Functional and Mechanistic Analysis of Mesenchymal Stem Cell Secretome to Ameliorate Ischemic Damage of Rodent Hearts in situ and Human Myocardium-on-a-Chip

心脏移植的使用受到供体器官供应严重短缺的限制，导致许多人死亡。 在合适的供体心脏可用之前，心脏移植候选人。1，2除了稀缺性之外， 器官采购组织协会收集的全国移植数据， 表明全国范围内大约70%的心脏同种异体移植物在移植期间被排斥， 2009-2011.由于严格的验收标准，这些心脏中的大多数被丢弃，其中之一是 从获取到移植的时间间隔有限。这一时间与 心肌缺血/再灌注（I/R）损伤，并限制了可接受的地理距离之间的 供体心脏移植和移植部位。总的来说，迫切需要制定有效的 通过增加器官数量来增加可移植物的方法， 的搜索.尽管运输时间延长，但I/R损伤的改善和器官中的损伤被认为是潜在的边缘 将改善移植物功能的保存，从而扩大供体库并增加获取。 人脂肪源性干/基质细胞（hASC）代表MSC的独特实用亚型，这是由于 它们的丰富性、从脂肪组织分离的简单性和它们在体外的快速扩增能力。We3 和其他人4已经表明，hASC产生旁分泌因子，在治疗中提供有益的效果。 多种病理状态在心肌梗死的背景下，我们已经表明hASC保留了 心肌功能，抑制细胞凋亡，并主要通过ASC分泌的因子刺激血管生成。 此外，我们以前报道过，用hASC预处理心脏移植后， 在离体心脏灌注系统中急性I/R损伤后的功能恢复。6我们的初步数据 表明缺血前输注ASC衍生的旁分泌因子也可改善缺血期间的心肌功能。 从冷缺血中恢复，显著保留了正常的分子模式“指纹”， 心肌转录组，如深度RNA测序所定义。这些RNASeq实验 表明冷缺血导致一组基因（Arnt/Bmal、Esrra、Per 2、Per 3、Cry 2）的显著破坏 控制心肌内的昼夜节律钟，这反过来又促使协调增加， 转录指导线粒体生物发生;这些破坏被特异性地抵消， hASC因子。 因此，我们提出假设，将hASC衍生因子输注到心脏中， 循环将改善缺血/再灌注诱导的模型供体心脏的功能恶化 离体以及人iPS衍生的心肌细胞在体外，通过可溶性 生长因子以及外泌体，它们通过保持正常的心肌细胞来限制对心肌细胞的损伤。 昼夜节律基因表达的模式和减轻有害线粒体的诱导 生物起源为了验证这一假设，我们将采用三个具体目标： 目标1。体外输注人脂肪干细胞的保护作用 条件培养基（ASC-CM），组分，对正常小鼠供体心脏冷保存 静态存储 目标二。识别ASC-CM及其外泌体和外泌体保护的特定细胞靶点 外泌体部分，使用人iPS衍生的人心肌细胞（iCM）、iPS衍生的内皮细胞（iCM）、 细胞（iEC）单独培养以及一起使用三维人类“心肌上的”， 芯片”（MOC）模型器官。 目标3。确定外泌体和外泌体的选定分子组分的相对作用， ASC-CM的外泌体部分在保护MOC器官构建体中的人iEC和iCM中的作用。

Use of heart transplantation is limited by severe shortage in donor organ supply, resulting in death of many heart transplantation candidates before a suitable donor heart becomes available.1, 2 In addition to the scarcity of total donor hearts, national transplant data collected by the Association of Organ Procurement Organizations indicates that nationwide approximately 70% of cardiac allografts were rejected for transplantation during 2009–2011. The majority of these hearts were discarded due to stringent acceptance criteria, one of which is the limited acceptable time between procurement and transplantation. This time correlates with progression of myocardial ischemia/reperfusion (I/R) injury, and constrains the acceptable geographic distance between the sites of donor heart explantation and transplantation. Overall, there is an urgent need to develop effective approaches to increase transplantable grafts by improving the numbers of organs which will fulfill acceptance criteria. Amelioration of I/R injury despite prolonged transport times and in organs felt to be potentially marginal will improve preservation of graft function, thus expanding the donor pool and increasing access. Human adipose-derived stem/stromal cells (hASC) represent a uniquely practical subtype of MSC, due to their abundance, the simplicity of isolation from adipose tissue and their rapid in vitro expansion capacity. We3 and others4 have shown that hASC produce paracrine factors that provide therapeutically beneficial effects in multiple pathological conditions. In the context of myocardial infarction, we have shown that hASC preserve myocardial function, inhibit apoptosis, and stimulate angiogenesis primarily through ASC-secreted factors.5 Moreover, we previously reported that pre-treatment of explanted hearts with hASC improved myocardial functional recovery following acute I/R injury in an ex-vivo heart perfusion system.6 Our preliminary data indicates that pre-ischemic infusion of ASC-derived paracrine factors also improves myocardial function during recovery from cold ischemia, with significant preservation of a normal molecular pattern “fingerprint” of the myocardial transcriptome, as defined by deep RNA sequencing. These RNASeq experiments specifically indicate that cold ischemia leads to prominent disruption of a set of genes (Arnt/Bmal, Esrra, Per2, Per3, Cry2) governing the circadian clock within the myocardium, which in turn prompts a coordinated increase in transcription directing mitochondrial biogenesis; and that these disruptions are specifically counteracted by hASC factors. Accordingly, we propose the hypothesis that infusion of hASC-derived factors into the cardiac circulation will ameliorate ischemia/reperfusion-induced functional deterioration of model donor hearts ex vivo as well as of human iPS-derived cardiomyocytes in vitro, by mechanisms mediated by soluble growth factors as well as exosomes, which limit damage to cardiomyocytes by preserving a normal pattern of circadian gene expression and mitigating the induction of deleterious mitochondrial biogenesis. To test this hypothesis, we will employ three specific aims: Aim 1. Evaluate the protective effect of extracorporeal infusion of human adipose stem cell conditioned medium (ASC-CM), fractions, on normal mouse donor heart preservation during cold static storage. Aim 2. Identify the specific cellular targets protected by ASC-CM as well as its exosomal and extra- exosomal fractions, using human iPS-derived human cardiomyocytes (iCM), iPS-derived endothelial cells (iEC) cultured individually as well as together using a three-dimensional human “myocardium-on- a-chip” (MOC) model organ. Aim 3. Determine the relative roles of selected molecular components of the exosomal and extra- exosomal fractions of ASC-CM in protecting the human iEC and iCM in the MOC organ construct.