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.

心脏移植的使用受到捐赠器官供应严重短缺的限制，导致许多人死亡 在适当的供体心脏之前，心脏移植候选者可用。1，2除了稀缺 在总体捐助者心中，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％的心脏同种异体移植被拒绝 2009- 2011年。这些心大多数由于严格的接受标准而被丢弃，其中之一就是 采购和移植之间可接受的时间有限。这次与进展 心肌缺血/再灌注（I/R）损伤，并约束可接受的地理距离 捐赠者心脏展开和移植的部位。总体而言，迫切需要有效 通过改善将满足接受的器官数量来增加移植移植的方法 标准。 I/R伤害的改善长时间的运输时间和在器官中可能是边缘的 将改善移植功能的保存，从而扩大供体池并增加访问权限。 人脂肪衍生的茎/基质细胞（HASC）代表MSC的独特实用亚型 它们的丰度，从脂肪组织中分离的简单性以及它们快速的体外扩张能力。韦3 其他4已表明HASC产生旁分泌因子，可在 多种病理条件。在心肌梗塞的背景下，我们已经证明了HASC保存 心肌功能，抑制凋亡并主要通过ASC分泌因子刺激血管生成。55 此外，我们以前报告说，用HASC改善了心脏的外植物的预处理 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，并明显保留正常分子模式的“指纹” 心肌转录组，由深RNA测序定义。这些RNASEQ实验专门 表明冷缺血会导致一组基因的明显破坏（ARNT/BMAL，ESRRA，PER2，PER3，CRY2） 管理心肌内的昼夜节律，这反过来促使 转录指导线粒体生物发生；这些破坏是由 HASC因素。 彼此之间，我们提出了以下假设：将HASC衍生因子输注到心脏 循环将改善模型供体心脏的缺血/再灌注诱导的功能定义 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诱导的模式 生物发生。为了检验这一假设，我们将采用三个具体目标： 目标1。评估人脂肪干细胞体外输注的保护作用 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及其外泌体和外部靶标 使用人类IPS衍生的人类心肌细胞（ICM），IPS衍生的内皮的外泌体分数 单独培养的细胞（IEC）使用三维人的“心肌”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在保护MOC器官构建体中人IEC和ICM方面的外泌体级分。