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Divya Sivaraman, Abdul Rasheed Vazhanthodi, Samer Hattar, Jackson James; RGC differentiation from ES cells: Influence of FGF2 and Notch signalling. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2233. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Embryonic stem (ES) cells forms a promising source of cells for therapeutic cell replacement in Glaucoma, characterized by degeneration of retinal ganglion cells (RGCs). But the critical limiting factors for this are the yield of ES cell derived RGCs and functional integration into ganglion cell layer (GCL). Here, we demonstrate enrichment of RGCs from ES cells and characterize optimal conditions for RGC transplantation in vivo for functional integration through an explant culture system.
D3 mouse ES cells were cultured and Embryoid bodies (EBs) generated were exposed to Retinoic Acid for neuronal induction. The ES cell derived neural progenitors (ES-NPs) were generated and differentiated by treating with FGF2 to generate RGCs. Enrichment of RGCs were done by exposing ES-NPs to DAPT (Notch signaling inhibitor) along with FGF2. Retinal lineage specification was analyzed by RT-PCR, Western blot and Relative luciferase assay. For optimizing transplantation and integration, an in vitro retinal explant culture system was used. Intra-vitreal transplantation was done with FGF2 induced GFP expressing ES-NPs into PN7 mice models and were examined immunohistochemically.
We report the ability of ES-NPs to differentiate along RGC lineage, confirmed by the expression of Ath5, Brn3b and Islet1 and positively regulated by FGF2, suggesting that FGF2 is sufficient for RGC differentiation. Expression of brn3b was seen increased gradually during RGC differentiation from ES-NPs and inhibition of Notch signalling increased RGC generation. An enhanced Brn3b promoter activity was found in the presence of FGF2 and DAPT. Retinal explant co-culture of RGC-5 cells with FGF2 alone and a combination of FGF8 and BDNF showed improved integration. The enhancement of RGCs integration in culture prompted us for in vivo transplantation experiments in PN7 animals, where FGF2 induced GFP expressing ES-NPs showed integration into the ganglion cell layer of retina as determined by co-expression of GFP and RGC markers Ath5 and Brn3b.
Our study provides evidences for the role of FGF2/Notch signalling in improved RGC generation from ES-NPs. Importantly, we obtained preliminary data that these RGCs integrate in the ganglion cell layer in vivo. Future studies will determine whether the transplanted RGCs will form functional integration by using electrophysiological and behavioural studies in glaucoma animal models.
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