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Anna La Torre, Xinlei Wang, Pengfei Zhang, Robert J Zawadzki, Edward N Pugh; Generation, Transplantation and In Vivo Imaging of Transplanted Embryonic Stem Cell-derived Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6072.
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© ARVO (1962-2015); The Authors (2016-present)
Several pathologies result in retinal ganglion cell (RGC) degeneration and irreversible blindness. Stem cell-based cell replacement therapies offer promising novel approaches for vision restoration after RGC loss. However, to date successful cell replacement of RGCs remains a major challenge. We combined methods to differentiate Embryonic Stem Cells (ESCs) into retinal organoids to obtain donor RGCs with state-of-the-art imaging technologies to follow individual ESC-derived RGCs after transplantation in living rodents to accurately measure survival, migration and integration with the host to pinpoint all the barriers to cell engraftment.
We used a 3D culturing system to direct mouse ESC colonies towards retinal fates following a stepwise differentiation process that recapitulates the normal retinal developmental timeline. These cultures progressively developed into retinal progenitor cells and post-mitotic retinal neurons, including RGCs. Next, ESC-derived RGCs were transduced with AAV2-CMV-eGFP and the GFP+ cells were purified by FACS and transplanted intravitreally in adult C57Bl/6J mice. Fundus video camera (FVC) and scanning laser ophthalmoscopic (SLO) imaging were used to track the transplanted cells over time in intact living mice.
We observed GFP+ transplanted cells surviving for long periods of time (up to 13 months) in the host retinas. Though absent immediately after transplantation, 4 months after the transplant, the GFP+ RGCs had sprouted linear processes, many of which exceeded 200 um of length, indicating that the developmental programs that lead to axogenesis can be activated in adult environments. Moreover, many of the transplanted GFP+ RGCs migrated substantially below the most superficial retinal blood vessels, suggesting possible migration in depth across the ILM.
Our data indicate that ESC-derived RGCs can survive and thrive in adult host retinas after transplantation. Further studies are underway to systematically identify the barriers to successful cell engraftment, and to test for successful synaptogenesis. A detailed understanding of the roadblocks of these technologies will lead us to specific interventions to treat RGC degenerations.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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