Abstract
Purpose :
Optic neuropathies, characterized by retinal ganglion cell (RGC) dysfunction and death, cause irreversible vision loss. RGC transplantation holds potential for restoring vision, but fewer than 1% of RGCs transplanted in mouse eyes persist for more than a few weeks, underscoring the need for novel approaches to improve donor neuron engraftment. Inhibition of phosphatase and TENs in homolog deleted on chromosome 10 (PTEN) increases RGC survival and axon regeneration after optic nerve injury. Here, we evaluated the effects of PTEN deletion on RGC survival and engraftment following transplantation.
Methods :
Human embryonic stem cells (hESCs) expressing tdTomato from the endogenous BRN3B locus were subject to PTEN deletion using CRISPR. Purified RGCs differentiated from parental (WT) hESCs or PTEN knockout (KO) hESCs were transplanted in immunosuppressed (daily s.c. cyclosporine) Lama1nmf223 mice (37,500 cells/eye). WT RGCs were injected intravitreally into one randomly selected eye and PTEN KO RGCs contralaterally. At 2wk post-transplantation, retinas were processed for confocal microscopy. Immunolabeling was performed for tdTomato (donor RGC marker), human nuclear antigen (to ensure donor origin), choline acetyltransferase (ChAT, to landmark IPL sublamina), and DAPI. Images were processed in ImageJ and Arivis Pro.
Results :
PTEN KO was confirmed in hESCs and differentiated RGCs by DNA sequencing, RT-PCR, and WB. PTEN KO RGCs demonstrated significantly greater survival at 2wks after transplantation compared to WT RGCs (510.6 ± 285.7 vs 191.0 ± 299.7 donor RGCs/retina, p=0.0014). PTEN KO RGCs grew longer and more complex neurites, with dendrites laminating within the IPL and axons coursing through the retinal nerve fiber layer and into the optic nerve head.
Conclusions :
PTEN KO significantly improves survival of RGCs following transplantation and augments neurite outgrowth, which may promote functional visual pathway integration. Analyses will characterize the dendritic architecture of donor RGCs and synaptogenesis with the retinal neurocircuitry. Genetic engineering of donor RGCs represents a strategy to achieve RGC repopulation in optic neuropathies through cell transplantation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.