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

Biomed Res. 2017;38(4):229-238. doi: 10.2220/biomedres.38.229.

Abstract

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.

MeSH terms

  • Animals
  • Biomarkers
  • Cell Differentiation* / genetics
  • Embryonic Stem Cells / cytology*
  • Embryonic Stem Cells / metabolism
  • Fibroblasts / cytology*
  • Fibroblasts / metabolism*
  • Fluorescent Antibody Technique
  • Gene Expression
  • Gene Knock-In Techniques
  • Gene Order
  • Gene Targeting
  • Genes, Reporter
  • Genetic Vectors / genetics
  • Mice
  • Microscopy, Fluorescence
  • Muscle, Smooth, Vascular / cytology
  • Myocytes, Smooth Muscle / cytology*
  • Myocytes, Smooth Muscle / metabolism*
  • Pericardium / cytology*
  • Pericardium / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Recombinant Fusion Proteins / genetics
  • T-Box Domain Proteins / genetics
  • T-Box Domain Proteins / metabolism*

Substances

  • Biomarkers
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • T-Box Domain Proteins
  • Tbx18 protein, mouse