Neuregulin/ErbB signaling regulates cardiac subtype specification in differentiating human embryonic stem cells.
Neuregulin/ErbB signaling regulates cardiac subtype specification in differentiating human embryonic stem cells.
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DOI：
10.1161/circresaha.110.223917
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发表时间：
2010-09-17
影响因子：
20.1
通讯作者：
Laflamme MA
中科院分区：
文献类型：
--
作者：
Zhu WZ;Xie Y;Moyes KW;Gold JD;Askari B;Laflamme MA
人胚胎干细胞衍生的心肌细胞（hESC-CM）表现出“工作”室或细胞样表型。为了产生最佳的hESC-CM制剂，用于最终在基于细胞的治疗中的临床应用，我们将需要控制它们分化成这些专门的心脏亚型。证明hESC-CM中完整的神经调节蛋白-1 β（NRG-1β）/ErbB信号传导，并检验该信号传导途径调节hESC-CM培养物中心脏亚型丰度的假设。所有实验均采用使用我们最近报道的定向分化方案产生的hESC-CM培养物。为了支持后续的动作电位表型分析方法，并提供一个更高通量的方法来确定心脏亚型，我们首先开发并验证了一种新的遗传标记，识别胚胎型hESC-CM。接下来，将对照hESC-CM制备物与在外源性NRG-1β、抗NRG-1β中和抗体或ErbB拮抗剂AG 1478存在下分化的那些进行比较。我们使用了三种独立的方法来确定所得到的人群中心脏亚型的比例：电流钳下的直接动作电位表型，上述遗传标记的激活，以及通过RT-PCR进行的亚型特异性标记表达。使用所有三个终点，我们发现NRG-1β/ErbB信号传导的抑制大大增加了显示结节表型的细胞比例。NRG-1β/ErbB信号调节分化hESC-CM培养物中结型细胞与工作型细胞的比例，并且推测在早期人类心脏发育期间具有类似的功能。我们推测，通过操纵NRG-1β/ErbB信号传导，将有可能产生用于梗死修复的富集工作型心肌细胞的制备物，或者相反，用于生物起搏器的潜在用途的结细胞。
Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) exhibit either a “working” chamber or a nodal-like phenotype. To generate optimal hESC-CM preparations for eventual clinical application in cell-based therapies, we will need to control their differentiation into these specialized cardiac subtypes. To demonstrate intact neuregulin-1β (NRG-1β)/ErbB signaling in hESC-CMs and test the hypothesis that this signaling pathway regulates cardiac subtype abundance in hESC-CM cultures. All experiments employed hESC-CM cultures generated using our recently reported directed differentiation protocol. To support subsequent action potential phenotyping approaches and provide a higher-throughput method of determining cardiac subtype, we first developed and validated a novel genetic label that identifies nodal-type hESC-CMs. Next, control hESC-CM preparations were compared to those differentiated in the presence of exogenous NRG-1β, an anti-NRG-1β neutralizing antibody, or the ErbB antagonist AG1478. We used three independent approaches to determine the ratio of cardiac subtypes in the resultant populations: direct action potential phenotyping under current-clamp, activation of the aforementioned genetic label, and subtype-specific marker expression by RT-PCR. Using all three endpoints, we found that inhibition of NRG-1β/ErbB signaling greatly enhanced the proportion of cells showing the nodal phenotype. NRG-1β/ErbB signaling regulates the ratio of nodal- to working-type cells in differentiating hESC-CM cultures and presumably functions similarly during early human heart development. We speculate that, by manipulating NRG-1β/ErbB signaling, it will be possible to generate preparations of enriched working-type myocytes for infarct repair, or, conversely, nodal cells for potential use in a biological pacemaker.