Clonality of mouse and human cardiomyogenesis in vivo
Clonality of mouse and human cardiomyogenesis in vivo
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DOI：
10.1073/pnas.0903089106
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发表时间：
2009-10-06
影响因子：
11.1
通讯作者：
Leri, Annarosa
中科院分区：
文献类型：
--
作者：
Hosoda, Toru;D'Amario, Domenico;Leri, Annarosa
对心脏祖细胞（CPCs）的克隆性和体内心肌细胞周转的分析需要对未分化细胞进行遗传标记，以便在特化后代中追踪单个母细胞的克隆标记。用携带EGFP的慢病毒感染小鼠心房和心尖的CPC壁龛，并在1-5个月后测定标记细胞的命运。在分离的CPC、心肌细胞、内皮细胞（ECs）和成纤维细胞中发现了一个共同的整合位点，记录了CPC的自我更新和多潜能性以及分化细胞群体的克隆起源。随后，在注射后2-4天评估CPCs的egfp -慢病毒感染程度，6个月后检测表达该报告基因的肌细胞数量。BrdU脉冲追踪方案也被引入作为心肌细胞周转分析的附加分析。在6个月的时间里，每个egfp阳性的CPC分裂约8次，产生230个心肌细胞；该值与BrdU标记的新形成细胞数一致。为了确定人类CPCs （hCPCs）是否具有自我更新和多能性，我们将这些细胞用egfp慢病毒转导，并在免疫抑制大鼠急性心肌梗死后注射。从再生心肌中收集的hcpc、肌细胞、内皮细胞和成纤维细胞在人类基因组中显示出共同的病毒整合位点。因此，我们的研究结果表明，成人心脏中含有大量调节心脏稳态和修复的常驻干细胞。
An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1-5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cells (ECs), and fibroblasts, documenting CPC self-renewal and multipotentiality and the clonal origin of the differentiated cell populations. Subsequently, the degree of EGFP-lentiviral infection of CPCs was evaluated 2-4 days after injection, and the number of myocytes expressing the reporter gene was measured 6 months later. A BrdU pulse-chasing protocol was also introduced as an additional assay for the analysis of myocyte turnover. Over a period of 6 months, each EGFP-positive CPC divided approximately eight times generating 230 cardiomyocytes; this value was consistent with the number of newly formed cells labeled by BrdU. To determine whether, human CPCs (hCPCs) are self-renewing and multipotent, these cells were transduced with the EGFP-lentivirus and injected after acute myocardial infarction in immunosuppressed rats. hCPCs, myocytes, ECs, and fibroblasts collected from the regenerated myocardium showed common viral integration sites in the human genome. Thus, our results indicate that the adult heart contains a pool of resident stem cells that regulate cardiac homeostasis and repair.