Ikeda Nobuhito, Nakazawa Natsumi, Kurata Yasutaka, Yaura Hisako, Taufiq Fikri, Minato Hiroyuki, Yoshida Akio, Ninomiya Haruaki, Nakayama Yuji, Kuwabara Masanari, Shirayoshi Yasuaki, Hisatome Ichiro
Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Institute of Regenerative Medicine and Biofunction, Tottori University Graduate School of Medical Science.
Department of Physiology II, Kanazawa Medical University.
Biomed Res. 2017;38(4):229-238. doi: 10.2220/biomedres.38.229.
Proepicardium (PE) cells generate cardiac fibroblasts, smooth muscle cells (SMCs) and endothelial cells that form coronary arteries. T-box18 (Tbx18) is a well-known marker of PE cells and epicardium. We examined whether Tbx18-positive cells differentiated from murine embryonic stem (ES) cells serve as PE progenitors to give rise to vascular SMCs and fibroblasts. To collect Tbx18-positive cells, we established Tbx18-EGFP knock-in mouse ES cells using the CRISPR/Cas9 system. We harvested the Tbx18-EGFP-positive cells on day 8, 10 and 14 after the initiation of differentiation; Tbx18 mRNA was enriched on day 8 to 14 and Snai2 mRNA was enriched on day 8 and 10, indicating successful collection of Tbx18-positive cells. Tbx18-EGFP-positive cells expressed the PE marker WT1 on day 8 and 10. They also expressed the SMC marker Acta2 and fibroblast markers Thy1 and Fsp1 on day 8 to 14, but did not express the endothelial cell marker PECAM or the cardiac cell marker CD166 or Myh7. In conclusion, Tbx18-positive cells represent a part of PE cells in the initial phase of differentiation and subsequently include SMCs as well as fibroblasts. These results indicate that Tbx18-positive cells serve as a PE progenitor to supply a variety of cells that contribute to the formation of coronary arteries.
心外膜前体细胞（PE）可生成心脏成纤维细胞、平滑肌细胞（SMC）和形成冠状动脉的内皮细胞。T-box18（Tbx18）是PE细胞和心外膜的一个知名标志物。我们研究了从小鼠胚胎干细胞分化而来的Tbx18阳性细胞是否作为PE祖细胞产生血管SMC和成纤维细胞。为了收集Tbx18阳性细胞，我们使用CRISPR/Cas9系统建立了Tbx18-EGFP基因敲入小鼠胚胎干细胞。在分化开始后的第8、10和14天收获Tbx18-EGFP阳性细胞；Tbx18 mRNA在第8至14天富集，Snai2 mRNA在第8和10天富集，表明成功收集到了Tbx18阳性细胞。Tbx18-EGFP阳性细胞在第8和10天表达PE标志物WT1。它们在第8至14天还表达SMC标志物Acta2以及成纤维细胞标志物Thy1和Fsp1，但不表达内皮细胞标志物PECAM或心肌细胞标志物CD166或Myh7。总之，Tbx18阳性细胞在分化初始阶段代表了PE细胞的一部分，随后包括SMC和成纤维细胞。这些结果表明，Tbx18阳性细胞作为PE祖细胞可提供多种有助于冠状动脉形成的细胞。
