Regulation of Human Embryonic Stem cell Neuro-retinal Differentiation

胚胎干细胞和诱导多能干细胞研究最重要的潜在用途之一是开发疾病模型。虽然许多疾病可以在小鼠中建模，但由于各种原因，其他疾病难以或不可能在非人类生物体中充分建模。例如，在视网膜中，有许多遗传性变性的小鼠模型，但这些模型更难产生。 像黄斑变性和多种形式的Usher病。因此，来自人类患者iPS细胞的疾病模型将在视网膜研究中具有很大的实用性。在过去的六年里，我们的研究小组和其他研究小组已经开发出了从人类胚胎干细胞和诱导多能干细胞高效生产视网膜细胞的方案。视网膜发育的早期阶段通过我们的hESC细胞定向分化的方案非常好地再现;然而，即使经过长时间的培养，细胞也未能达到我们在人胎儿或出生后发育的晚期阶段观察到的标记物的表达水平。因此，从iPSC创建疾病模型的一个重大挑战将是更好地理解和促进培养细胞向视网膜成熟阶段的进展。控制视网膜祖细胞发育时间的机制尚不清楚;然而，我们实验室最近的证据显示了miRNAs的关键作用。具体地说，我们发现视网膜祖细胞中Dicer的缺失导致它们无法从“早期”状态进展到“晚期”状态。因此，我们假设：（1）miRNAs调控了小鼠的发育时间， 视网膜祖细胞和（2）ESC衍生的视网膜细胞中异时途径的错误调节导致它们在体外不能以与体内相同的速率进展。我们建议以下列具体目标来检验这些假设。 目标1：确定特定miRNAs（及其调节的基因）的表达是否与视网膜祖细胞从早期到晚期的进展相关。目的2.确定小鼠ESC/iPSC衍生的视网膜祖细胞的发育进程是否受阶段特异性祖细胞miRNAs的控制。目标3.确定阶段特异性miRNA是否可以加速人ESC衍生的视网膜祖细胞的发育进程。这些研究的结果将使我们能够更好地控制hESC衍生的视网膜细胞的发育，并产生更合适的视网膜疾病模型。

One of the most important potential uses of embryonic stem cells and induced pluripotent stem cell research is in the development of disease models. Although many diseases can be modeled in mice, others are difficult or impossible to adequately model in non-human organisms for a variety of reasons. In the retina, for example, there are mouse models for many inherited degenerations, but these have been more difficult to generate for diseases like macular degeneration and many forms of Usher's disease. Therefore, disease models from human patient iPS cells would be of great utility in retinal research. In the past six years, our group, and others, have developed protocols for efficient production of retinal cells from human ESCs and iPSCs. The early stages of retinal development are extremely well recapitulated by our protocol of directed differentiation of hESC cells; however, the cells fail to attain the level of expression of markers that we observe in the late stages of human fetal or postnatal development, even with prolonged culture periods. Therefore, a significant challenge for creation of disease models from iPSCs will be to better understand and promote the progression of the cultured cells to mature stages of retina. The mechanisms that control the developmental timing of retinal progenitor cells are not clear; however, recent evidence from our lab shows a key role for miRNAs. Specifically, we found that the loss of Dicer in retinal progenitor cells leads to their failure to progress from the "early" state to the "late" state. We therefore hypothesize (1) that miRNAs regulate developmental timing in retinal progenitor cells and (2) that misregulation of the heterochronic pathway in ESC-derived retinal cells accounts for their failure to progress in vitro at the same rate as in vivo. We propose to test these hypotheses with the following specific aims. Aim 1: Determine whether the expression of specific miRNAs (and the genes they regulate) correlates with the progression of retinal progenitors from the early to late stage. Aim 2.Determine whether the developmental progression of mouse ESC/iPSC-derived retinal progenitors is controlled by stage-specific progenitor miRNAs. Aim 3. Determine whether the developmental progression of human ESC derived retinal progenitors can be accelerated by stage-specific miRNAs. The results of these studies will enable us to better control the development of hESC-derived retinal cells and produce more appropriate models of retinal disease.