Readily Available Stem Cell-Based Vascular Grafts for Emergent Surgical Care

用于紧急手术护理的现成干细胞血管移植物

• 批准号: 10414459
• 负责人: Yibing Qyang
• 金额: $ 6.19万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10414459
• 关键词: BCL2 gene; Caliber; Caring; Cell Line; Cell physiology; Cells; Data; Development; Disease; Doxycycline; Elderly; Endothelial Cells; Endothelium; Ensure; Event; Genes; Goals; Human; Immunocompromised Host; Implant; Lead; Liquid substance; Modeling; Operative Surgical Procedures; Patients; Polyglycolic Acid; Population; Problem Solving; Rattus; Research Personnel; Risk; Safety; Smooth Muscle Myocytes; Source; System; Thrombosis; Thymidine Kinase; Time; Tissues; Training; Traumatic injury; Vascular Graft; Vascular Smooth Muscle; Work; acute care; base; clinical application; efficacy testing; implantation; induced pluripotent stem cell; inducible gene expression; injury and repair; parent grant; patient population; repaired; scaffold; shear stress; stem cells; thrombotic; transcription activator-like effector nucleases; vascular injury; vascular tissue engineering

Project Summary Tissue engineered vascular grafts (TEVGs) for traumatic vascular injury repair in small diameter (2- 4mm) vessels can be made from human induced pluripotent stem cell derived vascular smooth muscle cells (hiPSC-VSMC) seeded onto a polyglycolic acid (PGA) scaffold. Furthermore, these TEVGs can be subsequently decellularized and stored long term for use in acute care for traumatic injury. For larger diameter vessel repair, it is adequate to implant an acellular scaffold and allow host cells to migrate from anastomotic edges to line the implanted vessel lumen. However, this model is not effective for smaller diameter vessels that are more prone to thrombosis. Researchers have attempted to solve this problem by coating the lumen of implanted vessels with endothelial cells (ECs) prior to implantation. However, data from implanted grafts suggests there is a short turnover time for implanted EC populations within these grafts. In the case of elderly or diseased patients, they may not retain quality EC function. Therefore, this patient population may display poor integration of host cells into the implanted tissue, which may lead to an increased risk of thrombotic and stenotic events. The purpose of this project is to generate a stable hiPSC line with controllable expression of the pro-survival factor Bcl-2 to generate hiPSC derived endothelial cells (hiPSC-ECs) as a readily available cell source to line the lumen of decellularized TEVGs prior to implantation. A stable hiPSC line with doxycycline inducible expression of the pro-survival factor Bcl-2 will be generated via transcription activator-like effector nucleases (TALEN) gene editing. Additionally, a robust “safety switch” system using ectopically expressed thymidine kinase (TK) will be employed to ensure cells may be removed post implantation should unwanted effects occur. Decellularized TEVGs will have their lumen coated with hiPSC-ECs made from this gene edited Bcl-2+TK+ cell line. These endothelialized TEVGs will undergo fluid shear stress training to enhance hiPSC-EC function prior to implantation into an immunocompromised rat model as an aortic interposition graft to test the efficacy of this doxycycline inducible Bcl-2 system. This platform will allow for a readily available cell line to be used to produce a long lasting hiPSC-EC lumen that can maintain patency of the graft as it fully integrates with patients who may have subpar EC function.

项目摘要 组织工程化血管移植物修复小直径（2- 1 mm）创伤性血管损伤的实验研究 4 mm）血管可以由人诱导多能干细胞衍生的血管平滑肌制成。 肌肉细胞（hiPSC-VSMC）接种到聚乙醇酸（PGA）支架上。而且这些 TEVG随后可以脱细胞化并长期储存，用于创伤患者的急性护理。 损伤对于较大直径的血管修复，植入脱细胞支架并允许宿主 细胞从吻合边缘迁移到植入的血管腔中。然而，这种模式是 对更容易血栓形成的较小直径血管无效。研究人员 试图通过用内皮细胞覆盖植入血管的管腔来解决这个问题 (ECs)在植入之前。然而，来自植入移植物的数据表明， 在这些移植物内植入EC群体的时间。在老年人或患病患者的情况下， 他们可能无法保持高质量的EC功能。因此，该患者人群可能表现出不良的 宿主细胞整合到植入组织中，这可能导致血栓形成风险增加 和狭窄事件。该项目的目的是生成稳定的hiPSC生产线， 表达促存活因子Bcl-2以产生hiPSC衍生的内皮细胞（hiPSC-EC） 作为容易获得的细胞来源，在植入前衬在脱细胞TEVG的内腔中。一 具有多西环素诱导的促存活因子Bcl-2表达的稳定hiPSC系将 通过转录激活因子样效应物核酸酶（TALEN）基因编辑产生。此外，还 使用异位表达的胸苷激酶（TK）的稳健“安全开关”系统将用于 确保如果发生不希望的效应，则可以在植入后移除细胞。脱细胞 TEVG将使其管腔涂覆有由该基因编辑的Bcl-2+TK+细胞系制成的hiPSC-EC。 这些内皮化的TEVG将经历流体剪切应力训练以增强hiPSC-EC功能 在作为主动脉间置移植物植入免疫功能低下的大鼠模型中之前， 这种强力霉素诱导的Bcl-2系统的功效。这个平台将允许一个随时可用的细胞 用于产生持久的hiPSC-EC管腔的线，该管腔可以保持移植物的通畅性， 与EC功能可能低于标准的患者完全整合。