Dataset Information

Microarray characterization of human embryonic stem cell--derived retinal cultures.

ABSTRACT:

Purpose

A number of protocols have been published to induce retinal determination from human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC). Although all these studies have shown some degree of expression of markers of retinal cells, fewer than 30 markers are typically used to characterize the ESC-derived retinal cells. Hence, it is not known whether they express all the genes present in normal developing retinal cells. To assess the efficiency of their retinal determination protocol at the transcriptome level and to understand the changes in human retinal gene expression patterns during development, the authors conducted a microarray-based analysis comparing human retina to hESC-derived retinal cells.

Methods

The authors extracted total RNA from 60-day, 80-day, and 96-day human fetal retina and hESC-derived retinal cells at 3 weeks and 9 weeks after induction. RNA was subjected to analysis using a commercial microarray. Data were normalized using Affymetrix Power Tools and analyzed using commercial microarray software.

Results

On K-median clustering analysis, the authors found that overall there was a very high correlation between genes expressed in human fetal retina and those in ESC-derived retinal cultures. The cultures were at similar developmental ages to the corresponding fetal retinal ages. They found only 1% of the genes on the array to be expressed at a higher level in ESC-derived retinal cells than in fetal retina, and most of these were expressed in the retinal pigment epithelium and ciliary epithelium.

Conclusions

In sum, gene array profiling provides an effective method for characterization of the efficiency of directed differentiation of hESCs to retinal cells.

SUBMITTER: Lamba DA

PROVIDER: S-EPMC3175935 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.