Neuroprotective effects of SIRT1 in human RGCs derived from iPSCs following oxidative stress induction at early and late stages of differentiation.

BACKGROUND

Neurodegeneration of retinal ganglion cells (RGCs) is the primary cause of irreversible vision loss in optic neuropathies. Preclinical in vivo studies have shown the neuroprotective potential of SIRT1 gene therapy in various optic neuropathy mouse models. This study seeks to build on these findings by utilizing a human cell culture model of induced pluripotent stem cell (iPSC)-derived RGCs to assess the neuroprotective effects of SIRT1 expression.

METHODS

RGCs were differentiated from control human iPSCs using a previously defined in vitro methodology with small molecules and peptide modulators. Cell morphology and identity was confirmed by Brn3a and RBPMS immunostaining as well as qPCR analysis of RGC-specific markers. SIRT1 and GFP (control) were expressed using AAV vectors driven by the RGC-selective SNCG promoter. Oxidative stress was induced using tert-butyl hydroperoxide solution (TBHP) or growth factor withdrawal, and neuroprotection was assessed via MTT assay.

RESULTS

By day 42 of differentiation, cultures were enriched with Brn3a + cells (>80 %) that demonstrated morphologic features of mature RGCs, and these RGC properties were maintained up to day 90. SIRT1 expression induced significant neuroprotection to human iPSC-derived RGCs following TBHP-induced oxidative stress and with depletion of growth factors. Rescue effects were observed within three weeks of SIRT1 expression, with improved RGC survival compared to controls (∼20 %), at two timepoints: Days 42 and 90.

CONCLUSIONS

These findings support the therapeutic success of SIRT1-based gene therapy in an in vitro culture model of human RGCs and confirm those obtained in mouse models of optic neuropathies.