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: 9898148
• 负责人: KEITH LEONARD MARCH
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 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 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898148
• 关键词: 3-Dimensional; ARNTL gene; Accounting; Acute; Adipose tissue; Adult; Aftercare; Apoptosis; Biogenesis; Blood Circulation; Blood flow; Cardiac; Cardiac Myocytes; Cells; Cessation of life; Circadian Rhythms; Conditioned Culture Media; Cultured Cells; Data; Dependence; Deterioration; Disease; Dose; Endothelial Cells; Excision; Exposure to; 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; Stromal Cells; System; Testing; Therapeutic; Therapeutic Effect; Time; Transplantation; Transportation; Veterans; angiogenesis; cellular targeting; circadian; circadian pacemaker; curative treatments; exosome; experimental study; graft function; heart allograft; heart damage; heart function; heart preservation; improved; induced pluripotent stem cell; insight; loss of function; novel; organ on a chip; overexpression; paracrine; preservation; protective effect; stem; stem cells; 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.

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，心脏移植候选者1，2除了稀缺性之外 在捐赠心脏总数中，器官采购组织协会收集的国家移植数据 表明在全国范围内，大约70%的同种异体心脏移植在 2009-2011年。这些心脏中的大多数由于严格的接受标准而被丢弃，其中之一是 从购买到移植之间可接受的有限时间。这一时间与 心肌缺血/再灌注(I/R)损伤，并限制了 供心移植和移植的部位。总体而言，迫切需要制定有效的 通过增加可接受器官的数量来增加可移植移植物的途径 标准。I/R损伤的改善，尽管运输时间延长，器官感觉可能是边缘的 将改善移植物功能的保存，从而扩大捐赠者池并增加获取途径。 人类脂肪来源的干细胞/基质细胞(HASC)代表了MSC的一种独特的实用亚型，原因是 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张能力。WE3 和其他4人已经表明，HASC产生旁分泌因子，在治疗上对 多种病理情况。在心肌梗死的情况下，我们已经证明了HASC保留了 主要通过ASC分泌的因子发挥心肌功能、抑制细胞凋亡和刺激血管生成。 此外，我们先前报道，用HASC对移植心脏进行预处理可以改善心肌 体外心脏灌流系统急性I/R损伤后的功能恢复6我们的初步数据 提示缺血前输注ASC来源的旁分泌因子也能改善心肌功能 从冷缺血中恢复，并显著保留了正常的分子模式 心肌转录组，根据深RNA测序的定义。这些RNAseq实验特别 表明冷缺血导致一系列基因(ARNT/BMAL、ESRRA、PER2、PER3、Cry2)的显著中断 控制心肌内的生物钟，这反过来又促使 转录指导线粒体的生物发生；并且这些干扰被特异性地通过 HASC因素。 因此，我们提出假设，HASC衍生因子注入心脏 循环将改善模型供者心脏缺血/再灌注所致的功能恶化 体外培养和体外培养人诱导性多能干细胞来源的心肌细胞，其机制为 生长因子和外体，通过保持正常的心肌细胞来限制对心肌细胞的损伤 昼夜节律基因的表达模式和减轻有害线粒体的诱导 生物发生学。为了验证这一假设，我们将采用三个具体目标： 目的1.评价体外输注人脂肪干细胞的保护作用 条件培养液(ASC-CM)及其组分对正常小鼠供心冷藏保存的影响 静态存储。 目的2.鉴定ASC-CM及其胞外体和胞外区保护的特异性细胞靶点。 胞外部分，使用人iPS来源的人心肌细胞(ICM)，iPS来源的内皮细胞 细胞(IEC)单独培养和联合培养，使用三维人体“心肌上的细胞”。 A-CHIP“(MOC)模型器官。 目的3.确定胞外体和胞外部分分子组分的相对作用。 ASC-CM胞外部分在MOC器官结构中对人IEC和ICM的保护作用。