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: 10394875
• 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 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10394875
• 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; Circadian gene expression; Conditioned Culture Media; Data; Dependence; Deterioration; Disease; Dose; Endothelial Cells; Excision; Exposure to; Fingerprint; 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.

心脏透明的使用受到供体器官酱的严重短缺的限制 心脏透明候选者在合适的供体心脏之前。1，2除了稀缺 在捐助者心中，由器官采购组织协会收集的国家跨地数据 表明在全国范围内，大约70％的心脏同种异体移植被拒绝 2009年 - 2011年。 这次采购和移植之间的可接受时间有限。 心肌缺血/再灌注（I/R）损伤，并约束可接受的地理距离 捐赠者心脏膨胀和移植的位置总体上是迫切需要有效的 通过器官的数量来增加接受的器官的数量，以增加接受的方法 标准。 将改善移植功能的保存，从而扩大供体池并增加访问权限。 人类脂肪衍生的茎/基质细胞（HASC）代表MSC的UniQuly实用亚型 它们的丰度，从脂肪组织组织中分离的简单性是它们的快速体外膨胀能力 和其他4显示，thasc产生了旁分泌因素，该因素在治疗中受益于 多种病理状况。 心肌功能，渗入凋亡和刺激血管生成主要通过ASC分泌因子。55 此外，我们以前报道了用hascardial治疗植光心脏的治疗 急性I/R损伤后的功能恢复。6我们的初步数据 表明ASC衍生的旁分泌因子的缺血前输注也可以改善心肌功能 从冷缺血中恢复，并明显保存 由深RNA测序定义的心肌转录组 表明冷缺血导致AT基因的明显破坏（Arnt/Bmal，Esra，Per3，Cry2） 管理心肌内的昼夜节律，这反过来促使 转录方向的米奇氏菌生物发生； 哈斯克因素。 我们提出了这样一个假设，即输注HASC衍生因素对心脏 循环将取消缺血/再灌注诱导的模型供体心脏的功能恶化 通过可溶性介导的机制，体外和人IPS衍生的心肌细胞的体外心肌细胞 生长因子和外泌体，通过保持正常来限制对心肌细胞的损害 昼夜节律表达和减轻有害线粒体的产业的模式 生物发生。 目标1。评估人体外输注人脂肪干细胞的保护作用 有条件的培养基（ASC-CM），分数，在寒冷的正常小鼠供体心脏保存下 静态存储。 AIM 2。确定由ASC-CM保护的特定细胞靶标以及ATS外泌体和外部的特定细胞靶标 使用人类IPS衍生的人类心肌细胞（ICM），IPS衍生的内皮细胞，外泌体果实 单独培养的细胞（IEC）使用树维的人“心肌N- n-”一起培养 A-Chip”（MOC）模型器官。 AIM 3。确定外泌体和外部选定分子成分的相对作用 ASC-CM在MOC器官构建中保护人IEC和ICM方面的外泌体果实。