Screening for Molecules that Promote Photoreceptor Synaptogenesis

项目摘要 NEI大胆目标计划是一项大胆的努力，旨在“通过神经元再生恢复视力 以及眼睛和视觉系统中的神经连接。”哺乳动物的主要障碍之一 光感受器移植实验一直是，并将继续是，整合效率低 以及感光细胞群移植后的突触形成。为了解决这一 路障，并响应RFA-EY-15-002（针对“基于发现的方法， 未知的因素对神经元的再生至关重要，引导它们的轴突到达目标， 功能连接”），我们建议将联合收割机最先进的干细胞方法学与高含量的 筛选方法以鉴定促进人类免疫的新的小分子和分子途径， 光感受器轴突生长和突触形成。为了实现这一宏伟目标，我们 我召集了一个多学科的研究小组，他们在人类视网膜干细胞方面有多年的经验 细胞生物学，视网膜细胞和分子生物学，高含量筛选（HCS）检测开发和药物 筛选、轴突引导、突触生物学和微流体学。 研究小组的成员已经进行了筛选，成功识别出 促进鼠视网膜神经节细胞（RGC）和其它视网膜神经元的神经突生长的分子， 并且在人干细胞衍生的神经元培养物中增加突触形成。对于这个项目，我们 我建议扩展这项先前的工作，并在体外开发强大的和可重复的神经突生长， 使用从人多能干细胞（hPSC）获得的光感受器（PR）的突触发生测定 衍生的三维视泡样结构，然后使用这些测定来鉴定和 表征生物学和临床相关分子。更具体地说，SA 1将侧重于 开发和执行一种双层体外筛选，旨在识别影响 hPSC-PR轴突生长和/或指导; SA 2将集中于筛选以鉴定增强hPSC-PR轴突生长和/或指导的分子。 hPSC-PR突触标志物表达; SA 3将专注于开发检测方法，以确认 功能性PR突触形成。成功完成这些目标和里程碑将产生 这是第一个体外人体检测系统，旨在快速筛选和严格测试分子， 促进hPSC-PR连接的能力。这个平台不仅要加快努力， 功能性PR替代患者，但也可以作为一个有价值的人类临床前模型系统。

PROJECT SUMMARY 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.