Leveraging the HIF-alpha pathway to improve the engraftment and therapeutic efficacy of human nanowired cardiac organoids

项目概述：心脏病占美国每年死亡人数的近四分之一， 强调了对修复受损心脏的疗法的迫切需求。人诱导多能干细胞- 衍生的心肌细胞（hiPSC-CM）已经成为心脏修复的强大细胞来源，但其 注射后的存活率和植入率差限制了其潜力。为了应对这些挑战，我们 实验室率先开发了由导电材料组成的人类心脏类器官， 硅纳米线（e-SiNW）、hiPSC-CM和支持细胞。我们的初步体内研究表明， 体外培养的心脏类器官成功移植到缺血/再灌注（I/R）损伤的大鼠心脏中并发育 与非心脏类器官相比，具有更有组织的收缩结构。尽管取得了这些进展，但 移植后7天，超过一半（约30%）的注射类器官仍植入梗死心脏， 可以归因于体外类器官的不充分的预血管化和缺氧/缺血预处理。 为了解决这个问题，我们探索了HIF-a的药理学稳定性作为促进HIF-a表达的策略。 类器官内的预血管化和缺血耐受性。我的初步体外实验数据显示， Molidustat（脯氨酰羟化酶结构域（PHD）抑制剂）治疗显著改善了内皮网络 和管腔形成（即，CD 31+覆盖率增加约150%）。虽然这些结果 有希望，进一步的研究是必要的，以揭示表型和基因型的变化，HIF-α稳定 心脏类器官以及它们与移植效率的关系。本提案的目标是 确定HIF-α稳定对血管成熟、心功能、细胞和组织水平的影响 心脏类器官的代谢和转录组学变化（目的1），并证明治疗疗效 HIF-α稳定的类器官在心肌I/R损伤大鼠模型中的作用（目的2）。这个问题的核心假设是 这一观点认为，心脏类器官中HIF-α信号转导的稳定性改善了心肌细胞的存活和植入。 hiPSC-CM在梗死心肌中的表达，并增强其促进心脏功能恢复的能力， 受伤的心该提案的创新之处在于，我们将首次研究缺氧模拟是如何进行的 试剂预处理人工程化心脏组织以增强hiPSC-CM的移植效率。 我的长期目标是开发临床适用的心脏再生疗法， 疾病因此，我们将追求以下具体目标：1）确定药理学HIF-α 稳定化重新编程和预处理人类心脏类器官用于缺血保护，以及2）确定 HIF-α稳定化对移植物-宿主吻合术、长期植入和 受损心脏中的人类心脏类器官。这项研究将为紧急临床应用提供信息 缺氧模拟剂治疗心血管疾病，并将有助于促进我们的人类心脏类器官 大型动物研究平台，以加速其临床转化。

PROJECT SUMMARY: Heart disease accounts for nearly 1 in 4 deaths in the United States each year, highlighting the urgent need for therapies that can repair damaged hearts. Human induced pluripotent stem cell- derived cardiomyocytes (hiPSC-CMs) have emerged as a powerful cell source for cardiac repair, but their potential has been limited by poor survival and engraftment after injection. To address these challenges, our lab has pioneered the development of nanowired human cardiac organoids composed of electrically conductive silicon nanowires (e-SiNWs), hiPSC-CMs, and supporting cells. Our preliminary in vivo studies showed that nanowired cardiac organoids successfully engraft in ischemia/reperfusion (I/R) injured rat hearts and develop more organized contractile structures compared to non-nanowired cardiac organoids. Despite this progress, less than half (~30%) of injected organoids remained engrafted in infarcted hearts 7 days post-transplantation, which can be attributed to inadequate prevascularization and hypoxic/ischemic preconditioning of the organoids in vitro. To address this, we have explored pharmacological stabilization of HIF-a as a strategy to promote prevascularization and ischemic tolerance within the organoids. My preliminary in vitro data showed that treatment with Molidustat, a prolyl hydroxylase domain (PHD) inhibitor, significantly improved endothelial network and lumen formation (i.e., ~150% increase of CD31+ coverage) within the cardiac organoids. While these results are promising, further investigation is necessary to reveal phenotypic and genotypic changes in HIF-α stabilized cardiac organoids and how they correlate with transplantation efficiency. The goals of this proposal are to determine the effects of HIF-α stabilization on vascular maturation, cardiac function, cell and tissue-level metabolism, and transcriptomic changes in cardiac organoids (Aim 1), and to demonstrate therapeutic efficacy of HIF-α stabilized organoids in a rat model of myocardial I/R injury (Aim 2). The central hypothesis of this proposal is that stabilization of HIF-α signaling in cardiac organoids improves the survival and engraftment of hiPSC-CMs in infarcted myocardium and enhances their capacity to promote cardiac functional recovery in injured hearts. The proposal is innovative in that, for the first time, we will investigate how hypoxia mimetic agents precondition human engineered cardiac tissue to enhance the transplantation efficiency of hiPSC-CMs. My long-term goal is to develop clinically applicable cardiac regenerative therapies to treat cardiovascular diseases. Accordingly, we will pursue the following specific aims: 1) Determine how pharmacological HIF-α stabilization reprograms and preconditions human cardiac organoids for ischemic protection, and 2) Determine the effects of HIF-α stabilization on graft-host anastomosis, long-term engraftment, and therapeutic efficacy of nanowired human cardiac organoids in injured hearts. This research will inform emergent clinical applications of hypoxia mimetic agents to treat cardiovascular disease and will help advance our human cardiac organoid platform towards large animal studies to accelerate their clinical translation.