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

总结 用于梗死后心脏修复的细胞疗法显示出有限和混合的结果，最可能是由于患者的 个体细胞特征或移植细胞所经历的梗塞环境。识别 有助于患者特异性反应的潜在分子和细胞组分以及最小化 移植细胞的环境压力可能导致心脏修复新策略的发展。 此外，许多研究已经表明，至少一些基于细胞的疗法的效果可以是有效的。 这主要归因于包装在外泌体内的分泌细胞因子。这些外来体是 被认为是细胞间信息的关键介质，并在损伤诱导的组织修复中发挥直接作用 多个生理系统的过程。我们小组特别感兴趣的是 来自心血管疾病患者的外泌体，这些患者在细胞治疗研究中和在利用 这些外泌体货物在开发组织工程化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测试平台， 患者细胞-外泌体相互作用以及与临床试验反应的关联。我们将从骨髓中 在一些实施方案中，骨髓间充质干细胞（MSC）、MSC外泌体和人诱导多能干细胞（hiPSC）来自骨髓间充质干细胞（MSC）、MSC外泌体和人诱导多能干细胞（hiPSC）。 心血管细胞治疗研究网络（CCTRN）中的健康个体和患者-FOCUS clinical 6个月时被归类为改善者的试验，[即，改善左心室射血分数、收缩末期容积 和最大耗氧量]，或作为非改善者（在3个结果中下降）。心脏微组织， 含有来源于健康对照hiPSC的心脏细胞，在天然和梗死样条件下培养， 用改良剂的外泌体治疗微组织功能、心肌细胞成熟、细胞存活和 将在改善剂、安慰剂和非改善剂外泌体之间比较增殖。同时，高 通量测定将用于鉴定前3种临床改良剂的外泌体货物的差异， 非改良剂，并与体外反应相关。我们预计改良剂的外泌体将含有强效的 在梗死样应激下增强细胞和微组织成熟和功能的有益因子。接下来，病人- 含有由前3名改善者骨髓产生的hiPSC衍生的心脏细胞的特定微组织 而非改善者将用自体或同种异体（改善者、非改善者或健康）外泌体治疗 在类似梗塞的条件下。通过将患者特异性"梗死"心脏微组织暴露于自体或自体移植物中， 同种异体外泌体，我们可以在受控的体外环境中评估患者特异性细胞-外泌体相互作用 模拟临床症状最终，这些研究将为细胞外泌体提供新的见解 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 为开发具有上级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.