RPE-derived factors modulate photoreceptor differentiation: A possible role in the retinal stem cell niche

Abstract

A photoreceptor cell line, designated 661W, was tested for its response to growth factors secreted by retinal pigment epithelial cells including basic fibroblast growth factor, epidermal growth factor, and nerve growth factor. Early passaged 661W cells expressed high levels of retinal progenitor markers such as nestin and Pax6, but not opsin or glial fibrillary acidic protein. 661W cells grown in FGF-2 or EGF exhibited a multiple-process morphology with small phase-bright nuclei similar to neurons, whereas cells cultured in nerve growth factor (NGF) or retinal pigment epithelium (RPE)-conditioned medium (RPE-CM) displayed rounded profiles lacking processes. 661W cells grown in FGF-2 were slightly elevated, but not significantly above, control cultures; but cells treated with RPE-CM or NGF were fewer, approximately 63% and 49% of control, respectively. NGF immunodepletion of RPE-CM strongly suppressed the inhibitory activity of RPE-CM on cell proliferation. Cells treated with FGF-2, but not NGF, upregulated their expression of opsin. All treatment conditions resulted in almost 100% viability based on calcium AM staining. Cells grown on extracellular matrix proteins laminin, fibronectin, and/or collagen resembled those grown on untreated dishes. This study showed that early passaged 661W cells displayed characteristics of retinal progenitor cells. The 661W cells proliferated and appeared to mature morphologically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited proliferation and morphological differentiation of 661W cells, possibly inducing cell cycle arrest. These findings are consistent with reports that the RPE modulates photoreceptor differentiation and retinal progenitor cells via secreted factors and may play a role in the regulation of the retinal stem cell niche.

RPE-secreted factors: influence differentiation in human retinal cell line in dose- and density-dependent manner

“…To summarize, our studies confirm and are closest to that of Sheedlo et al [58]. However, in some aspects, our results on RPE-conditioned medium on photoreceptor differentiation differ from theirs.…”

“…No dramatic differences were seen in the expression of other antigens. Actin levels in treated and untreated cultures were similar Thus, our studies confirm some aspects of their [58] findings but differ in others. These differences could be related to differences in species.…”

“…Previously, Sheedlo et al [58] reported that the mouse photoreceptor cell line GG1W [59] has the characteristics of retinal progenitor cells in which mature photoreceptors could be generated in response to bFGF, but the same was inhibited by NGF and RPE-secreted conditioned medium. Our results in more than one aspect complement their studies.…”

Topographical control of ocular cell types for tissue engineering

“…These cells, typically derived from fetal eyes, have the capacity to differentiate into multiple cell types including photoreceptors and RPE [123]. Because environmental factors such as orientation and paracrine cell signaling have been shown to play a critical role in retinal cell differentiation, topography may be able to induce progenitors differentiation towards a particular fate and/or into a physiologically-relevant structure [124–126]. …”

“…Long-term culture results in a loss of photoreceptor morphology and function in favor of fibroblastic markers. However this could be due in part to the vast environmental differences between their in vitro and native in vivo culture conditions [126]. Native photoreceptors are embedded in a three-dimensional matrix that is soft and in close proximity to RPE producing potent neurotrophic factors.…”