Retinal Progenitor Production From Human Embryonic Stem Cells: The Influence of Basic Fibroblast Growth Factor

Abstract

Purpose: : Members of the fibroblast growth factor (FGF) family are important in the development of the neural retina in mammals, birds and amphibians. In particular basic FGF (bFGF) initiates the differentiation of the neural retina in the chick. Established culture techniques of human embryonic stem cells (HESCs) often includes bFGF, it is thus important to investigate the influence of this factor in the derivation of ocular cells from HESCs.

Methods: : Cell culture: Undifferentiatied HESCs were cultured on mouse embryonic fibroblasts. Embryoid bodies (EBs) were formed by treating the undifferentiated colonies with type IV collagenase. The EBs were then grown in culture dishes for 6 days in the presence of standard HESC media (knockout DMEM+serum) with the addition of the factors noggin, Dickkopf 1, and insulin-like growth factor-1. These were added at the concentrations defined as optimal by Lamba et al., (2006) PNAS 103:12769-74. In order to asses the influence of bFGF on differentiation, half of the EBs were also treated with bFGF (5ng/ml). Immunocytochemistry: EBs were fixed, and analyzed with various primary antibodies. Real-time quantitative PCR (QPCR): Total RNA was extracted from EBs, DNase treated and cDNA transcribed. QPCR was performed for various genes with power SYBR and the results were normalised to the geometric mean of 3 stable internal control genes.

Results: : Genes assessed by QPCR were found to be present in both + and -bFGF groups and their levels were not significantly altered in either group. Genes assessed included markers for: undifferentiated HESCs (Nanog and Oct4), differentiating neuro-epithelium (Nestin), Eye field transcription factors (Lhx2, Pax6, and Six3), photoreceptor transcription factors (Crx, Otx2, Retinoid related orphan receptor β, Retinoid X receptor γ and Nrl) as well as the photopigment short wavelength sensitive opsin (S-opsin). Similar results were obtained at the protein level with cells positive for Nestin, Otx2, Sox2, βIII tubulin, Six3, Pax6 and S-opsin-like immunoreactivity present in both groups.

Conclusions: : At a concentration commonly used in HESC culture protocols, bFGF did not influence the expression of either retinal determination genes or undifferentiated HESC markers. However the factors and media conditions used here do appear capable of supporting a retinal progenitor phenotype in three dimensional aggregate cultures of HESCs.

Keywords: opsins • transcription • transcription factors