Regulation of Human Embryonic Stem Cell Neuro-Retinal Differentation

在过去的二十年中，在视网膜电图的方法的开发方面取得了稳步进展。 修复动物模型。以胎儿视网膜为供体，效果最好 迄今为止的结果表明，未成熟的神经元在移植方案中存活得更好， 比成熟的神经元。由于对使用胎儿人体组织的伦理和实际关注， 将动物模型中的努力转化为人类疾病的治疗将需要一种新的来源， 发育中的视网膜神经元为此，我们已经开发了定向人类胚胎干细胞的方法， (hES)视网膜祖细胞的命运。我们使用了两种不同的联邦批准的人类 胚胎干细胞，并将它们置于基于先前眼睛分子研究的协议中， 发展我们发现，在我们的方案中，培养物中高达80%的细胞显示出特征性基因， 在视网膜测定（RD）培养基中三周后的视网膜祖细胞谱。许多细胞 也在体外分化成功能性视网膜神经元，初步实验表明， 来源于hES细胞的神经元和祖细胞将在移植后存活并分化， 退化的老鼠视网膜我们现在建议在以下三个目标中扩展这些研究。AIM 1. 确定hES细胞衍生的视网膜祖细胞中是否存在能力变化的时钟。 确定Notch/delta信号传导是否调节hES细胞衍生的视网膜祖细胞的分化。 AIM 2.为了确定Notch/delta信号传导是否调节hES细胞来源的视网膜神经元的分化， 祖先AIM 3.开发方法，通过以下方式将来自人ES细胞的视网膜神经元分类为不同类型： 特异性启动子-GFP构建体的表达。目标4.为了确定hES细胞衍生的视网膜 在移植到小鼠模型中后，祖细胞可以分化成功能性光感受器。 Leber先天性黑蒙和视网膜色素变性，使用电生理分析。如果我们 成功挽救和/或恢复动物的功能（通过ERG和行为分析评估） RP和LCA模型，结果将是人类胚胎干细胞可以用于原理的证明 用于这些视网膜疾病的细胞替代疗法。

Over the past twenty years, there has been steady progress made in the development of methods for retinal repair in animal models. The best results have been obtained when fetal retina has been used as donor tissue, and results to date indicate that immature neurons survive the transplantation protocols much better than mature neurons. Due to ethical and practical concerns with the use of fetal human tissues, the translation of the efforts in animal models to the treatment of human disease will require a new source of developing retinal neurons. To this end, we have developed methods for directing human embryonic stem (hES) cells to the retinal progenitor fate. We have used two different lines of Federally approved human embryonic stem cells, and subjected them to a protocol based on previous molecular studies of eye development. We find that with our protocol up to 80% of the cells in the cultures show characteristic gene profiles of retinal progenitors after three weeks in Retinal Determination (RD) medium. Many of the cells also differentiate into functional retinal neurons in vitro, and preliminary experiments show that the retinal neurons and progenitors derived from hES cells will survive and differentiate following transplantation to degenerating mouse retinas. We now propose to extend these studies in the following three Aims. AIM 1. To determine whether the clock of changing competence is present in hES cell derived retinal progenitors. To determine whether Notch/delta signaling regulates differentiation of hES cell-derived retinal progenitors. AIM 2. To determine whether Notch/delta signaling regulates differentiation of hES cell-derived retinal progenitors. AIM 3. Develop methods to sort retinal neurons derived from human ES cells into types by expression of specific promoter-GFP constructs. AIM 4. To determine whether hES cell-derived retinal progenitors can differentiate into functional photoreceptors following transplantation to mouse models of Leber's congenital amaurosis and Retinitis Pigmentosa, using electrophysiological analyses. If we are successful in rescuing and/or restoring function (as assessed by ERG and behavioral analyses) to animal models of RP and LCA, the results will be a proof of principle that human embryonic stem cells can be used in cell replacement therapies for these retinal diseases.