Hypoxia-induced reprogramming to RPE stem cells

描述（申请人提供）：在尾动物和无尾动物中，视网膜再生的一个主要组成部分是RPE向神经视网膜的转分化。但哺乳动物的再生能力有限，这使它们容易受到视网膜损伤和致盲疾病的影响。最近的研究在人类RPE培养物中发现了一个RPE干细胞群（RPESC）。这些RPESC具有不受限制的增殖能力，其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，但它们不能诱导RHO等光受体标记物。因此，这些RPESC可能不是哺乳动物RPE转分化为光感受器的中间物。我们检查了成年小鼠RPE培养物中具有RPESC分化能力的细胞，但我们未能识别出这样的细胞。然而，我们发现具有RPESC特性的细胞可以通过缺氧依赖途径有效且稳定地从RPE （iRPESC）诱导，类似于描述的维持和诱导MSC的途径。缺氧导致RPE损伤，并与新生血管和AMD有关。iRPESC重编程途径的关键是缺氧诱导因子1a （Hif1a）超诱导到足以结合并激活Oct4干细胞基因启动子的阈值。Oct4反过来诱导Dnmt1， Dnmt1沉默细胞周期阻断细胞周期蛋白依赖的激酶抑制剂，导致无限制增殖。这些iRPESC能够抵抗缺氧，重要的是，与人类的RPESC相反，它们能够分化为Rho+细胞——实际上，这种分化比胚胎干细胞或诱导多能干细胞更有效。此外，iRPESC不经历典型的上皮-间质转化（EMT），当RPE放置在培养物中时，这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）是由于功能性RPE和光感受器的丧失而突出，因此iRPESC进行光感受器分化和抵抗emt引发的表型丧失的能力表明，该细胞具有独特的治疗潜力。在这个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优化iRPESC的光感受器分化的实验提供基础，并在iRPESC扩展时保持功能RPE表型，以便将来我们可以开始在移植实验中测试分化的iRPESC的有效性。该分子分析的第二点是确定最终可用于体内iRPESC检测的途径标记，并了解将来可能用于刺激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.