Investigating the role of VSX2 (CHX10) in human retinogenesis using iPS cells

Abstract

Purpose: We sought to examine the role of VSX2 in human retinal cell differentiation using induced pluripotent stem cell (iPSC) technology.

Methods: iPSCs were created from 1) a microphthalmic patient with a homozygous mutation in the homeodomain region of VSX2 and 2) an unaffected sibling. The resulting VSX2 mutant and wildtype (WT) iPSCs were differentiated into optic vesicle-like structures (OVs) as previously described. Thereafter, isolated mutant and WT OV cultures were analyzed at multiple time points during differentiation by RNA-seq, qRT-PCR, Western blot, and ICC. Furthermore, WT VSX2 was ectopically expressed in mutant VSX2 hiPSC cultures in an effort to rescue the observed phenotype.

Results: At very early stages of retinal differentiation, prior to VSX2 expression, VSX2 mutant and WT iPSC cultures were indistinguishable. At day 20 of differentiation, OVs isolated from mutant and WT iPSCs had similar retinal gene expression profiles. However, genes involved in WNT and FGF signaling were up- and downregulated, respectively, in mutant OVs compared to WT OVs. By day 30, differences in retinal gene expression were evident and, furthermore, mutant OVs showed reductions in proliferation and growth rate compared to WT OVs. Unlike WT OVs, most mutant OVs maintained MITF expression and became pigmented with time in conjunction with the expression of characteristic RPE genes. With the exception of bipolar cells, mutant OVs did generate neuroretinal cells over time, although overall amounts were reduced and maturation was delayed for some cell types. Importantly, much of the mutant OV phenotype could be rescued by ectopic expression of WT VSX2.

Conclusions: OVs derived from VSX2 mutant hiPSCs display a phenotype in culture consistent with findings in human patients and animal models. Overall, the ability to isolate and differentiate unlimited amounts of differentiating OVs from iPSCs facilitates in depth analyses and shows promise for investigating roles of key factors during retinal development.

Keywords: 698 retinal development • 688 retina • 721 stem cells