Transcriptomic profiling of murine ES and iPS cells, embryoid bodies, and ES and IPS cell-derived cardiomyocytes. Mus musculus

In this study we have compared functional and molecular properties of highly purified murine induced pluripotent stem (iPS) cell- and embryonic stem (ES) cell-derived cardiomyocytes (CM). In order to obtain large amounts of purified CM, we have generated a transgenic murine iPS cell line, which expresses puromycin resistance protein N-acetyltransferase and EGFP under the control of the cardiomyocyte-specific α-myosin heavy chain promoter (alphaMHC-Puro-IRES-GFP, aPiG). We demonstrate that murine aPIG-iPS and aPIG-ES cells differentiate into spontaneously beating CM at comparable efficiencies. When selected with puromycin both cell types yielded more than 97% pure population of CMs. Both aPIG-iPS and aPIG-ES cell-derived CM express typical cardiac transcripts and structural proteins and possess similar sarcomeric organization. Action potential recordings revealed that iPS- and ES cell-derived CM respond to beta-adrenergic and muscarinic receptor modulation, express functional voltage-gated sodium, calcium and potassium channels and possess comparable current densities. Comparison of global gene expression profiles of CM generated from iPS and ES cells revealed that both cell types cluster close to each other but are highly distant to undifferentiated ES or iPS cells as well as unpurified iPS and ES cell-derived embryoid bodies (EB). Both iPS and ES cell-derived CMs express genes and functional categories typical for CM. They are enriched in genes involved in transcription and genes coding for structural proteins involved in cardiac muscle contraction and relaxation. They also express genes involved in heart and muscle developmental processes, ion export and ion binding processes and various metabolic processes for ATP synthesis. These CMs downregulate genes involved in immune response, cell cycle and cell division, thus demonstrating the CMs population is mitotically inactive. Most surface signaling pathways are also downregulated. Thus, a transgenic aPiG-iPS cell line can provide a robust supply of highly purified and functional CMs for future in vitro and in vivo studies. Overall design: Seven different experimental groups were included into analysis: undifferentiated murine ES cells (1) and undifferentiated murine iPS cells (2), murine ES cell-derived embyroid bodies (3) and murine iPS cell-derived embryoid bodies at day 16 of differentiation (4), murine ES cell-derived cardiomyocytes (5) and murine iPS cell-derived cardiomyocytes (6) at day 16 of differentiation (they were generated by puromycin selection for 7 days prior to RNA isolation). Adult mouse tail tip fibroblasts (7) were used as a control for iPS cells. Total RNA samples were prepared from three independent biological replicates in groups 1-6. In group 7, single RNA probes were analyzed as three technical replicates.

本研究比较了高纯度小鼠诱导多能干细胞（induced pluripotent stem cell, iPS）与胚胎干细胞（embryonic stem cell, ES）来源的心肌细胞（cardiomyocytes, CM）的功能与分子特性。为获取大量纯化的心肌细胞，我们构建了转基因小鼠iPS细胞系，该细胞系在心肌特异性α-肌球蛋白重链启动子（α-myosin heavy chain promoter, αMHC）的调控下，表达嘌呤霉素抗性蛋白N-乙酰转移酶与增强绿色荧光蛋白（enhanced green fluorescent protein, EGFP），即αMHC-Puro-IRES-GFP（简称aPiG）。研究证实，小鼠aPiG-iPS与aPiG-ES细胞的分化效率相当，均可自发形成搏动的心肌细胞。经嘌呤霉素筛选后，两种细胞系来源的心肌细胞纯度均超过97%。aPiG-iPS与aPiG-ES细胞来源的心肌细胞均表达典型的心脏转录本与结构蛋白，且肌节组织结构相似。动作电位记录结果显示，iPS与ES细胞来源的心肌细胞可对β肾上腺素能与毒蕈碱受体调控产生响应，表达功能性电压门控钠、钙与钾通道，且电流密度相当。对iPS与ES细胞来源的心肌细胞的全局基因表达谱进行比较后发现，两类细胞的聚类结果彼此接近，但与未分化的ES或iPS细胞、未纯化的iPS与ES细胞来源的胚状体（embryoid bodies, EB）均存在显著差异。iPS与ES细胞来源的心肌细胞均表达心肌细胞典型的基因与功能类别：富集于转录相关基因、心肌收缩与舒张相关的结构蛋白编码基因；同时表达参与心脏与肌肉发育过程、离子转运与离子结合过程，以及ATP合成相关各类代谢过程的基因。此类心肌细胞会下调免疫应答、细胞周期与细胞分裂相关基因，表明该心肌细胞群体处于有丝分裂失活状态；多数表面信号通路亦被下调。综上，转基因aPiG-iPS细胞系可稳定提供高纯度且功能完整的心肌细胞，为后续体外与体内研究提供可靠的细胞来源。实验设计：本分析共纳入7组不同的实验样本：①未分化的小鼠ES细胞、②未分化的小鼠iPS细胞、③小鼠ES细胞来源的胚状体、④分化第16天的小鼠iPS细胞来源的胚状体、⑤分化第16天的小鼠ES细胞来源的心肌细胞、⑥分化第16天的小鼠iPS细胞来源的心肌细胞（该类样本在RNA提取前经7天嘌呤霉素筛选）；另以成年小鼠尾尖成纤维细胞（⑦）作为iPS细胞的对照。第1至6组的总RNA样本均取自3次独立的生物学重复；第7组仅对1份RNA样本进行检测，但设置3次技术重复。