Metzele Roxana, Alt Christopher, Bai Xiaowen, Yan Yasheng, Zhang Zhi, Pan Zhizhong, Coleman Michael, Vykoukal Jody, Song Yao-Hua, Alt Eckhard
Department of Molecular Pathology, University of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030, USA.
FASEB J. 2011 Mar;25(3):830-9. doi: 10.1096/fj.09-153221. Epub 2010 Nov 8.
Various types of stem cells have been shown to have beneficial effects on cardiac function. It is still debated whether fusion of injected stem cells with local resident cardiomyocytes is one of the mechanisms. To better understand the role of fusion in stem cell-based myocardial regeneration, the present study was designed to investigate the fate of human adipose tissue-derived stem cells (hASCs) fused with neonatal rat cardiomyocytes in vitro. hASCs labeled with the green fluorescent probe Vybrant DiO were cocultured with neonatal rat cardiomyocytes labeled with the red fluorescent probe Vybrant DiI and then treated with fusion-inducing hemagglutinating virus of Japan (HVJ). Cells that incorporated both red and green fluorescent signals were considered to be hASCs that had fused with rat cardiomyocytes. Fusion efficiency was 19.86 ± 4.84% at 5 d after treatment with HVJ. Most fused cells displayed cardiomyocyte-like morphology and exhibited spontaneous rhythmic contraction. Both immunofluorescence staining and lentiviral vector labeling showed that fused cells contained separate rat cardiomyocyte and hASC nuclei. Immunofluorescence staining assays demonstrated that human nuclei in fused cells still expressed the proliferation marker Ki67. In addition, hASCs fused with rat cardiomyocytes were positive for troponin I. Whole-cell voltage-clamp analysis demonstrated action potentials in beating fused cells. RT-PCR analysis using rat- or human-specific myosin heavy chain primers revealed that the myosin heavy-chain expression in fused cells was derived from rat cardiomyocytes. Real-time PCR identified expression of human troponin T in fused cells and the presence of rat cardiomyocytes induced a cardiomyogenic protein expression of troponin T in human ASCs. This study illustrates that hASCs exhibit both stem cell (proliferation) and cardiomyocyte properties (action potential and spontaneous rhythmic beating) after fusion with rat cardiomyocytes, supporting the theory that fusion, even if artificially induced in our study, could indeed be a mechanism for cardiomyocyte renewal in the heart.
多种类型的干细胞已被证明对心脏功能具有有益作用。注射的干细胞与局部驻留心肌细胞的融合是否为其中一种机制仍存在争议。为了更好地理解融合在基于干细胞的心肌再生中的作用，本研究旨在体外研究与新生大鼠心肌细胞融合的人脂肪组织来源干细胞（hASC）的命运。用绿色荧光探针Vybrant DiO标记的hASC与用红色荧光探针Vybrant DiI标记的新生大鼠心肌细胞共培养，然后用融合诱导剂日本血凝病毒（HVJ）处理。同时掺入红色和绿色荧光信号的细胞被认为是与大鼠心肌细胞融合的hASC。HVJ处理后5天，融合效率为19.86±4.84%。大多数融合细胞呈现心肌细胞样形态并表现出自发节律性收缩。免疫荧光染色和慢病毒载体标记均显示融合细胞包含独立的大鼠心肌细胞核和hASC细胞核。免疫荧光染色分析表明，融合细胞中的人类细胞核仍表达增殖标志物Ki67。此外，与大鼠心肌细胞融合的hASC肌钙蛋白I呈阳性。全细胞膜片钳分析显示跳动的融合细胞存在动作电位。使用大鼠或人类特异性肌球蛋白重链引物的RT-PCR分析表明，融合细胞中的肌球蛋白重链表达源自大鼠心肌细胞。实时PCR鉴定了融合细胞中人类肌钙蛋白T的表达，并且大鼠心肌细胞的存在诱导了人ASC中肌钙蛋白T的心肌生成蛋白表达。本研究表明，hASC与大鼠心肌细胞融合后兼具干细胞特性（增殖）和心肌细胞特性（动作电位和自发节律性跳动），支持了融合即使在我们的研究中是人工诱导的，也确实可能是心脏中心肌细胞更新机制的理论。
