Abstract
Purpose: :
Several studies have shown that retinal cells can be derived from human ES and iPS cells. Although all of these studies have shown some degree of expression of markers of retinal cells, fewer than 50 markers are typically used to characterize the ES-derived retinal cells, and so it is not known whether they express all of the genes present in normal developing retinal cells. To assess the efficiency of our retinal differentiation protocol, as well as to understand the changes in human retinal gene expression patterns during development, we profiled the gene expression pattern across 3 ages of human retina, as well as human ES derived retinal cells from 2 different stages and human retina-derived photoreceptor cells that were purified by FACS after viral infection with a photoreceptor-specific reporter (IRBP-GFP).
Methods: :
We extracted total RNA from 62 day, 80 day and 96 day human fetal retina, FACS-purified photoreceptors from 90 day human fetal retina as well as retinal cells derived from human ES cell at 3 weeks and 2 months after induction. The RNA was subjected to analysis using the Affymetrix Human Gene 1.0 ST array. The data was normalized and analyzed using TM4 MEV software and STEM analysis software.
Results: :
Upon comparison, we found that over 70% of the 33,000 genes on the array showed similar levels of between the human retina and the ES cell-derived retinal cells. Nearly all photoreceptor-specific genes expressed in the fetal human retina were also expressed at comparable levels in the human ES-derived retinal cells. In addition, genes previously demonstrated to be expressed by retinal progenitors were also expressed in the retinal cells derived from human ES cells. Most of the differences in gene expression between the human ES derived retinal cells and the fetal human retina were due to the fact that the former also contained genes expressed in retinal pigmented epithelium and ciliary epithelium, while the latter did not.
Conclusions: :
In sum, gene array profiling provides an effective method for characterization of the efficiency of directed differentiation of human ES cells to retinal cells.
Keywords: gene microarray • retinal development • photoreceptors