Li Guang, Plonowska Karolina, Kuppusamy Rajarajan, Sturzu Anthony, Wu Sean M
Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA.
Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA Cardiovascular Medicine Division, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Development. 2015 Mar 1;142(5):846-57. doi: 10.1242/dev.116897. Epub 2015 Jan 29.
The transcriptional profiles of cardiac cells derived from murine embryos and from mouse embryonic stem cells (mESCs) have primarily been studied within a cell population. However, the characterization of gene expression in these cells at a single-cell level might demonstrate unique variations that cannot be appreciated within a cell pool. In this study, we aimed to establish a single-cell quantitative PCR platform and perform side-by-side comparison between cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) derived from mESCs and mouse embryos. We first generated a reference map for cardiovascular single cells through quantifying lineage-defining genes for CPCs, CMs, smooth muscle cells (SMCs), endothelial cells (EDCs), fibroblasts and mESCs. This panel was then applied against single embryonic day 10.5 heart cells to demonstrate its ability to identify each endocardial cell and chamber-specific CM. In addition, we compared the gene expression profile of embryo- and mESC-derived CPCs and CMs at different developmental stages and showed that mESC-derived CMs are phenotypically similar to embryo-derived CMs up to the neonatal stage. Furthermore, we showed that single-cell expression assays coupled with time-lapse microscopy can resolve the identity and the lineage relationships between progenies of single cultured CPCs. With this approach, we found that mESC-derived Nkx2-5(+) CPCs preferentially become SMCs or CMs, whereas single embryo-derived Nkx2-5(+) CPCs represent two phenotypically distinct subpopulations that can become either EDCs or CMs. These results demonstrate that multiplex gene expression analysis in single cells is a powerful tool for examining the unique behaviors of individual embryo- or mESC-derived cardiac cells.
源自小鼠胚胎和小鼠胚胎干细胞（mESCs）的心脏细胞的转录谱主要是在细胞群体水平上进行研究的。然而，在单细胞水平上对这些细胞中的基因表达进行表征可能会揭示细胞群体中无法察觉的独特差异。在本研究中，我们旨在建立一个单细胞定量PCR平台，并对源自mESCs和小鼠胚胎的心脏祖细胞（CPCs）和心肌细胞（CMs）进行并行比较。我们首先通过定量CPCs、CMs、平滑肌细胞（SMCs）、内皮细胞（EDCs）、成纤维细胞和mESCs的谱系定义基因，生成了心血管单细胞的参考图谱。然后将该图谱应用于胚胎第10.5天的单个心脏细胞，以证明其识别每个心内膜细胞和特定腔室CM的能力。此外，我们比较了不同发育阶段胚胎来源和mESC来源的CPCs和CMs的基因表达谱，结果表明，直到新生阶段，mESC来源的CMs在表型上与胚胎来源的CMs相似。此外，我们还表明，结合延时显微镜的单细胞表达分析可以解析单个培养的CPCs后代之间的身份和谱系关系。通过这种方法，我们发现mESC来源的Nkx2-5(+) CPCs优先分化为SMCs或CMs，而单个胚胎来源的Nkx2-5(+) CPCs代表两个表型不同的亚群，它们可以分化为EDCs或CMs。这些结果表明，单细胞多重基因表达分析是研究单个胚胎或mESC来源的心脏细胞独特行为的有力工具。
