Hypoxia-induced reprogramming to RPE stem cells

缺氧诱导的 RPE 干细胞重编程

• 批准号: 8819132
• 负责人: DOUGLAS Chase DEAN
• 金额: $ 22.05万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8819132
• 关键词: Adult; Binding; Cell Aging; Cell Cycle; Cell Differentiation process; Cells; Cessation of life; Cyclin-Dependent Kinase Inhibitor; Detection; Development; Disease; Effectiveness; Embryo; Environment; Epithelial; Foundations; Future; Generations; Genes; Goals; Health; Hemostatic function; Human; Hypoxia; Hypoxia Inducible Factor; In Situ; Injury; Knowledge; Left; Link; Maintenance; Mammals; Mesenchymal; Mesenchymal Stem Cells; Mitotic; Molecular; Molecular Analysis; Mus; Natural regeneration; Neural Retina; Neuroepithelial; Neuronal Differentiation; Neurons; Newts; Optics; Organ; Pathway interactions; Phenotype; Photoreceptors; Play; Population; Proliferating; Property; Protocols documentation; Rana; Repression; Resistance; Retinal; Retinal Diseases; Rodent; Role; Salamander; Stem cells; Structure of retinal pigment epithelium; Suspension substance; Suspensions; Testing; Therapeutic; Tissues; Transplantation; adult stem cell; design; diencephalon; embryonic stem cell; in vivo; induced pluripotent stem cell; inhibitor/antagonist; monolayer; neovascularization; neuroepithelium; pluripotency; prevent; progenitor; promoter; repaired; research study; response; restoration; retinal regeneration; retinal rods; rho; senescence; toad; transdifferentiation

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.

描述（由申请人提供）：在有尾目和无尾目动物中，视网膜再生的主要组成部分是RPE向神经视网膜的转分化。但哺乳动物的再生能力有限，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 RPE 干细胞 (RPESC) 群体。这些 RPESC 显示出不受限制的增殖，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。尽管 RPESC 分化并表达神经元标记，但它们未能诱导光感受器标记，例如 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。我们检查了成年小鼠 RPE 培养物中具有 RPESC 分化能力的细胞，但我们未能鉴定出此类细胞。然而，我们发现具有 RPESC 特性的细胞可以通过缺氧依赖性途径从 RPE (iRPESC) 有效且稳定地诱导出来，类似于 MSC 的维持和诱导。缺氧会导致 RPE 损伤，并与新生血管形成和 AMD 相关。该 iRPESC 重编程途径的关键是将缺氧诱导因子 1a (Hif1a) 超诱导至足以结合并激活 Oct4 干细胞基因启动子的阈值。 Oct4 反过来诱导 Dnmt1，后者沉默细胞周期，阻断细胞周期蛋白依赖性激酶抑制剂，从而导致不受限制的增殖。这些 iRPESC 能够抵抗缺氧，重要的是，与人类 RPESC 不同，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 不会经历 RPE 培养时所见的典型上皮间质转化 (EMT)，因此有可能在细胞扩增时保留 RPE 表型。由于 AMD 等致盲性疾病的特点是功能性 RPE 和光感受器的丧失，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 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 的光感受器分化并在 iRPESC 扩增时维持功能性 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确定最终可用于体内检测 iRPESC 的途径标记，并了解未来可能用于刺激 RPE 原位生成 iRPESC 的因素。