Utilizing advanced biomedical imaging to determine the role of cardiac stem cells in physiological remodeling of the heart

干细胞在生理和病理应激后具有修复心脏的潜力。更好地了解干细胞生物学可能会促进这一过程，为心力衰竭提供有效的治疗方法。我们最近发现了内源性心脏干细胞（eCSCs），这是生理重构中心脏再生所必需和充分的。该项目将使用尖端的体内生物医学成像技术同时监测心脏功能，并跟踪标记有谱系特异性生物发光报告基因的eCSCs在生理重塑中的命运。这种新颖的体内eCSC谱系追踪方法以前从未进行过，它将揭示eCSC激活和心脏再生之间的关键联系。项目目标1:0-18个月通过干细胞标记驱动荧光素酶的Cre重组获得体内谱系追踪小鼠，生物发光荧光素酶可用于跟踪活体小鼠体内的细胞。利用Cre-Lox重组技术，我们将交叉现有的小鼠系，使荧光素酶的表达仅限于表达c-kit和Sca-1的CSCs及其后代（Vandeputte et al.2014, Neurobiology of Disease）。这将为体内eCSCs的命运/扩增的无创串行监测提供一个强大的模型。单次注射异丙肾上腺素可引起弥漫性心肌损伤，心肌细胞减少8-10%，导致急性心力衰竭（takotsubo样应激性心肌病），在28天内可在结构和功能上自发逆转（1,2）。我们已经证明c-kit阳性的eCSCs在这种生理重塑中负责心肌细胞更新(1)。在这里，我们将利用新的MRI、光学和核成像技术，对心肌肥大、水肿、收缩、纤维化、炎症和细胞死亡进行连续评估，全面表征异丙肾上腺素模型。这些研究独立于Aim-1的结果，并将使用现有的转基因小鼠模型进行串联，这些模型允许通过c-kit表达的诱导cre谱系追踪来绘制eCSCs的命运图谱（Heger et al.2014）。Eur J immuno1), Sca-1 （Uchida et al.2013）。干细胞Rep)和Wt1 （Chong et al.2011）。干细胞)。这些小鼠的组织切片将通过免疫组织化学和共聚焦显微镜在细胞水平上分析心肌再生和重塑(1)。目标3:18-36个月心脏干细胞在生理重塑中的作用的高级生物医学成像目标1和目标2将结合起来，在异丙肾上腺素诱导的生理重塑后，利用生物发光成像同时追踪eCSCs的体内谱系，并利用MRI、核和光学成像监测心功能。

Strategic Research Priority: World Class BioscienceAbstract Stem-cells have the potential to repair the heart after physiological and pathological stress. Better understanding of stem-cell biology may enhance this process, providing effective treatments for heart-failure. We recently identified endogenous cardiac stem-cells (eCSCs) which are necessary and sufficient for cardiac regeneration in physiological remodeling. This project will use cutting-edge in-vivo biomedical imaging to simultaneously monitor cardiac function and follow the fate of eCSCs tagged with a lineage specific bioluminescent reporter-gene in response to physiological remodeling. This novel in-vivo eCSC lineage-tracing approach has never been undertaken previously and will inform on the critical link between eCSC activation and cardiac regeneration. Project Aim 1: 0-18 monthsDerive an in vivo lineage tracing mouse through stem cell marker driven Cre recombination of luciferaseBioluminescent luciferase can be used to track cells within living mice. Using Cre-Lox recombination technology we will cross existing mouse lines so that luciferase expression is restricted to c-kit and Sca-1 expressing CSCs and their progeny (Vandeputte et al.2014, Neurobiology of Disease). This will provide a powerful model for non-invasive serial monitoring of the fate/expansion of eCSCs in vivo. Aim-2: 0-18 months Advanced biomedical imaging of physiological remodeling following acute injection of isoproterenol A single injection of isoproterenol induces diffuse myocardial damage with a drop out of 8-10% cardiomyocytes, resulting in acute cardiac failure (Takotsubo-like stress cardiomyopathy), which is both structurally and functionally spontaneously reversible within 28 days(1,2). We have shown that c-kit positive eCSCs are responsible for cardiomyocyte renewal in this physiological remodeling(1). Here, we will fully characterize the isoproterenol model using novel MRI, optical and nuclear imaging techniques that serially assess myocardial hypertrophy, edema, contractility, fibrosis, inflammation and cell death. These studies are independent of the outcome of Aim-1 and will be undertaken in tandem using existing transgenic mouse models that allow fate mapping of eCSCs through inducible CRE-lineage tracing by their expression of c-kit (Heger et al.2014.Eur J Immunol), Sca-1 (Uchida et al.2013.Stem Cell Rep) and Wt1 (Chong et al.2011.Cell Stem Cell). Tissue sections from these mice will be analysed at the cellular level for myocardial regeneration and remodeling by immunohistochemistry and confocal microscopy(1). Aim-3: 18-36 monthsAdvanced biomedical imaging of the role of cardiac stem cells in physiological remodelingAims 1 and 2 will be combined allowing simultaneous in vivo lineage tracing of eCSCs using bioluminescence imaging and monitoring of cardiac function using MRI, nuclear and optical imaging after isoproterenol-induced physiological remodeling.