Regulation of WNT Signaling by VSX2 During Optic Vesicle Patterning in Human Induced Pluripotent Stem Cells

Few gene targets of Visual System Homeobox 2 (VSX2) have been identified despite its broad and critical role in the maintenance of neural retina (NR) fate during early retinogenesis. We performed VSX2 ChIP-seq and ChIP-PCR assays on early stage optic vesicle-like structures (OVs) derived from human iPS cells (hiPSCs), which highlighted WNT pathway genes as direct regulatory targets of VSX2. Examination of early NR patterning in hiPSC-OVs from a patient with a functional null mutation in VSX2 revealed mis-expression and upregulation of WNT pathway components and retinal pigmented epithelium (RPE) markers in comparison to control hiPSCOVs. Furthermore, pharmacological inhibition of WNT signaling rescued the early mutant phenotype, whereas augmentation of WNT signaling in control hiPSC-OVs phenocopied the mutant. These findings reveal an important role for VSX2 as a regulator of WNT signaling and suggest that VSX2 may act to maintain NR identity at the expense of RPE in part by direct repression of WNT pathway constituents

Lentiviral mediated RPE65 gene transfer in healthy hiPSCs-derived retinal pigment epithelial cells markedly increased RPE65 mRNA, but modestly protein level.

The retinal pigment epithelium (RPE) is a monolayer of cobblestone-like epithelial cells that accomplishes critical functions for the retina. Several protocols have been published to differentiate pluripotent stem cells into RPE cells suitable for disease modelling and therapy development. In our study, the RPE identity of human induced pluripotent stem cell (hiPSC)-derived RPE (iRPE) was extensively characterized, and then used to test a lentiviral-mediated RPE65 gene augmentation therapy. A dose study of the lentiviral vector revealed a dose-dependent effect of the vector on RPE65 mRNA levels. A marked increase of the RPE65 mRNA was also observed in the iRPE (100-fold) as well as in an experimental set with RPE derived from another hiPSC source and from foetal human RPE. Although iRPE displayed features close to bona fide RPE, no or a modest increase of the RPE65 protein level was observed depending on the protein detection method. Similar results were observed with the two other cell lines. The mechanism of RPE65 protein regulation remains to be elucidated, but the current work suggests that high vector expression will not produce an excess of the normal RPE65 protein level