RESUMO
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.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Pericárdio/citologia , Proteínas com Domínio T/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Imunofluorescência , Expressão Gênica , Técnicas de Introdução de Genes , Ordem dos Genes , Marcação de Genes , Genes Reporter , Vetores Genéticos/genética , Camundongos , Microscopia de Fluorescência , Músculo Liso Vascular/citologia , Pericárdio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas com Domínio T/genéticaRESUMO
BACKGROUND: The prion protein (PrP) might be useful as a tool to collect cardiac progenitor cells derived from embryonic stem (ES) cells. It is also possible that PrP(+) cells include undifferentiated cells with a capacity to develop into tumors. METHODS: PrP(+) cells isolated from embryoid bodies (EB) formed by mouse AB1 ES cells were examined using RT-PCR analysis and clonogeneic cell assay. To assess their potential to differentiate into cardiomyocytes, Nkx2.5(GFP/+) (hcgp7) cells, another ES cell line that carries the GFP reporter gene in the Nkx2.5 loci, were used. RESULTS: PrP(+) cells isolated from EB of day 7 and 14 did not express pluripotency markers, but expressed cardiac cell markers, while PrP(+) cells isolated from EB of day 21 expressed pluripotency markers. Cultured PrP(+) cells isolated from EB of day 21 expressed pluripotency markers to form colonies, whereas those isolated from EB of day 7 and 14 did not. To exclude proliferating cells from PrP(+) cells, stage specific embryo antigen 1 (SSEA1) was employed as a second marker. PrP(+)/SSEA1(-) cells did not proliferate and expressed cardiac cell markers, while PrP(+)/SSEA1(+) did proliferate. CONCLUSION: PrP(+) cells isolated from EB included undifferentiated cells in day 21. PrP(+)/SSEA1(-) cells included cardiomyoctes, suggesting PrP and SSEA1 may be useful as markers to enrich the fraction of cardiomyocytes.
