Differentiation of Human Embryonic/Induced-Pluripotent Stem Cells to Retinal Ganglion Cells.

The generation of retinal ganglion cells (RGCs) differentiated from human embryonic stem cell (hESC) or induced-pluripotent stem cells (iPSC) could aid with understanding of human RGC development, neuronal biology, drug discovery, potential cell-based therapies, and gene regulation. Here, we present a protocol for differentiation of hESC to RGCs using a 40-day protocol, significantly shorter than typical retinal organoids while still yielding cells with RGC-enriched markers and show physiological and morphological properties typical of RGCs.

Programmed switch in the mitochondrial degradation pathways during human retinal ganglion cell differentiation from stem cells is critical for RGC survival.

Retinal ganglion cell (RGC) degeneration is the root cause for vision loss in glaucoma as well as in other forms of optic neuropathy. A variety of studies have implicated abnormal mitochondrial quality control (MQC) as contributing to RGC damage and degeneration in optic neuropathies. The ability to differentiate human pluripotent stem cells (hPSCs) into RGCs provides an opportunity to study RGC MQC in great detail. Degradation of damaged mitochondria is a critical step of MQC, and here we have used hPSC-derived RGCs (hRGCs) to analyze how altered mitochondrial degradation pathways in hRGCs affect their survival. Using pharmacological methods, we have investigated the role of the proteasomal and endo-lysosomal pathways in degrading damaged mitochondria in hRGCs and their precursor stem cells. We found that upon mitochondrial damage induced by the proton uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP), hRGCs more efficiently degraded mitochondria than did their precursor stem cells. We further identified that for degrading damaged mitochondria, stem cells predominantly use the ubiquitine-proteasome system (UPS) while hRGCs use the endo-lysosomal pathway. UPS inhibition causes apoptosis and cell death in stem cells, while hRGC viability is dependent on the endo-lysosomal pathway but not on the UPS pathway. These findings suggest that manipulation of the endo-lysosomal pathway could be therapeutically relevant for RGC protection in treating optic neuropathies associated with mitophagy defects. Endo-lysosome dependent cell survival is also conserved in other human neurons as we found that differentiated human cerebral cortical neurons also degenerated upon endo-lysosomal inhibition but not with proteasome inhibition.