Professor Rutgers University New York, New York, United States
Introduction:: The number of visually impaired individuals will grow to 25 million adults by 2050 in the United States [1]. Extracellular matrices (ECMs) create microenvironments that provide structural and chemical signals that assist cells with differentiation, migration, intercellular connectivity, and survival. Past studies have shown that ECMs have an impact on the mechanism of cell adhesion and migration, which are necessary for cell transplantation [2]. We have linked adhesion and migration of retinal progenitor cells (RPCs) to Top2b, an enzyme that is critical for post-mitotic differentiation and cell migration in terminally differentiated retinal neurons. Our project is the first to examine mechanisms of Top2b regulation in combination with substrates used for transplantable matrixes. Our data illustrates significant morphological changes of RPCs upon matrixes used for transplantation.
Materials and Methods:: RPCs (R28, ATCC) were cultured in minimum essential medium (Quality Biologics) for a maximum of 15 passages. The culture was genetically transfected either upregulated knockdown Top2b expression or upregulated Top2b. RPCs migration was observed in transwell assays (Costar) towards fibroblast growth factor-8 (FGF-8), vascular endothelial growth factor (VEGF), and stromal derived growth factor-a (SDF1-a) after 6 hours. RPCs morphological changes were observed in 24-well plates (Costar) coated with Laminin (MEMD Millipore Corp.), Poly-L-Lysine (Sigma Aldrich), and Collagen IV (Sigma Aldrich) over a 24-hour period.
Results, Conclusions, and Discussions:: RPCs that were treated with growth factors were more likely to migrate. Knockdown RPCs were found to migrate significantly less than RPCs in every growth factor, while overexpressed RPCs were found to migrate less than RPCs (Fig. 1). Overexpressed RPCs had a significantly lower CSI than RPCs, while knockdown RPCs had the largest CSI for all the matrices (Fig. 2). In this study, we characterized the effect of Top2b manipulation on the migration and morphology of RPCs upon transplantable matrixes. The ability to manipulate Top2b expression in combination with transplantation in the retina has tremendous potential for restorative vision therapies.
Acknowledgements (Optional): : This work was supported by the National Institutes of Health (R21 EY031439-01), and the National Science Foundation (CBET 0939511).
