Phenotypic characteristics of retinal progenitor cells isolated from hPAP–positive rat embryonic retina

Abstract

Abstract: : Purpose: To characterize the multiple phenotypic characteristics of retinal progenitor cells (RPCs) isolated from embryonic human placental alkaline phosphatase (hPAP)–positive rat retina. Materials and Methods: hPAP positive retinal progenitor cells were isolated from embryonic day (E) 17 retina, and were cultured and expanded in serum–free defined culture media supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The in vitro expanded retinal progenitor cells were analyzed to determine the co–expression of nestin with neural or glial cell markers. Cells at passage (P) 2 and P6 were induced for 8 days with all–trans retinoic acid. Double immunostaining for various combinations of neuronal or glial–specific markers: nestin, calbindin, protein kinase C (PKC), rhodopsin, neurofilament 200 (NF200), GFAP and cellular retinaldehyde binding protein (CRALBP), was performed to characterize the phenotypic profile of the induced cell population. Results: Co–expression of nestin with the neuronal cell marker, MAP–2 or the glial cell marker, GFAP was observed in cultured E17 derived hPAP positive RPCs prior to induction. Following induction of in vitro expanded retinal progenitor cells at P2 and P6, a variety of additional retinal specific neuronal and glial cell markers could be detected, including PKC, calbindin, rhodopsin, CRALBP, NF200, MAP–2, and nestin; PKC and rhodopsin positive cells were the predominant cell population. Co–expression of several markers was also observed in many cells including calbindin and NF 200, Nestin and MAP–2, rhodopsin and MAP–2, and CRALBP and nestin. The hPAP expression remained stable in these cultured RPCs following differentiation. Conclusions: Co–expression of nestin with neuronal or glial cell marker in cultured hPAP positive RPCs, suggests that RPCs in culture may already be lineage–restricted. RPCs appear to be capable of being induced to yield a variety of differentiated retinal cell phenotypes, in particular photoreceptors and rod bipolar cells. Supported by: Foundation Fighting Blindness, Anonymous Sponsor, Foundation for Retinal Research, Fletcher Jones Foundation, NIH EY03040.

Keywords: retinal development • retinal culture • proliferation