Modulating senescence in induced pluripotent stem cell-derived endothelial cells for vascular disease modeling and therapy

调节诱导多能干细胞衍生内皮细胞的衰老用于血管疾病建模和治疗

• 批准号: 9329121
• 负责人: Katherine Elizabeth Hekman
• 金额: $ 6.31万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9329121
• 关键词: Acetylesterase; Anticoagulation; Attenuated; Autophagocytosis; Biomedical Engineering; Blood Vessels; CDKN2A gene; Cell Aging; Cell Cycle; Cell Cycle Checkpoint; Cell Line; Cell Therapy; Cell physiology; Cells; Cellular Morphology; Cellular biology; Complement; Data; Development; Development Plans; Diagnostic; Digestion; Disease; Disease model; Endothelial Cells; Environment; Evaluation; Feedback; Foundations; Goals; Heart; Human; Human Papillomavirus; Hyperplasia; Individual; Intervention; Investigation; Knowledge; Lead; Limb structure; Longevity; Measures; Mediating; Mediator of activation protein; Messenger RNA; Methodology; Molecular; Morphology; Nutrient; Organ; Oxygen; Pathway interactions; Patients; Phenotype; Process; Proteins; Public Health; Publishing; Recycling; Regenerative Medicine; Research Methodology; Research Personnel; Resveratrol; Retinoblastoma Protein; SIRT1 gene; Science; Scientist; Signal Pathway; Sirtuins; Small Interfering RNA; Source; Stem cells; Stress; Subfamily lentivirinae; Surgeon; Surrogate Markers; TP53 gene; Technology; Therapeutic; Therapeutic Intervention; Therapeutic Uses; Time; Tissues; Training; Tumorigenicity; Umbilical vein; Vascular Diseases; Vascular Endothelial Cell; Vascular Graft; adenylate kinase; attenuation; blood vessel occlusion; body system; career; career development; cellular transduction; design; functional restoration; healing; human disease; improved; improved functioning; induced pluripotent stem cell; injured; knock-down; multidisciplinary; mutant; overexpression; oxidative damage; phenotypic biomarker; premature; regenerative; response; senescence; small hairpin RNA; small molecule; stem cell biology; success; telomere; tissue repair; tool; tumorigenic

PROJECT SUMMARY Induced pluripotent stem cells (iPSCs) are potential diagnostic and therapeutic tools for disease modeling and regenerative medicine, but remain severely limited by reprogramming induced senescence (RIS), which is degenerative changes within cellular progeny resulting in progressive loss of the mature phenotypes possessed by fully functional endogenous cells. RIS is a stress-induced, or premature senescence and is therefore potentially amenable to modulation via intervention in regulatory mechanisms. Several key pathways have been repeatedly implicated in triggering RIS, including cell cycle checkpoint regulators and mediators of autophagy, which is the process of degrading and recycling intracellular contents. Despite this knowledge, investigations aimed at harnessing these pathways to produce phenotypically stable and translatable, long-lived progeny from iPSCs for eventual diagnostic and therapeutic use have met limited success. Our lab demonstrated and published that RIS can be manipulated in iPSC-derived endothelial cells (iPSC-ECs), resulting in stabilization of phenotype and prolongation of function via overexpression of Sirtuin 1 (SIRT1), an NAD-dependent de-acetylase known to modulate mediators of senescence by downregulating the cell cycle regulator p53 and by inducing autophagy. Given this evidence, and in conjunction with additional supportive data, we hypothesize that direct attenuation of SIRT1 targets at critical time-dependent intervals during the reprogramming process will yield iPSC-ECs that display robust and stable endothelial-like phenotypes with direct translatable potential. In Aim 1 we will transiently suppress p53 and p16INK4a with targeted siRNA to knockdown their expression and will uncover the specific time-dependent windows during reprogramming to maximize endothelial function of iPSC-derived cells while circumventing RIS. In Aim 2 we will induce autophagy with the small molecule AMP kinase activators ML246 and Rg2 at critical time points during iPSC induction and EC-differentiation of iPSC-ECs to suppress the switch to RIS and promote longevity of iPSC-ECs and durability of their phenotype and function. The scientific aims reinforce the training goal, which is to develop the investigator’s career niche in vascular and stem cell biology and to develop tools that will form the foundation of an academic career as a vascular surgeon-scientist. The scientific goal of this project is to identify key time points and strategies to intervene during reprograming to overcome RIS in iPSC-ECs, and to generate phenotypically stable and long lived endothelial-like cells. Knowledge gained from this investigation will have broad applications to generate stable iPSC-derived cells for other organ systems beyond the vasculature alone. Durable and functional iPSC-derived cells, such as those that accurately recapitulate endothelial function, would be tools for personalized disease modeling for vascular disease and may be used for therapeutic intervention in regenerative or bioengineering applications such as cell therapy to help injured tissues repair or to replace tissues when damage is too far advanced.

项目摘要 诱导多能干细胞（iPSC）是疾病建模的潜在诊断和治疗工具 和再生医学，但仍然受到重编程诱导衰老（RIS）的严重限制， 细胞后代中的退行性变化，导致所具有的成熟表型的逐渐丧失 由功能齐全的内源性细胞组成。RIS是一种应激诱导的或过早衰老，因此 潜在地易于通过干预调节机制进行调节。几个关键途径已经被 反复参与触发RIS，包括细胞周期检查点调节剂和自噬介质， 这是降解和循环细胞内内容物的过程。尽管有这些知识，调查 旨在利用这些途径产生表型稳定和可翻译的长寿后代， 用于最终诊断和治疗用途的iPSC取得了有限的成功。我们的实验室证明， 发表了RIS可以在iPSC衍生的内皮细胞（iPSC-EC）中操作，导致细胞的稳定。 通过NAD依赖性去乙酰化酶Sirtuin 1（SIRT 1）的过表达延长表型和功能 已知通过下调细胞周期调节因子p53和通过诱导 自噬鉴于这一证据，并结合其他支持性数据，我们假设， 在重编程过程中，SIRT 1靶在关键的时间依赖性间隔处的衰减将产生 iPSC-EC显示具有直接翻译潜力的稳健和稳定的内皮样表型。目标1 我们将用靶向siRNA瞬时抑制p53和p16 INK 4a的表达， 在重编程过程中的特定时间依赖性窗口，以最大化iPSC衍生的内皮功能， 细胞，同时规避RIS。在目标2中，我们将用小分子AMP激酶激活剂诱导自噬 ML 246和Rg 2在iPSC诱导和iPSC-EC的EC分化过程中的关键时间点抑制iPSC的表达。 并促进iPSC-EC寿命及其表型和功能的持久性。科学 目的强化培训目标，即发展研究者在血管和干细胞领域的职业定位 生物学和开发工具，将形成作为血管外科医生科学家的学术生涯的基础。 该项目的科学目标是确定关键的时间点和策略，在重新编程期间进行干预， 克服iPSC-EC中的RIS，并产生表型稳定和长寿命的内皮样细胞。 从这项研究中获得的知识将具有广泛的应用，以产生稳定的iPSC衍生细胞，用于 除了脉管系统之外的其他器官系统。持久的和功能性的iPSC衍生细胞，例如那些 准确再现内皮功能，将是血管疾病个性化建模的工具， 并且可用于再生或生物工程应用中的治疗性干预， 治疗，以帮助受伤的组织修复或更换组织时，损害太远先进。