2011 Fiscal Year Final Research Report
Establishment of efficient differentiation method of retinal ganglion cells from human iPS cells.
| Project/Area Number |
22791689
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Ophthalmology
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| Research Institution | Keio University |
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Principal Investigator |
YUKI Kenya 慶應義塾大学, 医学部, 助教(非常勤) (00365347)
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| Project Period (FY) |
2010 – 2011
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| Keywords | iPS細胞 / 緑内障 / retina homeobox gene / 網膜神経節細胞 |
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| Research Abstract |
[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.
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