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在疾病模型的开发中。虽然许多疾病可以在老鼠身上建模，但由于各种原因，其他疾病很难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，但这些模型更难生成 黄斑变性和多种亚瑟氏病等疾病。因此，人类患者iPS细胞的疾病模型在视网膜研究中将是非常有用的。在过去的六年里，我们的团队和其他人开发了从人类ESCs和IPSCs高效生产视网膜细胞的方案。我们的hESC定向分化方案很好地概括了视网膜发育的早期阶段；然而，即使延长了培养时间，这些细胞也无法达到我们在人类胎儿或出生后发育后期观察到的标志物的表达水平。因此，从IPSCs建立疾病模型的一个重大挑战将是更好地了解和促进培养细胞向视网膜成熟期的进展。控制视网膜前体细胞发育时机的机制尚不清楚；然而，我们实验室最近的证据表明，miRNAs具有关键作用。具体地说，我们发现视网膜前体细胞中DICER的缺失导致它们无法从早期状态发展到晚期状态。因此，我们假设(1)miRNAs在 视网膜前体细胞和(2)ESC来源的视网膜细胞中异时性通路的错误调节是它们在体外无法以与体内相同的速度进展的原因。我们建议用以下具体目标来检验这些假设。 目的1：确定特定miRNAs(及其调控基因)的表达是否与视网膜前体细胞从早期到晚期的进展相关。目的2.确定小鼠ESC/IPSC来源的视网膜前体细胞的发育过程是否受阶段特异性前体细胞miRNAs的控制。目的3.确定阶段特异性miRNAs能否加速人胚胎干细胞来源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来源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.