Antibody-based selective isolation of atrial and ventricular cardiomyocytes by differential expression of integrin α6 (CD49f). Mus musculus

Central questions like cardiomyocyte subtype emergence during cardiogenesis or availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, characterization and implementation of pure cardiomyocyte subtypes is still challenging due to technical limitations. Our aim was to identify surface markers enabling the selective detection and purification of atrial and ventricular cardiomyocytes from mouse hearts. In a surface marker screen we found differential expression of CD49f in atrial and ventricular embryonic cardiomyocytes (E13.5). By flow cytometry we could correlate a high CD49f expression with MLC-2a on the single cell level; a low CD49f expression corresponded to MLC-2v. Based on the persisting differential CD49f expression we developed purification protocols for cardiomyocytes subtypes from the developing mouse heart. Flow sorting of E15.5 hearts into ErbB-2+/CD49flow and ErbB-2+/CD49fhigh cells led to a selective depletion (CD49flow) or enrichment of MLC-2a+ cells (CD49fhigh). We found a corresponding CD49f-dependent distribution of MLC-2a when pre-enriched neonatal cardiomyocytes (P2) were flow-sorted into CD49flow and CD49fhigh. Atrial and ventricular identity was confirmed by expression profiling and patch clamp analysis of sorted embryonic hearts, which unequivocally demonstrated that the sorted cells were viable and functional. For the first time, we introduce a non-genetic, antibody-based approach to specifically isolate atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This newly gained capability of obtaining highly pure, viable cells will facilitate in-depths characterization of the individual cellular subsets and will aid translational research and therapeutic applications. Overall design: The dataset comprises four different cardiomyocytes subtypes from the developing mouse heart. Embryonic (E15.5) hearts were dissociated and flow-sorted into ErbB-2+/CD49flow and ErbB-2+/CD49fhigh cardiomyocytes. Neonatal (P2) hearts were dissociated, contaminating non-myocytes were removed by MACS depletion, and the purified cardiomyocytes were flow-sorted into CD49flow and CD49fhigh cardiomyocytes. Four biological replicates were available for each sample groups. Microarray analysis was conducted on the Agilent Whole Mouse Genome Oligo Microarray 8x60K platform.

诸如心脏发生过程中心肌细胞亚型的起源，以及用于细胞替代治疗的心肌细胞亚型可及性这类核心科学问题，均有赖于心房与心室心肌细胞的选择性鉴定与纯化。然而，受限于技术瓶颈，纯心肌细胞亚型的鉴定与应用仍存在诸多挑战。本研究旨在筛选可用于选择性鉴定并纯化小鼠心脏来源心房与心室心肌细胞的表面标志物。通过表面标志物筛选实验，我们发现CD49f在小鼠胚胎（E13.5）心房与心室心肌细胞中存在差异表达。借助流式细胞术，我们可在单细胞层面将CD49f高表达与肌球蛋白轻链2a（MLC-2a）关联起来；而CD49f低表达则对应肌球蛋白轻链2v（MLC-2v）。基于CD49f持续存在的差异表达特征，我们建立了从发育中小鼠心脏中分离心肌细胞亚型的纯化方案。将E15.5胚胎心脏解离后，通过流式分选出ErbB-2阳性/CD49f低表达（ErbB-2+/CD49flow）与ErbB-2阳性/CD49f高表达（ErbB-2+/CD49fhigh）的细胞群，前者（CD49flow）可选择性去除MLC-2a阳性细胞，后者（CD49fhigh）则可富集MLC-2a阳性细胞。当对预富集的新生小鼠（P2）心肌细胞进行流式分选，分选出CD49flow与CD49fhigh细胞群时，我们同样观察到MLC-2a的分布依赖于CD49f的表达水平。通过对分选后的胚胎心脏细胞进行表达谱分析与膜片钳检测，我们证实了分选细胞的心房/心室身份，且明确证明分选所得细胞具有活性与功能。本研究首次提出一种基于抗体的非遗传方法，可从不同发育阶段的小鼠心脏中特异性分离心房与心室心肌细胞。这种可获取高纯度活性心肌细胞的新方法，将推动单一细胞亚群的深度表征研究，并为转化研究与治疗应用提供助力。

实验整体设计：本数据集包含发育中小鼠心脏的4种不同心肌细胞亚型。将胚胎（E15.5）心脏解离后，通过流式分选出ErbB-2阳性/CD49f低表达与ErbB-2阳性/CD49f高表达的心肌细胞群。新生（P2）心脏解离后，先通过磁珠分选（MACS）去除污染的非肌细胞，再将纯化后的心肌细胞流式分选为CD49flow与CD49fhigh两个细胞群。每个样本组均设置4个生物学重复。本研究采用安捷伦（Agilent）全小鼠基因组寡核苷酸芯片8x60K平台进行微阵列分析。