Hypoxia-induced reprogramming to RPE stem cells

缺氧诱导的 RPE 干细胞重编程

• 批准号: 8671540
• 负责人: DOUGLAS Chase DEAN
• 金额: $ 18.75万
• 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 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8671540
• 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; 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; public health relevance; 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urodeles和anurans中，视网膜再生的主要组成部分是RPE对神经视网膜的转变。但是，哺乳动物的再生受到限制，使它们容易受到视网膜损伤和盲目疾病的影响。最近的研究确定了人类RPE培养物中的RPE干细胞（RPESC）人群。这些RPESC表现出无限制的增殖及其分化潜力非常相似的间充质干细胞（MSC），它们与RPE共享一个常见的神经上皮起源。尽管RPESC有区别以表达神经元标记，但它们未能诱导像Rho这样的光感受器标记。因此，这些RPESC可能不代表哺乳动物RPE转差向感光体的中间体。我们检查了成年小鼠RPE的培养物的RPESC分化能力的细胞，但我们无法识别此类细胞。但是，我们发现，具有RPESC特性的细胞可以通过低氧依赖性途径有效且稳定地从RPE（IRPESC）诱导，类似于用于维持和诱导MSC的细胞。缺氧会导致RPE损害，并与新血管形成和AMD有关。该IRPESC重编程途径的关键是将缺氧诱导因子1a（HIF1A）的诱导到足以结合和激活OCT4干细胞基因启动子的阈值。 OCT4反过来诱导DNMT1，这使细胞周期阻塞细胞周期依赖性激酶抑制剂导致无限制增殖。这些IRPESC对缺氧具有抗性，重要的是，与人RPESC相反，它们分化为Rho+细胞 - 将这种分化与Rho+细胞的分化比在胚胎干细胞或诱导多能干细胞的情况下更有效。此外，IRPESC不会在将RPE放入培养物中时观察到的典型上皮 - 间质转变（EMT），从而为随着细胞的扩展提供了保留RPE表型的潜力。由于诸如AMD之类的盲疾病是通过功能性RPE和感光体的丧失突出的，因此IRPESC经历光感受器分化并抵抗EMT引发的表型丧失的能力表明了细胞的独特治疗潜力。在该R21提案的两年期间，我们旨在研究分子水平的IRPESC重编程途径。这些研究的目的是为实验提供基础，旨在优化与IRPESC的光感受器分化，并随着IRPESC的扩展而保持功能RPE表型，因此将来我们可以开始测试分化IRPESC在移植实验中的有效性。该分子分析的第二点是识别最终可以用于检测体内IRPESC的途径标记，并了解将来可能使用的因素来刺激IRPESC从原位刺激IRPESC的生成。