Effects of specific FGF isoforms on retinal progenitor cell fate in a human pluripotent stem cell model of retinal development

Abstract

Purpose: Fibroblast growth factor (FGF) signaling is known to play a key role in retinal development. Using a human pluripotent stem cell (hPSC) model of retinogenesis, we previously showed that FGF receptor 1 (FGFR1)-mediated signaling modulates Visual System Homeobox 2 (VSX2) expression in differentiating optic vesicle-like (OV) structures. In the present study, we sought to determine the role of specific FGF isoforms in the proliferation and differentiation of neural retina progenitor cells (NRPCs) from hPSCs.

Methods: The WA09 human embryonic stem cell (hESC) line, along with hiPSC lines derived from a patient harboring a mutation in VSX2 and an unaffected sibling, were directed towards a retinal fate using our previously described protocol (1) to produce and isolate 3-D cultures of OV and non-OV (forebrain) structures. Genome-wide expression data obtained from microarray, RNA-Seq and/or real-time quantitative RT-PCR analyses was used to ascertain the presence and expression levels of specific FGF isoforms during NRPC development in hESCs and mutant and WT VSX2 hiPSC lines. We then examined the effect of exogenous administration of selected recombinant FGF isoforms and/or neutralizing antibodies on NRPC proliferation and differentiation.

Results: Gene expression studies revealed high expression of FGF3, 8, 9, and 19 in OV structures derived from WA09 and WT VSX2 hiPSC lines with maximal expression of FGF3 and 8 at d20 and FGF9 and 19 at d30. In comparison studies at d20 of differentiation, FGF8, 9, and 19 expression was higher in OV vs. non-OV structures, and FGF3, 9, and 19 was higher in WT vs. mutant VSX2 hiPSC lines. Treatment of non-OV cultures with FGF9, but not FGF8 or 19, led to increased expression of VSX2. Conversely, treatment of OV cultures with FGF8 and 9 neutralizing antibody resulted in reduced expression of VSX2. Interestingly, only recombinant FGF9 treatment resulted in increased expression of genes associated with NRPC proliferation in both WT and mutant VSX2 hiPSC lines.

Conclusions: These results highlight FGF9 as a key FGF isoform involved in the regulation of VSX2 expression and NRPC behavior in our hPSC-based in vitro model of human retinogenesis.

Keywords: 721 stem cells • 698 retinal development • 543 growth factors/growth factor receptors