Dwarf gecko as a model for studying retinal neurogenesis (P1.9-012)

Objective: To develop a novel model for retinal neurogenesis in vitro. Background: The fovea is a photoreceptor-dense specialization of the human neural retina that is responsible for high acuity color vision. Foveate model organisms are limited. Lizards such as the dwarf gecko possess a fovea. Thus, the gecko may provide a more accessible platform for studying foveal diseases than primate models due to its simple rearing. Hence, we sought to develop an in vitro model of neurogenesis in the gecko Lygodactylus picturatus. Design/Methods: From the distal 2mm tail of one male and one female gecko, we derived a primary fibroblast cell line. Growth of cells was optimized by considering variable culture conditions, including temperature. Sendai virus transduction efficiency was assessed at different cell densities with the CytoTune EmGFP Sendai Fluorescence Reporter. Reprogramming with Yamanaka factors (OCT3/4. KLF4, cMYC and SOX2) was performed using Cytotune 2.0 Sendai virus system. Characterization of pluripotent stem cells is being performed by morphometry, immunohistochemistry, and differentiation potential. Results: Primary fibroblasts were derived from gecko tail and successfully cultured at 31°C. Compared to human fibroblasts, gecko fibroblasts have a higher cytoplasmic:nuclear ratio and a slower growth rate, with passage rate occurring approximately every 12–14 days. Fibroblasts displayed a smaller diameter, compared to human primary fibroblasts. Efficient transduction with GFP-expressing Sendai virus (MOI = 5) was observed at multiple cell densities (2 ×106, 2.5 ×106 and 3 ×106). Transduction with Sendai virus containing human Yamanaka factors resulted in rounded colony formation. Cell morphology from suspected colonies demonstrate a high nuclear:cytoplasmic ratio with decreased polarity. Characterization via immunohistochemical and differentiation testing is underway. Conclusions: By establishing a cell line in vitro from L. picturatus, we can study the conservation across species of genes involved in human retinal neurogenesis and potentially model neurodevelopmental and neurodegenerative disease states. Disclosure: Dr. Mendez has nothing to disclose. Dr. Wegerski has nothing to disclose. Dr. Li has nothing to disclose. Dr. Fufa has nothing to disclose. Dr. Hufnagel has nothing to disclose.