Cao Yinyin, Xu Jin, Wen Junxiang, Ma Xiaojing, Liu Fang, Li Yang, Chen Weicheng, Sun Liqun, Wu Yao, Li Shuolin, Li Jian, Huang Guoying
Pediatric Heart Center, Children's Hospital of Fudan University, Shanghai, China.
Clinical Laboratory Center, Children's Hospital of Fudan University, Shanghai, China.
Cell Physiol Biochem. 2018;50(2):538-551. doi: 10.1159/000494167. Epub 2018 Oct 11.
BACKGROUND/AIMS: Ventricular septal defects (VSDs) are one of the most common types of congenital heart malformations. Volume overload resulting from large VSDs can lead to heart failure (HF) and constitutes a major cause of pediatric HF with a series of often-fatal consequences. The etiology of VSD with HF is complex, and increasing evidence points toward a genetic basis. Indeed, we identified an L2483R mutation in the ryanodine receptor type 2 (RyR2) in a 2-month-old male patient with VSD with HF.
We generated integration-free induced pluripotent stem cells from urine samples (UiPSCs) of this patient using Sendai virus containing the Yamanaka factors and characterized these cells based on alkaline phosphatase activity, pluripotency marker expression, and teratoma formation. Then, we induced the derived UiPSCs to rapidly and efficiently differentiate into functional cardiomyocytes through temporal modulation of canonical Wnt signaling with small molecules. Real-time PCR and immunofluorescence were used to verify the expression of myocardium-specific markers in the differentiated cardiomyocytes. The ultrastructure of the derived myocardial cells was further analyzed by using transmission electron microscopy.
The established UiPSC lines were positive for alkaline phosphatase activity, retained the RyR2 mutation, expressed pluripotency markers, and displayed differentiation potential to three germ layers in vivo. The UiPSC-derived cells showed hallmarks of cardiomyocytes, including spontaneous contraction and strong expression of cardiac-specific proteins and genes. However, compared with cardiomyocytes derived from H9 cells, they had a higher level of autophagy, implying that autophagy may play an important role in the development of VSD with HF.
The protocol described here yields abundant myocardial cells and provides a solid platform for further investigation of the pathogenesis, pharmacotherapy, and gene therapy of VSD with HF.
背景/目的：室间隔缺损（VSD）是最常见的先天性心脏畸形类型之一。大的室间隔缺损导致的容量超负荷可引发心力衰竭（HF），是小儿心力衰竭的主要原因之一，常伴有一系列致命后果。室间隔缺损合并心力衰竭的病因复杂，越来越多的证据表明其存在遗传基础。事实上，我们在一名2个月大的患有室间隔缺损合并心力衰竭的男性患者中，发现了ryanodine受体2型（RyR2）的L2483R突变。
我们使用含有山中因子的仙台病毒从该患者的尿液样本（UiPSCs）中生成了无整合诱导多能干细胞，并基于碱性磷酸酶活性、多能性标志物表达和畸胎瘤形成对这些细胞进行了表征。然后，我们通过小分子对经典Wnt信号进行时间调控，诱导所获得的UiPSCs快速有效地分化为功能性心肌细胞。采用实时PCR和免疫荧光法验证分化心肌细胞中心肌特异性标志物的表达。通过透射电子显微镜进一步分析所获得心肌细胞的超微结构。
所建立的UiPSC系碱性磷酸酶活性呈阳性，保留了RyR2突变，表达多能性标志物，并在体内显示出向三个胚层的分化潜能。UiPSC衍生的细胞表现出心肌细胞的特征，包括自发收缩以及心脏特异性蛋白质和基因的强表达。然而，与源自H9细胞的心肌细胞相比，它们具有更高水平的自噬，这意味着自噬可能在室间隔缺损合并心力衰竭的发生发展中起重要作用。
本文所述方案可产生大量心肌细胞，并为进一步研究室间隔缺损合并心力衰竭的发病机制、药物治疗和基因治疗提供了坚实的平台。
