Hypoxia-induced reprogramming to RPE stem cells

描述（由申请人提供）：在有尾目动物和无尾目动物中，视网膜再生的主要成分是RPE向神经视网膜的转分化。但哺乳动物的再生能力有限，使它们容易受到视网膜损伤和致盲疾病的影响。最近的研究在人RPE的培养物中鉴定了RPE干细胞（RPESC）群体。这些RPESC显示出不受限制的增殖和分化潜力，与间充质干细胞（MSC）非常相似，后者与RPE具有共同的神经上皮起源。虽然RPESC分化表达神经元标记物，但它们未能诱导感光细胞标记物如RHO。因此，这些RPESC可能不代表哺乳动物RPE转分化为光感受器的中间体。我们检查了培养的成年小鼠视网膜色素上皮细胞的分化能力的RPESC的细胞，但我们未能确定这样的细胞。然而，我们发现具有RPESC特性的细胞可以通过低氧依赖性途径从RPE（iRPESC）有效且稳定地诱导，类似于针对MSC的维持和诱导所描述的。缺氧导致RPE损伤，并与新血管形成和AMD有关。这种iRPESC重编程途径的关键是缺氧诱导因子1a（Hif 1a）的超诱导，达到足以结合和激活Oct 4干细胞基因启动子的阈值。Oct 4反过来诱导Dnmt 1，其沉默细胞周期阻断细胞周期蛋白依赖性激酶抑制剂，导致不受限制的增殖。这些iRPESC具有耐缺氧性，重要的是，与人RPESC相反，它们分化为Rho+细胞--事实上，这种向Rho+细胞的分化比胚胎干细胞或诱导多能干细胞更有效。此外，iRPESC不经历当将RPE置于培养物中时所见的典型上皮-间充质转化（EMT），从而提供了在细胞扩增时保留RPE表型的潜力。由于致盲性疾病如AMD通过功能性RPE和光感受器两者的丧失而突出，iRPESC经历光感受器分化和抵抗EMT引发的表型丧失的能力表明细胞具有独特的治疗潜力。在R21提案的两年期间，我们的目标是在分子水平上研究iRPESC重编程途径。这些研究的目的是为设计用于优化来自iRPESC的光感受器分化并在iRPESC扩增时维持功能RPE表型的实验提供基础，因此将来我们可以开始测试分化的iRPESC在移植实验中的有效性。该分子分析的第二点是鉴定最终可用于体内检测iRPESC的途径标记物，并了解未来可能用于刺激RPE原位产生iRPESC的因素。

DESCRIPTION (provided by applicant): In urodeles and anurans a major component of retinal regeneration is transdifferentiation of RPE to neural retina. But regeneration is limited in mammals, leaving them susceptible to retinal injury and blinding diseases. Recent studies identified a population of RPE stem cells (RPESC) among cultures of human RPE. These RPESC show unrestricted proliferation and their differentiation potential closely resembled mesenchymal stem cells (MSC), which share a common neuroepithelial origin with RPE. Although RPESC differentiated to express neuronal markers, they failed to induce photoreceptor markers such as RHO. Thus, these RPESC may not represent an intermediate in transdifferentiation of mammalian RPE into photoreceptors. We examined cultures of adult mouse RPE for cells with the differentiation capacity of RPESC, but we failed to identify such cells. However, we found that cells with properties of RPESC can be efficiently and stably induced from RPE (iRPESC) through a hypoxia-dependent pathway, similar to that described for maintenance and induction of MSC. Hypoxia causes RPE damage and is linked to neovascularization and AMD. Key to this iRPESC reprogramming pathway is superinduction of hypoxia inducible factor 1a (Hif1a) to a threshold sufficient to bind and activate the Oct4 stem cell gene promoter. Oct4 in turn induces Dnmt1 which silences cell cycle blocking cyclin dependent kinase inhibitors leading to unrestricted proliferation. These iRPESC are resistant to hypoxia, and importantly, as opposed to human RPESC, they differentiate into Rho+ cells-indeed this differentiation to Rho+ cells is more efficient than seen with embryonic stem cells or induced pluripotent stem cells. Furthermore, iRPESC do not undergo the typical epithelial-mesenchymal transition (EMT) seen when RPE are placed in culture, providing the potential for retaining an RPE phenotype as the cells are expanded. Because blinding diseases such as AMD are highlighted by loss of both functional RPE and photoreceptors, the ability of the iRPESC to undergo photoreceptor differentiation and to resist EMT-initiated loss of phenotype suggest a unique therapeutic potential for the cells. During the two year period of this R21 proposal, we aim to investigate the iRPESC reprogramming pathway on a molecular level. The purpose of these studies is to provide a foundation for experiments designed to optimize photoreceptor differentiation from iRPESC and to maintain a function RPE phenotype as iRPESC are expanded, so in the future we can begin testing the effectiveness of the differentiated iRPESC in transplantation experiments. A second point of this molecular analysis is to identify pathway markers that can ultimately be used for detection of iRPESC in vivo, and to understand factors that might be used in the future to stimulate iRPESC generation from RPE in situ.