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Jian Ge, Deng Fei, Liu Ying, Xiong Yunfan; Derivation, Characterization and Retinal Neural Induction of Human Tenon’s -Derived iPS Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1365.
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© ARVO (1962-2015); The Authors (2016-present)
To determine if induced pluripotent stem cells (iPSCs) derived from human Tenon’s capsule fibroblasts (HTFs) could express retinal progenitor cell (RPC)-related genes with the capacity to directly differentiate into retinal neurons in vitro.
HTFs harvested from fresh samples were reprogrammed by retroviral transduction to iPSCs. The HTF-derived iPSCs (TiPSCs) were characterized for pluripotency by morphology, gene expression, surface antigens, alkaline phosphatase activity analysis and a teratoma formation assay. Human ESC colonies were used as the positive control. The resulting TiPSCs were induced to differentiate into retinal cells by stepwise treatment with the defined factor combination of Dkk1,Noggin,Lefty-A, DAPTand overexpression of ATOH7, which follows the human retinal development timeline. The induced retinal cells were analyzed with phase contrast microscopy, real-time PCR, immunofluorescence, FACS analysis, and calcium imaging analysis.
The resulting TiPS colonies were indistinguishable from human ESC colonies according to standard criteria. Upon retinal differentiation, embryoid bodies (EBs) were formed from TiPSCs by suspension culture in serum-free medium with the increase of RPC-related gene expressions such as Pax6, Sox2 and Nestin. In matrigel-coated culture, acquisition of neuroepithelial colonies at day 10 with an early eye field fate was enhanced through the addition of DKK1, NOGGIN and Lefty-A, as determined by qPCR and immunofluorescence. Further overexpression of ATOH7 combined with DAPT-treated cultures, TiPSCs can generate retinal neural cells that express Chx10,Brn3b, Atoh7, Syn,Crx,Mitf,GFAP,etc.
These findings demonstrate that iPSCs can be generated from HTFs and through a stepwise neural differentiation strategy, the TiPSCs can generate retina-specific cells in vitro, which should aid in the investigation of ophthalmological regenerative medicine.
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