Sepac Ana, Bosnjak Zeljko J, Seiwerth Sven, Sikiric Suncana, Dzombeta Tihana Regovic, Kulic Ana, Karsaj Jelena Marunica, Sedlic Filip
Department of Pathology, University of Zagreb School of Medicine, Zagreb, Croatia.
Int J Dev Biol. 2021;65(10-11-12):537-543. doi: 10.1387/ijdb.210115fs.
Human induced pluripotent stem cells (hiPSCs) need to be thoroughly characterized to exploit their potential advantages in various aspects of biomedicine. The aim of this study was to compare the efficiency of cardiomyogenesis of two hiPSCs and two human embryonic stem cell (hESC) lines by genetic living cardiomyocyte labeling. We also analyzed the influence of spontaneous beating on cardiac differentiation.
H1 and H9 hESC lines and C2a and C6a hiPSC lines were induced into directed cardiac differentiation. Cardiomyogenesis was evaluated by the analysis of cell cluster beating, cardiac protein expression by immunocytochemistry, ability of cells to generate calcium transients, and cardiomyocyte quantification by the myosin light chain 2v-enhanced green fluorescent protein gene construct delivered with a lentiviral vector.
Differentiation of all cell lines yielded spontaneously beating cell clusters, indicating the presence of functional cardiomyocytes. After the cell dissociation, H1-hESC-derived cardiomyocytes exhibited spontaneous calcium transients, corresponding to autonomous electrical activity and displayed ability to transmit them between the cells. Differentiated hESC and hiPSC cells exhibited striated sarcomeres and expressed cardiac proteins sarcomeric α-actinin and cardiac troponin T. Cardiomyocytes were the most abundant in differentiated H1 hESC line (20% more than in other tested lines). In all stem cell lines, cardiomyocyte enrichment was greater in beating than in non-beating cell clusters, irrespective of cardiomyogenesis efficiency.
Although C2a and C6a hiPSC and H9 hESC lines exhibited efficient cardiomyogenesis, H1 hESC line yielded the greatest cardiomyocyte enrichment of all tested lines. Beating of cell clusters promotes cardiomyogenesis in tested hESCs and hiPSCs.
人类诱导多能干细胞（hiPSC）需要进行全面表征，以挖掘其在生物医学各个方面的潜在优势。本研究的目的是通过基因活体心肌细胞标记比较两种hiPSC和两种人类胚胎干细胞（hESC）系的心肌发生效率。我们还分析了自发搏动对心脏分化的影响。
将H1和H9 hESC系以及C2a和C6a hiPSC系诱导进行定向心脏分化。通过分析细胞簇搏动、免疫细胞化学检测心脏蛋白表达、细胞产生钙瞬变的能力以及用慢病毒载体递送的肌球蛋白轻链2v增强绿色荧光蛋白基因构建体对心肌细胞进行定量来评估心肌发生。
所有细胞系的分化均产生了自发搏动的细胞簇，表明存在功能性心肌细胞。细胞解离后，H1-hESC来源的心肌细胞表现出自发钙瞬变，对应于自主电活动，并显示出在细胞间传递的能力。分化的hESC和hiPSC细胞表现出横纹肌小节，并表达心脏蛋白肌节α-肌动蛋白和心肌肌钙蛋白T。分化的H1 hESC系中心肌细胞最为丰富（比其他测试系多20%）。在所有干细胞系中，无论心肌发生效率如何，搏动细胞簇中的心肌细胞富集程度均高于非搏动细胞簇。
尽管C2a和C6a hiPSC系以及H9 hESC系表现出高效的心肌发生，但H1 hESC系在所有测试系中产生的心肌细胞富集程度最高。细胞簇的搏动促进了测试的hESC和hiPSC中的心肌发生。
