The NEI Audacious Goals Initiative is a bold effort to ?to restore vision through regeneration of neurons and neural connections in the eye and visual system.? One of the major roadblocks in mammalian photoreceptor transplantation experiments has been, and continues to be, the low efficiency of integration and synapse formation following transplantation of photoreceptor populations. In order to address this roadblock, and in response to RFA-EY-15-002 (which is directed at ?discovery-based approaches to identify unknown factors critical to the regeneration of neurons, guiding their axons to targets, and making new functional connections?), we propose to combine state-of-the-art stem cell methodologies with high content screening approaches to identify novel small molecules and molecular pathways that promote human photoreceptor axonal outgrowth and synapse formation. To accomplish this ambitious goal, we have assembled a multidisciplinary group of investigators who have years of experience in human retinal stem cell biology, retinal cell and molecular biology, high content screening (HCS) assay development and drug screening, axonal guidance, synaptic biology, and microfluidics. Members of the research team have already carried out screens that have successfully identified molecules that promote neurite outgrowth of murine retinal ganglion cells (RGCs) and other retinal neurons, and that increase synapse formation in cultures of human stem cell-derived neurons. For this project, we propose to extend this prior work and develop robust and reproducible in vitro neurite outgrowth and synaptogenesis assays using photoreceptors (PRs) obtained from human pluripotent stem cell (hPSCs) derived 3-dimensional optic vesicle-like structures, and then to use these assays to identify and characterize biologically and clinically relevant molecules. More specifically, SA1 will focus on the development and execution of a two-tiered in vitro screen designed to identify molecules that influence hPSC-PRs axon outgrowth and/or guidance; SA2 will focus on a screen to identify molecules that enhance hPSC-PR synaptic marker expression; and SA3 will focus on the development of assays to confirm functional PR synapse formation in culture. Successful completion of these aims and milestones will yield the first in vitro human assay system designed to rapidly screen and rigorously test molecules for their ability to promote hPSC-PR connectivity. This platform should not only accelerate efforts to achieve functional PR replacement in patients, but could also serve as a valuable human preclinical model system.
Based on transplantation experiments that have been performed to date, one of the major challenges to restoring vision to blind patients by transplanting stem cell-derived photoreceptor cells is the very low efficiency of donor cell integration and synapse formation within the recipient retina. In the spirit of NEI?s Audacious Goals program, designed ?to restore vision through regeneration of neurons and neural connections in the eye and visual system,? we propose to combine state-of-the-art stem cell methodologies with high content screening approaches to discover novel small molecules and molecular pathways that promote human photoreceptor axonal outgrowth and synapse formation. If successful, the discovery of such molecules and pathways would greatly aid in the development of viable approaches to restore vision to individuals suffering from photoreceptor damage and degeneration.
