Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA.
Stem Cell Reports. 2015 Dec 8;5(6):1239-1249. doi: 10.1016/j.stemcr.2015.10.021.
Isolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. Here we show that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We designed MBs (IRX4 MBs) to target mRNA encoding Iroquois homeobox protein 4 (Irx4), a transcription factor specific for vCMs. To purify mESC vCMs, IRX4 MBs were delivered into cardiomyogenically differentiating mESCs, and IRX4 MBs-positive cells were FACS-sorted. We found that, of the cells isolated, ~98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca(2+) transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional ventricular CMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies.
由于缺乏特异性的表面标记物，分离心室肌细胞（vCMs）一直具有挑战性。在这里，我们展示了可以使用针对特定细胞内 mRNA 的分子信标（MBs）从分化的小鼠胚胎干细胞（mESCs）中纯化 vCMs。我们设计了 MBs（IRX4 MBs）来靶向编码同源异形盒蛋白 4（Irx4）的 mRNA，Irx4 是 vCMs 的特异性转录因子。为了纯化 mESC vCMs，将 IRX4 MBs 递送至心肌生成性分化的 mESCs 中，并通过 FACS 对 IRX4 MBs 阳性细胞进行分选。我们发现，通过电生理分析，在分离的细胞中，约 98%显示出 vCM 样动作电位。通过自发搏动、Ca(2+) 瞬变和 vCM 特异性蛋白的表达，这些 MB 纯化的 vCMs 持续保持其 CM 特征。我们的研究表明，通过不修饰宿主基因的细胞分选来分离纯 vCMs 是可行的。通过基于 MB 的方法产生的同质且功能正常的心室 CM 可用于疾病研究、药物发现和基于细胞的治疗。
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