Differentiation and morphogenesis in pellet cultures of developing rat retinal cells

Purpose: We previously developed a reaggregate cell culture system (pellet cultures) in which retinal neuroepithelial cells proliferate and give rise to rod photoreceptor cells (rods) in vitro (Watanabe and Raff, Neuron, 4, 461-467, 1990). In the present study, we analyzed cell differentiation and morphogenesis in pellet cultures using both cell-type-specific markers with immunofluorescence staining (IF) and electron microscopy (EM). Methods: We centrifuged 150000 embryonic day 15 (E15) retinal cells in a conical tube, giving cell pellets that were then cultured on polycarbonate filters floating in D-MEM supplemented with 10 % FCS. After 15 to 17 days in culture, pellets were processed for IF with cell-type-specific antibodies and EM. To confirm the generation of major retinal cell types in vitro, pellets were exposed to BrdU in culture. Results: We demonstrate that, in addition to rods, the other major retinal cell types, including amacrine, bipolar, Müller, and ganglion cells are all present in the pellets, where most can develop from dividing precursor cells in vitro. These different cell types become organized into two distinct structures - dark (D) and pale (P) rosettes. The cellular composition of these structures indicate that D correspond to the outer nuclear layer and P to the inner nuclear layer of the normal retina. EM studies indicate that the thin layer of neuronal processes surrounding D corresponds to the outer plexiform layer, while the central region of P corresponds to the inner plexiform layer of the normal retina. Other features of normal retinal development also occur in the pellets, including programmed cell death and the formation of inner and outer rod cell segments and synapses. Conclusions: Pellet cultures provide a convenient way to study various aspects of retinal development where one can control the size and the cellular composition of the initial reaggregate.

Duke Scholars

Author James T. Voyvodic Radiology