HUMAN TISSUE ENGINEERED PULSATILE CONDUITS USING ENGINEERED HEART TISSUE OF DEFINED COMPOSITION AND FIBER ORIENTATION

摘要： 从人诱导多能干细胞衍生的心室肌制备的工程化心脏组织（EHT） 心肌细胞（hiPSC-VCM）可以用作心血管治疗的有前景的工具。单个 心室心脏缺陷是一类先天性疾病，其中只有一个心室正常发育。 这可能导致富氧血液和贫氧血液在循环过程中混合， 对身体组织的氧气供应不足。此外，这类缺陷会增加应变， 在收缩时的单心室。患有这种疾病的儿童通常用 Fontan手术使血流从上级和下腔静脉直接进入肺动脉 动脉内衬有骨髓来源的干细胞的合成移植物已被用作血管导管， 连接下腔静脉和肺动脉。然而，这些合成 移植物不能提供帮助血液循环的泵送活性。这个项目的目的是产生 组织工程脉动导管（TEPC）通过产生收缩力来帮助循环，因此 增加的驱动压力，以帮助通过肺部系统的流动。采用的设计策略 TEPC生产使用脱细胞人脐动脉作为脱细胞血管支架， 其机械特性允许其维持下腔静脉中的通畅血流。这个脚手架是 用hiPSC-VCM衍生的EHT包裹，以为导管提供收缩力。脱细胞猪心 组织被用作产生EHT的支架，因为它具有允许 在组织内产生心肌纤维的可控排列。hiPSC-VCM的共培养EHT 制备心脏成纤维细胞并测试收缩力输出。初步数据显示， 将成纤维细胞引入组织中对收缩输出具有积极作用。微血管网络 将产生支持较厚的TEPC肌肉层，并增加整体TEPC压力的产生。 TEPC将接受由物理和电暗示组成的体外训练方案，以增强 它们的收缩性和电处理性能。流动生物反应器将在TEPC内提供拉伸 管腔以给予组织机械压力，其模拟将在大鼠下腔静脉中经历的压力。 放置在该生物反应器系统内的电极将提供场刺激，该场刺激旨在模拟 人类发育环境，以诱导出更成熟的电特性从hiPSC- VCM。为了测试移植物的基本存活，将使用裸大鼠模型来评估移植物的基本参数。 作为下腔静脉间置移植物成功植入。完善和优化这 在这项研究中开发的用于生产TEPC的稳健设计策略将为测试 未来的治疗潜力。最终，这项工作将在开发改进的 治疗单心室缺陷的患者。

Abstract: Engineered heart tissue (EHT) made from human induced pluripotent stem cell-derived ventricular cardiomyocytes (hiPSC-VCMs) can be used as a promising tool for the cardiovascular therapeutics. Single ventricle heart defects are a class of congenital disorder where only a single ventricle properly develops. This can result in mixing of oxygen-rich blood and oxygen-poor blood during circulation, leading to inefficient oxygen supply to tissues of the body. Additionally, this class of defects places an increased strain on the single ventricle during contraction. Children born with this disease are commonly treated with the Fontan procedure to re-route blood flow from the superior and inferior vena cava directly into the pulmonary artery. Synthetic grafts lined with bone marrow derived stem cells have been used as vascular conduits to make the connection between the inferior vena cava and the pulmonary artery. However, these synthetic grafts cannot provide pumping activity to help circulate blood. The purpose of this project is to generate tissue engineered pulsatile conduits (TEPCs) to aid in circulation by producing contractile force, and thus an increased driving pressure, to aid in flow through the pulmonary system. The design strategy employed for TEPC production uses decellularized human umbilical artery as an acellular vascular scaffold because its mechanical characteristics allow it to maintain patent blood flow in the inferior vena cava. This scaffold is wrapped with hiPSC-VCM derived EHTs to provide contractile force for the conduit. Decellularized pig heart tissue is used as a scaffold for generating EHTs because it has an inherent fiber structure that allows for generation of controllable alignment of myocardial fibers within the tissue. Co-culture EHTs of hiPSC-VCMs and cardiac fibroblasts were made and tested for contractile force output. Preliminary data shows the introduction of fibroblasts into the tissue has a positive effect on contractile output. Microvascular networks will be generated to support thicker TEPC muscle layers and increase overall TEPC pressure generation. TEPCs will be subjected to an in vitro training regimen consisting of physical and electrical cues to enhance their contractility and electrical handling properties. A flow bioreactor will provide stretch within the TEPC lumen to give the tissue mechanical pressures that mimic what will be experienced in rat inferior vena cava. Electrodes placed within this bioreactor system will provide field stimulation that is intended to mimic the human developmental environment to coax out more mature electrical characteristics from the hiPSC- VCMs. To test for basic survival of the graft, a nude rat model will be used to assess basic parameters for successful engraftment as an inferior vena cava interposition graft. Refinement and optimization of this robust design strategy for producing TEPCs developed in this study will lay the groundwork for testing the construct’s therapeutic potential in the future. Ultimately, this work will make strides in developing improved treatment for patients with single ventricle defects.