Regulation of Human Embryonic Stem Cell Neuro-Retinal Differentation

在过去的二十年里，视网膜治疗方法的发展取得了稳步的进展。 在动物模型中修复。以胎儿视网膜为供体效果最佳。 组织，到目前为止的结果表明，未成熟神经元在移植方案中存活得更好 而不是成熟神经元。出于对使用胎儿人体组织的伦理和实际问题的关注， 将动物模型中的努力转化为治疗人类疾病将需要一个新的来源 发育中的视网膜神经元。为此，我们开发了指导人类胚胎干细胞的方法 (HES)细胞对视网膜祖细胞的命运。我们已经使用了两种不同的联邦批准的人类 胚胎干细胞，并让它们接受基于先前眼睛分子研究的方案 发展。我们发现，在我们的方案中，培养的细胞中高达80%的细胞显示出特征基因 视网膜祖细胞在视网膜测定(RD)培养液中培养三周后的分布情况。许多细胞 也可以在体外分化为有功能的视网膜神经元，初步实验表明，视网膜 HES细胞来源的神经元和前体细胞移植到 退化的小鼠视网膜。我们现在建议将这些研究扩大到以下三个目标。目标1。 以确定在HES细胞来源的视网膜祖细胞中是否存在改变能力的时钟。 目的：确定Notch/Delta信号通路是否调节HES细胞来源的视网膜前体细胞的分化。 目的2.确定Notch/Delta信号是否调节HES细胞来源的视网膜的分化 祖先。目的3.开发从人类胚胎干细胞中分离视网膜神经元的方法 特异性启动子-GFP构建物的表达。目的4.确定HES细胞来源的视网膜 将祖细胞移植到小鼠模型后，可以分化为有功能的光感受器 Leber‘s先天性黑色素和视网膜色素变性，使用电生理分析。如果我们是 成功抢救和/或恢复动物的功能(通过ERG和行为分析进行评估) RP和LCA的模型，结果将证明人类胚胎干细胞可以使用的原则 在这些视网膜疾病的细胞替代疗法中。

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.