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

间充质干细胞分泌组改善啮齿动物原位心脏和人心肌芯片缺血损伤的功能和机制分析

• 批准号: 9352535
• 负责人: KEITH LEONARD MARCH
• 金额: --
• 依托单位: RLR VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9352535
• 关键词: ARNTL gene; Accounting; Acute; Adipose tissue; Adult; Aftercare; Apoptosis; Biogenesis; Biological Preservation; Blood Circulation; Blood flow; Cardiac; Cardiac Myocytes; Cells; Cessation of life; Circadian Rhythms; Conditioned Culture Media; Cultured Cells; Data; Dependence; Deterioration; Dimensions; Disease; Dose; Endothelial Cells; Excision; Fingerprint; Gene Expression; Genes; Genetic Transcription; Geographic Locations; Geography; Growth Factor; Heart; Heart Transplantation; Heart failure; Human; Hypoxia; In Situ; In Vitro; Individual; Inflammation; Inflammatory; Infusion procedures; Injury; Ischemia; Lead; Life; Mediating; Mesenchymal; Mesenchymal Stem Cells; Mitochondria; Modality; Modeling; Molecular; Mus; Myocardial; Myocardial Infarction; Myocardial Ischemia; Myocardium; Organ; Organ Donor; Organ Model; Organ Procurements; Pathologic; Patients; Pattern; Perfusion; Permeability; Preparation; Prevention; Recovery; Recovery of Function; Reperfusion Injury; Reperfusion Therapy; Reporting; Rodent; Role; Savings; Site; Source; Stem cells; Stromal Cells; System; Testing; Therapeutic; Therapeutic Effect; Time; Transplantation; Transportation; Veterans; angiogenesis; cellular targeting; circadian pacemaker; curative treatments; exosome; experimental study; graft function; heart allograft; heart function; heart preservation; improved; induced pluripotent stem cell; insight; loss of function; novel; overexpression; paracrine; protective effect; stem; transcriptome; transcriptome sequencing; treatment effect

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.

由于供体器官供应严重短缺，心脏移植的使用受到限制，导致许多人死亡 在合适的供体心脏出现之前就有心脏移植候选者。1, 2 除了稀缺之外 捐献心脏总数，器官采购组织协会收集的国家移植数据 表明大约 70% 的同种异体心脏移植物在移植过程中被排斥 2009-2011 年，由于严格的验收标准，其中大部分心脏被丢弃。 采购和移植之间可接受的时间有限，该时间与病情进展相关。 心肌缺血/再灌注（I/R）损伤，并限制了可接受的地理距离 总体而言，迫切需要开发有效的供体心脏摘除和移植部位。 通过增加满足接受度的器官数量来增加可移植移植物的方法 尽管运输时间较长，但 I/R 损伤的改善被认为是微不足道的。 将改善移植物功能的保存，从而扩大供体库并增加获取机会。 人脂肪干细胞/基质细胞 (hASC) 代表了 MSC 的一种独特实用的亚型，这是由于 它们的丰富性、从脂肪组织中分离的简单性以及它们的快速体外扩增能力。 等人 4 已经表明，hASC 产生旁分泌因子，在以下情况下提供治疗有益效果： 在心肌梗死的情况下，我们已经证明 hASC 可以保留。 主要通过 ASC 分泌因子调节心肌功能、抑制细胞凋亡并刺激血管生成。5 此外，我们之前报道过，用 hASC 预处理移植心脏可改善心肌 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，并显着保留了正常的分子模式“指纹” 心肌转录组，由深度 RNA 测序具体定义。 表明冷缺血会导致一组基因的显着破坏（Arnt/Bmal、Esrra、Per2、Per3、Cry2） 控制心肌内的生物钟，进而促进心肌细胞的协调增加 转录指导线粒体生物发生；并且这些破坏被特定地抵消 hASC 因素。 因此，我们提出以下假设：将 hASC 衍生因子输注至心脏 循环将改善模型供体心脏的缺血/再灌注引起的功能恶化 离体以及体外人 iPS 衍生的心肌细胞，通过可溶性介导的机制 生长因子和外泌体，通过保留正常的心肌细胞来限制对心肌细胞的损伤 昼夜节律基因表达模式和减轻有害线粒体的诱导 为了检验这个假设，我们将采用三个具体目标： 目的1.评价人脂肪干细胞体外输注的保护作用 条件培养基 (ASC-CM)，级分，对正常小鼠供体心脏在冷条件下保存的影响 静态存储。 目标 2. 识别 ASC-CM 及其外泌体和外泌体保护的特定细胞靶点 外泌体组分，使用人 iPS 衍生的人心肌细胞 (iCM)、iPS 衍生的内皮细胞 使用三维人类“心肌细胞”单独或一起培养细胞（iEC） a-chip”（MOC）模型器官。 目标 3. 确定外泌体和外泌体的选定分子成分的相对作用 ASC-CM 的外泌体部分在 MOC 器官构建体中保护人类 iEC 和 iCM。