Epigenetic Reprogramming of Retinal Neurons

描述(申请者提供)：视网膜变性每年影响全球数百万人。在过去的十年里，我的实验室一直在研究发育中的视网膜和视网膜增殖性疾病(如视网膜母细胞瘤)中增殖和分化的协调。最近，我们有了一个惊人的发现，从根本上改变了我们对视网膜发育的分子和细胞机制的理解，并可能对一些视网膜变性患者恢复视力的努力产生重大影响。我们发现，单个视网膜母细胞瘤细胞同时表达多种发育程序。这是通过解除受RB1蛋白直接或间接调控的表观遗传程序来实现的。为了进一步探索这一令人兴奋的发现，我们开发了一个新的实验系统，通过使用4个因子(Oct4、Klf4、Sox2和Myc)和体细胞核移植来量化单个视网膜神经元的表观遗传重编程。我们发现，重编程的表观遗传障碍在不同类型的视网膜细胞中差异很大，而且它们是发育阶段特有的。此外，我们还利用Sasai三维培养系统首次证明了小鼠IPSCs可以形成视杯和分化的视网膜。这些实验最令人兴奋的结果之一是，我们的来自视网膜神经元的IPSC系绕过了通过前神经外胚层规范的正常过渡。相反，它们保留了视网膜的表观遗传记忆，并仅形成分化为层状视网膜的视网膜前体细胞。来自视网膜神经元的IPSCs保持其表观遗传的视网膜记忆至少50代，而由遗传相同的MEF产生的IPSCs在这个系统中很少产生视网膜。我们现在已经证明，来自视网膜IPSCs的光感受器前体可以整合到视网膜中；因此，由成熟的视网膜神经元产生的IPSCs可能为细胞替代疗法提供可再生的光感受器前体来源，以恢复视网膜退行性变患者的视力。这一创新的研究方案将促进我们对表观遗传学在视网膜发育中的作用的理解，并开始阐明参与这一过程的分子机制和特定细胞类型的靶基因。它还将为未来光感受器替代疗法治疗视网膜变性的临床试验提供使用视网膜来源的ipscs的关键临床前数据。

DESCRIPTION (provided by applicant): Retinal degeneration affects millions of people around the world each year. For the past decade, my lab has studied the coordination of proliferation and differentiation in the developing retina and in proliferative diseases of the retna such as retinoblastoma. Recently, we made a startling discovery that has fundamentally altered our understanding of the molecular and cellular mechanisms of retinal development and may also have a major impact on efforts to restore vision in some patients with retinal degeneration. We discovered that individual retinoblastoma tumor cells express multiple developmental programs simultaneously. This occurs through deregulation of the epigenetic programs that are directly or indirectly regulated by the RB1 protein. To explore this exciting finding further, we developed a novel experimental system to quantify the epigenetic reprogramming of individual retinal neurons by using 4 factors (Oct4, Klf4, Sox2, and Myc) and somatic cell nuclear transfer. We discovered that the epigenetic barriers to reprogramming dramatically differ across retinal cell types, and they are developmental stage-specific. Moreover, we have used the Sasai 3-dimensional culture system to show for the first time that mouse iPSCs can form the optic cup and differentiated retinae. One of the most exciting results from these experiments is that our iPSC lines derived from retinal neurons bypass the normal transition through anterior neuroectodermal specification. Instead, they retain retinal epigenetic memory and form exclusively retinal progenitor cells that differentiate into laminated retinae. The iPSCs derived from retinal neurons retain their epigenetic retinal memory for at least 50 passages, whereas iPSCs generated from genetically identical MEFs rarely produce retinae in this system. We have now shown that the photoreceptor precursors derived from retinal iPSCs can integrate into the retina; thus, iPSCs generated from mature retinal neurons may provide a renewable source of photoreceptor precursors for cell-replacement therapies to restore vision in those who suffer from retinal degeneration. This innovative research proposal will advance our understanding of the role of epigenetics in retinal development and begin to elucidate the molecular mechanisms and cell type-specific target genes involved in that process. It will also provide crucial preclinial data on the use of retinal-derived iPSCs for future clinical trials of photoreceptor-replacement therapy to treat retinal degeneration.