Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.
Subtype-specific differentiation of cardiac pacemaker cell clusters from human induced pluripotent stem cells.
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DOI：
10.1186/s13287-017-0681-4
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发表时间：
2017-10-16
影响因子：
7.5
通讯作者：
Thomas D
中科院分区：
文献类型：
--
作者：
Schweizer PA;Darche FF;Ullrich ND;Geschwill P;Greber B;Rivinius R;Seyler C;Müller-Decker K;Draguhn A;Utikal J;Koenen M;Katus HA;Thomas D
人类诱导多能干细胞 (hiPSC) 具有分化成多种心脏细胞类型的潜力。先前的实验工作主要针对具有心室心肌细胞特征的 hiPSC 衍生细胞的产生。针对起搏器细胞建模和替换的直接方法，我们寻求选择性地区分具有节点型特性的细胞。通过在无血清培养基中与内脏内胚层样细胞共培养，hiPSC 分化成自发跳动的簇。随后在特定的富含胎牛血清 (FBS) 的细胞培养基中进行培养，产生了起搏器型表型，并使用定量实时聚合酶链反应 (qRT-PCR)、免疫细胞化学和膜片钳电生理学对其进行了详细研究。进一步的研究包括药理刺激和与新生儿心肌细胞的共培养。 hiPSC 在无血清培养基中与内脏内胚层样细胞系 END-2 共培养，培养 10-12 天后产生自发跳动的簇。起搏器特异性基因 HCN4、TBX3 和 TBX18 在此早期发育阶段大量表达，而肌节基因产物的水平仍然较低。我们观察到，在搏动开始后立即将早期簇转移到富含 FBS 的细胞培养基中，可以抑制工作型心肌细胞分化。在这些条件下 6 周后，窦房结 (SAN) 标志基因仍保持在高水平，而工作型心肌转录本（NKX2.5、TBX5）较低。簇的特点是有规律的活动和强劲的搏动频率（70-90 次/分钟），并由自发的 Ca2+ 瞬变触发，再现了真正起搏细胞的钙时钟特性。它们对肾上腺素能/胆碱能刺激有反应，并且能够在共培养实验中调节新生大鼠心室肌细胞的速度。对簇中个体细胞的动作电位 (AP) 测量显示结节型 (63.4%) 和心房型 (36.6%) AP 形态，而未观察到心室 AP 构型。我们提供了一种基于培养基的新型非转基因方法，用于定向生成 hiPSC 衍生的起搏器型细胞，这些细胞成簇生长，并为疾病建模、药物测试和 SAN 的个体化基于细胞的替代疗法提供了潜力。本文的在线版本 (doi:10.1186/s13287-017-0681-4) 包含补充材料，可供授权用户使用。
Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties. hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes. hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10–12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70–90 beats/min) and were triggered by spontaneous Ca2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed. We provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN. The online version of this article (doi:10.1186/s13287-017-0681-4) contains supplementary material, which is available to authorized users.
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影响因子：
5
作者：
Kim, Jong J.;Yang, Lei;Lin, Bo;Zhu, Xiaodong;Sun, Bin;Kaplan, Aaron D.;Bett, Glenna C. L.;Rasmusson, Randall L.;London, Barry;Salama, Guy
通讯作者：
Salama, Guy
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
5.2
作者：
Huber, Bruno C.;Ransohoff, Julia D.;Ransohoff, Katherine J.;Riegler, Johannes;Ebert, Antje;Kodo, Kazuki;Gong, Yongquan;Sanchez-Freire, Veronica;Dey, Devaveena;Kooreman, Nigel G.;Diecke, Sebastian;Zhang, Wendy Y.;Odegaard, Justin;Hu, Shijun;Gold, Joseph D.;Robbins, Robert C.;Wu, Joseph C.
通讯作者：
Wu, Joseph C.
影响因子：
1.2
作者：
Hoffmann, Sandra;Schmitteckert, Stefanie;Rappold, Gudrun A.
通讯作者：
Rappold, Gudrun A.
影响因子：
4.8
作者：
Most, P;Boerries, M;Remppis, A
通讯作者：
Remppis, A