Generation of functional murine cardiac myocytes from induced pluripotent stem cells
Generation of functional murine cardiac myocytes from induced pluripotent stem cells
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DOI：
10.1161/circulationaha.108.778795
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发表时间：
2008-07-29
期刊：
影响因子：
37.8
通讯作者：
Martin, Ulrich
中科院分区：
文献类型：
--
作者：
Mauritz, Christina;Schwanke, Kristin;Martin, Ulrich
诱导多能干细胞（iPS）与胚胎干细胞（ES）几乎没有区别，最近在诱导多能干细胞（iPS）的产生方面取得了突破，促进了小鼠疾病和人类患者特异性干细胞系的产生。本研究的目的是表征小鼠iPS细胞克隆的心脏分化潜力，并将其与已建立的小鼠胚胎干细胞系进行比较。方法和结果：采用胚胎干细胞标准的基于胚胎体的分化方案，诱导多能干细胞和胚胎干细胞分别分化24天。虽然与胚胎干细胞系相比，所分析的iPS细胞克隆显示出跳动胚状体形成的延迟和效率较低，但分化导致平均55%的iPS细胞胚状体自发收缩。分子、结构和功能水平的分析表明，iPS细胞衍生的心肌细胞具有胚胎干细胞衍生的心肌细胞的典型特征。逆转录聚合酶链式反应分析显示，中胚层、心脏中胚层和心肌细胞中典型的标记基因表达，包括Brachyury、中胚层后向因子1 （Mesp1）、GATA2之友（FOG- 2）、gata结合蛋白4 （GATA4）、NK2转录因子相关、基因座5 （Nkx2.5）、T-box 5 （Tbx5）、T-box 20 （Tbx20）、心房利钠因子（ANF）、肌球蛋白轻链2心房转录物（MLC2a）、肌球蛋白轻链2心室转录本（MLC2v）、α -肌球蛋白重链（α - mhc）和心肌肌钙蛋白T在iPS细胞分化培养中的作用。免疫细胞学证实了心肌细胞典型蛋白的表达，包括肌凝蛋白α -肌动蛋白、肌动蛋白、心肌肌钙蛋白T、MLC2v和连接蛋白43。iPS细胞心肌细胞表现出细胞内自发的节律性Ca2+波动，Ca2+瞬态振幅与ES细胞心肌细胞相当。在iPS细胞衍生的心肌细胞簇内同时释放Ca2+表明细胞的功能偶联。多电极阵列的电生理学研究证明了这些细胞中β -肾上腺素能和毒蕈碱信号级联的功能和存在。结论：iPS细胞分化为功能性心肌细胞。与胚胎干细胞相比，iPS细胞可以衍生出具有自体功能的心肌细胞，用于细胞心肌成形术和心肌组织工程。
Background - The recent breakthrough in the generation of induced pluripotent stem ( iPS) cells, which are almost indistinguishable from embryonic stem ( ES) cells, facilitates the generation of murine disease - and human patient specific stem cell lines. The aim of this study was to characterize the cardiac differentiation potential of a murine iPS cell clone in comparison to a well- established murine ES cell line.Methods and Results - With the use of a standard embryoid body - based differentiation protocol for ES cells, iPS cells as well as ES cells were differentiated for 24 days. Although the analyzed iPS cell clone showed a delayed and less efficient formation of beating embryoid bodies compared with the ES cell line, the differentiation resulted in an average of 55% of spontaneously contracting iPS cell embryoid bodies. Analyses on molecular, structural, and functional levels demonstrated that iPS cell - derived cardiomyocytes show typical features of ES cell - derived cardiomyocytes. Reverse transcription polymerase chain reaction analyses demonstrated expression of marker genes typical for mesoderm, cardiac mesoderm, and cardiomyocytes including Brachyury, mesoderm posterior factor 1 ( Mesp1), friend of GATA2 ( FOG- 2), GATA-binding protein 4 ( GATA4), NK2 transcription factor related, locus 5 ( Nkx2.5), T-box 5 (Tbx5), T- box 20 ( Tbx20), atrial natriuretic factor ( ANF), myosin light chain 2 atrial transcripts ( MLC2a), myosin light chain 2 ventricular transcripts (MLC2v), alpha-myosin heavy chain (alpha-MHC), and cardiac troponin T in differentiation cultures of iPS cells. Immunocytology confirmed expression of cardiomyocyte-typical proteins including sarcomeric alpha-actinin, titin, cardiac troponin T, MLC2v, and connexin 43. iPS cell cardiomyocytes displayed spontaneous rhythmic intracellular Ca2+ fluctuations with amplitudes of Ca2+ transients comparable to ES cell cardiomyocytes. Simultaneous Ca2+ release within clusters of iPS cell - derived cardiomyocytes indicated functional coupling of the cells. Electrophysiological studies with multielectrode arrays demonstrated functionality and presence of the beta- adrenergic and muscarinic signaling cascade in these cells.Conclusions - iPS cells differentiate into functional cardiomyocytes. In contrast to ES cells, iPS cells allow derivation of autologous functional cardiomyocytes for cellular cardiomyoplasty and myocardial tissue engineering.