RPE-derived factors modulate photoreceptor differentiation: A possible role in the retinal stem cell niche Harold J. Sheedlo & T. J. Bartosh & Zhaohui Wang & Bhooma Srinivasan & Anne M. Brun-Zinkernagel & Rouel S. Roque Received: 25 April 2007 / Accepted: 11 July 2007 / Published online: 9 October 2007 / Editor: J. Denry Sato # The Society for In Vitro Biology 2007 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 progen- itor 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, ∼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 morpho- logically expressing rod photoreceptor phenotype in response to FGF-2. In contrast, NGF and RPE-CM inhibited prolifer- ation 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. Keywords Basic fibroblast growth factor . Epidermal growth factor . Nerve growth factor . Photoreceptor cells . Retinal pigment epithelium . Retinal progenitor cells Introduction Mammalian cell lines have been developed over the years enabling researchers to study the effects of growth factors and to develop possible transplantation therapies, particu- larly in nervous tissue, to restore normal function. Among these cell lines are retinal cultures that have been immortalized by viral transformation using SV40 large-T- antigen, Rous sarcoma virus (RSV), E1A, human papillo- mavirus E6/E7, or the myc oncogene; whereas others arose spontaneously including human and rat retinal pigment epithelial cells (Dunn et al. 1996; Sheedlo et al. 1997). Included in the virally transformed retinal cell lines are R28 rat retinal cells (Seigel 1996); QNR/D quail amacrine/ ganglion cells (Pessac et al. 1983); RGC-5 rat retinal ganglion cells (Krishnamoorthy et al. 2001), RMC E6/E7 rat Müller cells (Roque et al. 1997); and 661W mouse photoreceptor cells (661W) (Al-Ubaidi et al. 1992). The 661W photoreceptor cell line was generated from retinal tumors in transgenic mice expressing the viral oncogene simian virus 40 large tumor antigen. Inspite of their highly In Vitro Cell.Dev.Biol.—Animal (2007) 43:361–370 DOI 10.1007/s11626-007-9051-3 H. J. Sheedlo (*) : T. J. Bartosh : Z. Wang : B. Srinivasan : A. M. Brun-Zinkernagel : R. S. Roque Department of Cell Biology and Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA e-mail: hsheedlo@hsc.unt.edu