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

[目的]探讨流式细胞仪（FACS）收集视网膜前体细胞（Rx阳性）的有效方法。[方法]利用Nanog-GFP-IRES-Puro报告基因构建小鼠iPS（miPS）细胞。iPS细胞在诱导分化时GFP表达阴性。（Okita，Yamanaka等人，Nature 2007）。分离到一个在其中心含有小鼠Rax基因的细菌人工染色体（BAC）克隆。通过重组技术，我们将DsRed-Neo盒插入小鼠Rax基因的5` UTR。我们通过电穿孔将修饰的BAC导入miPS细胞。将小鼠iPS细胞解离成单个细胞，并将5×10^4个细胞/1 ml分化培养基（参见参考文献）接种到细菌级培养皿（10 ml）中。在悬浮培养物中，在1d内自发产生了miPS聚集体。在第3天更换培养基。向分化培养基中加入Dkk 1（100 ng/ml）、LeftyA（500 ng/ml）、5%FBS和激活素A。[结果] SFEB/DLFA培养9 d后，在拟胚体中观察到Rx-DsRED阳性细胞。通过流式细胞术选择Rx-DsRed+小鼠iPS细胞。流式细胞术后，通过Imager仅在Ds-Red切片中观察到RxDsRed阳性细胞。[结论]流式细胞仪检测获得Rx-DsRed阳性细胞，初步认为其为视网膜前体细胞。

[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.