Using a Cardiac Microtissue System to Evaluate and Replicate Clinical Therapy Responses using Patient Cell-Derived Exosomes

摘要 心肌梗死后修复的细胞疗法显示出有限且好坏参半的结果，很可能是由于患者的 单个细胞特征或移植细胞所经历的梗死环境。识别 有助于患者特异性反应的潜在分子和细胞成分以及将其降至最低的方法 移植细胞面临的环境压力可能会导致心脏修复新策略的发展。 此外，大量研究表明，至少一些基于细胞的疗法的效果可以 主要归因于包装在外切体内的分泌细胞因子。这些外显体是 被认为是细胞间信息的关键介质，在损伤诱导的组织修复中发挥直接作用 多个生理系统中的过程。我们小组特别关注的是 来自心血管疾病患者的外体，这些患者在细胞治疗研究中和在利用 这些外体货物在开发组织工程心脏补片以实现最佳功能 心肌修复。我们也有兴趣使用我们的体外心脏微组织系统作为试验床来评估 患者细胞-外切体相互作用及其与临床试验反应的关系。我们会提取骨髓 间充质干细胞(MSCs)、MSC外体和人诱导多能干细胞(HiPSCs) 心血管细胞治疗研究网络(CCTRN)中的健康人和患者-重点临床 试验在6个月时被归类为改善，[即改善左心室射血分数，收缩末期容量 和最大耗氧量]，或作为非改善剂(在3个结果中下降)。心脏微组织， 含有来自健康对照HiPSCs的心肌细胞，在自然和梗死样条件下培养将 用改良剂的外切体治疗。微组织功能、心肌细胞成熟、细胞存活和 增殖率将在改良剂、安慰剂和非改良剂外切体之间进行比较。与此同时，高 吞吐量分析将用于确定排名前3位的临床改进剂和 非改良剂，并与体外反应相关。我们预计改良剂的外切体将含有有效的 促进细胞和微组织在梗死样应激下成熟和功能的有益因素。下一位，病人- 含有前3名改良剂骨髓来源的hPSC来源的心肌细胞的特定显微组织 非改良剂将用自体或同种异体(改良剂、非改良剂或健康的)外切体治疗。 在类似梗死的情况下。通过将患者特定的“梗死”心脏微组织暴露于自体或 同种异体外切体，我们可以在受控的体外环境中评估患者特定的细胞-外切体相互作用 这模拟了临床情况。最终，这些研究将为细胞外切体提供新的见解。 脑梗塞中的相互作用以及它们的相对效力和与临床试验反应的关联，因此 为开发具有卓越再生性能的心脏补片提供了一种创新的新方法。

Summary Cell therapy for post-infarct cardiac repair has shown limited and mixed results, most likely due to patients' individual cell characteristics or to the infarct environment experienced by the transplanted cells. Identifying the potential molecular and cellular components that contribute to patient-specific responses and ways to minimize the environmental stress on transplanted cells may lead to the development of new strategies for cardiac repair. Additionally, numerous studies have shown that the effects of at least some cell-based therapies can be attributed primarily to secreted cellular factors that are packaged inside exosomes. These exosomes are considered critical mediators of intercellular information and play a direct role in injury-induced tissue repair processes in multiple physiological systems. Our group is particularly interested in the characterization of exosomes from cardiovascular disease patients who improved in a cell therapy study and in the utilization of these exosome cargos in the development of tissue-engineered cardiac patches for optimal functional myocardial repair. We are also interested in using our in vitro cardiac microtissue system as a testbed to evaluate patient cell-exosome interactions and associations with clinical trial responses. We will derive bone marrow mesenchymal stem cells (MSCs), MSC exosomes, and human induced pluripotent stem cells (hiPSCs) from healthy individuals and patients in the Cardiovascular Cell Therapy Research Network (CCTRN)-FOCUS clinical trial categorized as improvers at 6 months, [i.e., improved left ventricular ejection fraction, end-systolic volume and maximal oxygen consumption], or as non-improvers (declined in the 3 outcomes). Cardiac microtissues, containing cardiac cells derived from healthy control hiPSCs, cultured in native and infarct-like conditions will be treated with improvers' exosomes. Changes in microtissue function, cardiomyocyte maturation, cell survival and proliferation will be compared among improvers, placebo and non-improvers exosomes. Meanwhile, high throughput assays will be used to identify differences in the exosome cargos of the top 3 clinical improvers and non-improvers and correlated with in vitro responses. We expect that improver's exosomes will contain potent beneficial factors to enhance cell and microtissue maturation and function under infarct-like stress. Next, patient- specific microtissues containing hiPSC-derived cardiac cells generated from bone marrow of the top 3 improvers and non-improvers will be treated with autologous or allogenic (improver, non-improver or, healthy) exosomes under infarct-like conditions. By exposing patient-specific “infarcted” cardiac microtissues to autologous or allogeneic exosomes, we can evaluate patient-specific cell-exosome interactions in a controlled in vitro setting that mimics the clinical condition. Ultimately, these studies will provide new insights into cell-exosome interactions in an infarct as well as their relative potency and association with clinical trial responses, thus providing an innovative new approach to developing cardiac patches with superior regenerative properties.