Rat E9 Retinal Embryonic Cells Exhibit Immature Cell Marker Nestin With Limited Propagation In Vitro are Progenitor Cells

Abstract

Abstract: : Purpose: To assess the ability of rat embryonic retinal stem cells (SC) to survive, self–renew, and exhibit multi–potentiality in vitro. Methods: Presumptive SC colonies (n=48) isolated from optic vesicles of 9–day fetal rat primordial retina were maintained in a defined serum–free medium containing bFGF and EGF for 3.5 months. Fresh medium was exchanged in the cultures twice a week and cells were expanded into other dishes when growth approached confluency. Some SC were maintained in an identical medium that was conditioned by isolated 13–day fetal rat spinal cord stem cells (SCSC). Other SC were maintained in medium with EGF or bFGF alone. Results: All SC colonies had initial robust neurosphere formation and rapid self–renewal that was present only for the first two passages. In all colonies, a lack of proliferation followed even in the presence of conditioned SCSC medium. Most SC colonies contained abundant amounts of Nestin as determined by Western blotting with some exhibiting up to 36 times that of E13 rat SCSC. A sub–population of SC were A2B5 (+) but Nestin (–) as determined by indirect immunofluorescence. At 1 month, many SC had multiple short processes the size of cell soma and other SC had long processes up to 8 times cell body with structures that resembled growth cones. Conclusions: Rat E9 retinal SC contain immature cell marker Nestin, exhibit limited ability to self–renew, and terminally resemble astrocytes and neuronal cells morphologically. These characteristics are consistent with retinal progenitor cells. Lack of SC propagation with E13 rat SCSC conditioned medium suggests absence of exogenous self–renewal factors and a post–mitotic state. Both intrinsic and extrinsic mechanisms may account for the difference between retinal and SCSC populations that originate from the same neural tube. Unlike E13 SCSC, the present data suggests that earlier stages prior to E9 need to be evaluated to determine non–progenitor retinal SC.

Keywords: retinal culture • growth factors/growth factor receptors • proliferation