Longitudinal Analysis of Neurogenesis in Optic Vesicle-like Neurospheres Derived from the H9 and H1 Human ES Cell Lines

Abstract

Purpose: : To compare cell fate and differentiation potential of retinal cultures derived from two commonly used human embryonic stem cell (hESC) lines over time.

Methods: : H1 and H9 hESC lines of similar passage were differentiated toward a retinal lineage using a previously published protocol. Highly enriched populations of optic vesicle (OV) stage retinal progenitors from each line were manually separated from forebrain progenitor populations at day 20 and allowed to differentiate for up to an additional 120 days. The sequence and timing of expression of markers indicative of retinal development were determined via RT-PCR and immunocytochemistry over a total time course of 130 days.

Results: : A greater number of OV-like neurospheres were obtained from H1 hESCs than H9 hESCs at day 20 of differentiation. By contrast, the appearance of pigmented RPE within OV-like neurospheres occurred earlier and more frequently in H9 cultures than in H1 cultures. However, beginning at approximately day 50 of differentiation and throughout the remainder of the study, a similar pattern of neuroretinal marker expression was observed between both hESC lines. More specifically, markers of ganglion cell differentiation were observed initially, followed by the appearance of photoreceptor and retinal interneuron markers.

Conclusions: : Results from this study confirm that RPE and neuroretinal cell types can be obtained from differentiating H1 and H9 hESCs in a sequence reminiscent of normal human retinal development. However, differences were seen in the proclivity of these lines to produce multipotent retinal progenitors and RPE, as well as the timing of differentiation of certain retinal cell types. Therefore, caution should be used when directly comparing results obtained from different hESC lines.

Keywords: retinal development • differentiation