Duration 5min, Q&A 3min
Derivation, Characterization and Retinal Neural Induction of Human Tenon's - Derived iPS Cells
Direct reprogramming of somatic cells to a pluripotent state has been achieved by ectopic expression of Oct-3/4, Sox2, c-Myc, and Klf4. As an essential reprogramming factor, SOX-2 also plays important roles throughout retinal development. This study was conducted 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 were obtained anonymously from tissue explants taken during strabismus or glaucoma-filtering surgery or from the eye bank (donors aged 18 to 60 years) of ZhongShan Ophthalmic Center, Sun Yat-sen University. H9 human ES cells were received as a gift from Professor Mengfei Chen.DKK1, Lefty-A were purchased from R&D Systems; Noggin was purchased from PeproTechInc; DAPT was purchased from Merck Millipore Company.
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, DAPT, and 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.
Results and Conclusion
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 and Sox2. 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.
[ Keyword ]
reprogramming / human iPS / differentiation / retinal neuron
[ Conflict of Interest ]
[ Conflict of Interest (Potential conflict) ]
This study was supported by the National Basic Research Program of China (Project 973) (Grant 2007CB512207), the National Natural Science Foundation of China (Grants 30973226 and 81170846) .The funders have not any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.