Embryonic Stem Cell Approach to Retinal Ganglion Cell Replacement: an In Vivo Study

项目摘要 几种人类病理导致视网膜神经节细胞（RGC）变性和不可逆的视力丧失。 基于干细胞的细胞替代疗法为视力恢复提供了有前途的新方法。然而，在这方面， 在这些疗法可以进行临床试验之前，还有许多问题需要克服： 将胚胎干细胞（ESC）分化为视网膜命运产生适度数量的RGC，并且先前的研究表明， 有报道表明，只有一小部分RGC可以与宿主视网膜整合， 移植本文的主要目标是：（1）改进现有的协议， 在移植的最佳年龄将ESCs分化为RGC，以及（2）识别可能的障碍 到细胞移植 1)为了产生高产量的ESC衍生的RGC：我们的实验室和其他人已经实施了方法， 将胚胎干细胞分化为真正的视网膜祖细胞。我们将制定当前的协议， 通过系统地测试阵列信号线索和miRNA调节剂来增加RGC的产生 已知在正常发育期间参与RGC分化。通过使用各种方法 包括RNA测序和电生理学，我们将表征ESC衍生的RGC。我们会贴上标签， 纯化和移植来自不同年龄的RGCs，以确定最佳发育阶段， 成为移植的供体细胞。 2)为了分析移植后ESC衍生的RGC的整合潜力：我们将使用状态- 最先进的成像技术，用于在活体啮齿动物中移植后跟踪单个ESC衍生的RGC 以准确衡量生存、迁移和与宿主融合的情况。我们将采用小鼠模型， RGC变性以测定环境在RGC植入潜力中的作用。

Project Summary Several human pathologies lead to retinal ganglion cell (RGC) degeneration and irreversible vision loss. Stem cell-based cell replacement therapy offers promising novel approaches for vision restoration. However, there are many issues to overcome before these therapies can be clinically trialled: The current methods to differentiate embryonic stem cells (ESCs) into retinal fates yield modest numbers of RGCs, and previous reports have suggested that only a small fraction of RGCs can integrate with a host retina upon transplantation. The main goals of the present proposal are: (1) to improve the current protocols to efficiently differentiate ESCs into RGC fates at the optimal age for transplantation, and (2) to identify possible barriers to cell engraftment. 1) To generate high yields of ESC-derived RGCs: Our lab and others have implemented methods to differentiate embryonic stem cells into bona-fide retinal progenitors. We will develop the current protocols to increase the production of RGCs by systematically testing an array signaling cues and miRNA regulators known to be involved in RGC differentiation during normal development. By using a variety of approaches including RNA-sequencing and electrophysiology, we will characterize the ESC-derived RGCs. We will label, purify and transplant RGCs from an array of different ages to pinpoint the optimal developmental stage to become donor cells for transplantation. 2) To analyze integration potential of ESC-derived RGCs following transplantation: We will use state- of-the-art imaging technologies to follow individual ESC-derived RGCs after transplantation in living rodents to accurately measure survival, migration and integration with the host. We will employ murine models of RGC degeneration to assay the role of the environment in RGC engraftment potential.