Tbx18-positive cells differentiated from murine ES cells serve as proepicardial progenitors to give rise to vascular smooth muscle cells and fibroblasts

• Published in: Biomedical Research 2017/08/01, Vol.38(4), pp.229-238
• Main Authors: IKEDA, Nobuhito, NAKAZAWA, Natsumi, KURATA, Yasutaka, YAURA, Hisako, TAUFIQ, Fikri, MINATO, Hiroyuki, YOSHIDA, Akio, NINOMIYA, Haruaki, NAKAYAMA, Yuji, KUWABARA, Masanari, SHIRAYOSHI, Yasuaki, HISATOME, Ichiro
• Format: Article
• Language: English
• Subjects: Animals; Arteries; Biomarkers; Cell Differentiation - genetics; Coronary artery; Coronary vessels; CRISPR; Differentiation; Embryonic Stem Cells - cytology; Embryonic Stem Cells - metabolism; Endothelial cells; Epicardium; Fibroblasts; Fibroblasts - cytology; Fibroblasts - metabolism; Fluorescent Antibody Technique; Gene Expression; Gene Knock-In Techniques; Gene Order; Gene Targeting; Genes, Reporter; Genetic Vectors - genetics; Heart diseases; Mice; Microscopy, Fluorescence; mRNA; Muscle, Smooth, Vascular - cytology; Myocytes, Smooth Muscle - cytology; Myocytes, Smooth Muscle - metabolism; Pericardium - cytology; Pericardium - metabolism; Recombinant Fusion Proteins - genetics; RNA, Messenger - genetics; RNA, Messenger - metabolism; Rodents; Smooth muscle; Snail protein; Stem cells; T-Box Domain Proteins - genetics; T-Box Domain Proteins - metabolism
• ISSN: 0388-6107; 1880-313X
• 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.