2.5万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2011
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22791689/
关键词：
iPS細胞緑内障 retina homeobox gene 網膜神経節細胞 FACS 網膜前駆細胞 RxDsRed陽性細胞 ips細胞

项目摘要

[Purpose] To investigate the efficient way collecting Rx-positive Retinal progenitor cell by FACS.[Method] We used mouse iPS(miPS) cells which was inserted Nanog-GFP-IRES-Puro reporter construct. The GFP was negative when the iPS cell when it was induced differentiation.(Okita, Yamanaka et al. Nature 2007). A Bacterial artificial chromosome(BAC) clone containing the mouse Rax gene in its center was isolated. By using the recombination technique, we inserted a DsRed-Neo cassette into the 5` UTR of the mouse Rax gene. We introduced the modified BAC into miPS cells by electroporation. Mice iPS cells were dissociated to single cells, and 5×10^4 cells per 1 ml differentiation medium(see ref) were seeded into bacterial-grade dishes(10ml). miPS aggregates were generated spontaneuously in a suspension culture within 1d. Medium was changed on day 3. Dkk1(100 ng/ml) LeftyA(500 ng/ml), 5% FBS and activin-A were added to the differentiation medium.(Osakada, Takahashi et al et al Nature Protocol)[Results] Rx-DsRED positive cells were observed in embryoid body after 9 day in the SFEB/DLFA culture. Rx-DsRed+mouse iPS cells are selected by flow cytometry. After flow cytometry, RxDsRed positive cells were observed only in the Ds-Red section by Imager.[Conclsion] By using flowcytometry, we obtain Rx-DsRed positive cells, which are thought to be retinal progenitor cells.

[目的]探讨FACS法采集rx阳性视网膜祖细胞的有效方法。[方法]采用小鼠iPS（miPS）细胞，植入Nanog-GFP-IRES-Puro报告结构。诱导iPS细胞分化时，GFP呈阴性。Okita， Yamanaka等。自然2007)。分离了一株中心含有小鼠Rax基因的细菌人工染色体（BAC）克隆体。通过重组技术，我们将DsRed-Neo卡带插入小鼠Rax基因的5 ' UTR中。我们通过电穿孔将改性BAC导入miPS细胞。将小鼠iPS细胞分离成单个细胞，每1ml分化培养基（见参考）5×10^4个细胞接种到细菌级培养皿（10ml）中。在1d的悬浮培养中，miPS聚集体自发产生。第3天更换培养基。分化培养基中加入Dkk1（100 ng/ml）、LeftyA（500 ng/ml）、5% FBS和激活素a。（Osakada, Takahashi et al . Nature Protocol）【结果】SFEB/DLFA培养9天后，在胚状体中观察到Rx-DsRED阳性细胞。流式细胞术选择Rx-DsRed+小鼠iPS细胞。流式细胞术后，成像仪仅在Ds-Red切片上观察到RxDsRed阳性细胞。[结论]流式细胞术检测得到的Rx-DsRed阳性细胞可能为视网膜祖细胞。