Abstract
Purpose :
We previously demonstrated calpain activation and retinal ganglion cell (RGC) damage in human retinal explants cultured under hypoxia. Recently, functional RGCs were produced from human pluripotent stem cells (iPSCs)-derived optic vesicles (OV).The purpose of the current experiment was to investigate if human iPSCs-derived OV are suitable experimental models of human retina. For this purpose, activation of calpains and caspases by hypoxia was studied in human iPSCs-derived OV.
Methods :
RGCs were induced from human iPSCs as previously described. Dissociated iPSCs were cultured in V-bottomed, 96-well plates. On Day 27, aggregated embryoid bodies were cultured on adhesive substrates. Damage of elongated neurites in OV was evaluated after culture under hypoxia/reoxygenation. When tested, caspase-8 (IETD) and caspase-9 (LEHD) inhibitors, and calpain inhibitor (SNJ1945) were added along with hypoxia. Activation of caspase-3 was assayed by cytochemical staining after dissociation of mechanically isolated OV. Cell lysates were immunoblotted for caspase-3 and α-spectrin.
Results :
Hypoxia/reoxygenation induced damage in human iPSC-derived RGCs in a time dependent manner. Activated caspase-3-positive cells were observed after 12 h hypoxia/3 h reoxygenation. Immunoblotting confirmed activation of caspase-3. RGC degeneration and activation of caspase-3 were ameliorated by caspase-8 and caspase-9 inhibitors. Calpain inhibitor inhibited RGC damage, but not caspase-3 activation. Inhibition of caspase-3 was confirmed by observing proteolyzed α-spectrin. Calpain inhibitor ameliorated production of calpain-specific α-spectrin breakdown product (SBDP145), but not caspase-3-specific 120 kDa SBDP. Caspase-9 inhibitor prevented production of caspase specific SBDP, but not calpain-specific SBDP145.
Conclusions :
Calpains and caspases are involved in hypoxia/reoxygenation-induced damage to iPSCs-derived RGCs. Human iPSCs derived RGCs are useful for research where access to human eye tissues is limited.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.