PRODUCING PIG INDUCED PLURIPOTENT STEM CELLS FOR CARDIOVASCULAR TISSUE ENGINEERING

生产用于心血管组织工程的猪诱导多能干细胞

• 批准号: 10214683
• 负责人: Luke Daniel Batty
• 金额: $ 3.53万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10214683
• 关键词: Address; Alkaline Phosphatase; Animal Model; Autologous; Biocompatible Materials; Biomechanics; Bioreactors; Biosensing Techniques; Blood Vessels; Bypass; Cardiovascular Diseases; Cardiovascular system; Cattle; Cells; Clinical; Collaborations; Collagen; Culture Media; Data; Detection; Doxycycline; Ectopic Expression; Elastin; Engineering; Environment; Epigenetic Process; Ethical Issues; Ethics; Family suidae; Financial Hardship; Financial cost; Foundations; Future; Gene Expression; General Hospitals; Growth Factor; Heterochromatin; Human; Human body; Immune; Immunosuppression; Inbreeding; Individual; Knowledge; Letters; Massachusetts; Mechanics; Mind; Miniature Swine; Modeling; Operative Surgical Procedures; Patients; Persons; Physiology; Polyglycolic Acid; Pre-Clinical Model; Production; Proliferation Marker; Publications; Reproducibility; Research; Resistance; Safety; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Somatic Cell; Source; Specificity; Stains; Stem Cell Factor; System; Talin; Technology; Testing; Tissue Engineering; Transgenes; United States; Vascular Graft; Vascular Smooth Muscle; Western Blotting; base; cell type; clinical application; engineered stem cells; implantation; induced pluripotent stem cell; inducible gene expression; insight; knock-down; mechanical properties; nonhuman primate; pluripotency; pre-clinical; preclinical evaluation; prevent; promoter; scaffold; self-renewal; small hairpin RNA; stem cells; success; tissue stem cells; tool; transcriptome sequencing; transcriptomics; vascular tissue engineering; virtual

PROJECT SUMMARY Tissue-engineered vascular grafts (TEVGs) constructed with human vascular smooth muscle cells (VSMCs) provide a valuable tool for addressing rampant cardiovascular disease. Due to their self-renewal and patient- specificity, induced pluripotent stem cell (iPSCs) from conversion of a person’s own somatic cells by ectopic expression of stem cell factors are the preferred cell-source for TEVGs. Before clinical use, studies on a large animal model simulating patient’s iPSC-based TEVG implantation are important to evaluate safety and efficacy of iPSC-based TEVG. Pigs are an excellent model due to their similarity to human physiology, affordability, and lack of ethical issues, compared to non-human primate models. However, genuine pig iPSCs (piPSCs) free of ectopic reprogramming factors have not been developed. My group recently derived piPSCs from inbred Massachusetts General Hospital (MGH) miniature swine, whose pluripotency is dependent on the doxycycline (DOX)-inducible expression of reprogramming factors. My preliminary data suggest there is an enhancement of pig pluripotency when epigenetic or biomechanical stiffness signaling is modified. With true, inbred piPSCs, functional VSMCs could be derived that would enable us to produce piPSC-based TEVGs for testing in multiple inbred pigs, since inbreeding overcomes immune-incompatibility issue between individuals. With this in mind, my overarching hypothesis is that pig TEVGs can be derived from true piPSCs and maintain suitable mechanical properties for implantation. In Aim 1, I will reprogram pig cells into true iPSCs by modifying their heterochromatin state and biomechanical signaling through addition of factors demonstrated to be able to overcome resistance to reprogramming in publications and in my own data. I will gain mechanistic insight into pig pluripotency via transcriptomic, epigenetic, and mechanical biosensing analysis of true piPSCs compared to transgene dependent piPSCs. In Aim 2, I will derive VSMCs from true piPSCs based on our previous studies and use piPSC-VSMCs to optimize the growth factors and basal media for vessel engineering, based on proliferation, marker expression, and collagen synthesis. We will generate inbred piPSC-based TEVGs in a pulsatile bioreactor system and compare collagen and elastin content and mechanical properties between pig iPSC- VSMC and primary VSMC-based TEVGs. The success of this proposal will set the stage for testing iPSC-based TEVG in a preclinical large animal model, producing essential knowledge for patient-specific, autologous vascular graft treatment of cardiovascular diseases. With “pig to pig” preclinical evaluation of inbred iPSC-TEVG, we will obtain important knowledge from pigs, which will set the foundation for “human to human” clinical application in the future.

项目总结 人血管平滑肌细胞构建的组织工程血管移植物 为解决猖獗的心血管疾病提供了一个有价值的工具。由于他们的自我更新和耐心- 人自身体细胞异位转化的特异性诱导多能干细胞(IPSCs) 干细胞因子的表达是TEVGs的首选细胞来源。在临床使用之前，对一种大型 模拟患者基于IPSC的TEVG植入的动物模型对于评估安全性和有效性具有重要意义 基于IPSC的TEVG。猪是一个很好的模型，因为它们与人类的生理相似，负担得起，而且 与非人类灵长类动物模型相比，缺乏伦理问题。然而，真正的猪IPSCs(PiPSCs)是免费的 异位重编程因子的数量尚未开发出来。我的团队最近从近交系中获得了piPSCs 马萨诸塞州总医院(MGH)小型猪，其多能性依赖于强力霉素 (DOX)-重新编程因子的诱导性表达。我的初步数据显示， 当表观遗传或生物力学僵硬信号被修改时，猪的多能性。有了真正的近亲繁殖的PiPSC， 可以派生出功能性VSMC，使我们能够生产基于PIPSC的TEVG，用于在多个 近亲交配猪，因为近亲交配克服了个体之间的免疫不相容问题。考虑到这一点， 我的主要假设是，猪的TEVG可以从真正的PiPSC衍生而来，并保持适当的机械性能 用于植入的属性。在目标1中，我将通过修改猪细胞的异染色质将其重新编程为真正的IPSCs 通过添加被证明能够克服阻力的因子的状态和生物力学信号 在出版物和我自己的数据中重新编程。我将通过以下方式获得对猪的多能性的机械洞察力 真PiPSCs的转录、表观遗传学和机械生物传感分析与转基因的比较 依赖的PPSCs。在目标2中，我将根据我们之前的研究和使用，从真实的piPSCs中提取VSMC PIPSC-VSMCs用于优化血管工程的生长因子和基础培养液，基于增殖， 标记物表达和胶原蛋白合成。我们将在脉动中产生基于PIPSC的近交系TEVG 并比较了猪IPSC的胶原和弹性蛋白含量及力学性能。 VSMC和主要基于VSMC的TEVG。该提案的成功将为基于IPSC的测试奠定基础 临床前大型动物模型中的TEVG，为患者特定的、自体的 心血管疾病的血管移植治疗。通过对近交系IPSC-TEVG的临床前评估， 我们将从猪那里获得重要的知识，这将为人与人之间的临床奠定基础。 在未来的应用。