Differentiation Potential Of TBX18~+Epicardial Progenitor Cells(EPCS) Into Vascular Smooth Muscle Cells(SMCS) In The Mouse

Part one Fidelity of Cre Recombinase in the Tbx18-Cre knock-in mouseObjective:To assess the fidelity of Cre recombinase in the Tbx18-Cre knock-in mouse.Methods:Real-time PCR was established to assess the copy number of Cre and expression of Tbx18down stream genes. Junction PCR and gene sequencing were used to detect the specificity of Tbx18-Cre integration site. In situ hybridization, X-gal staining and EYFP fluorescence analysis were employed to study Tbx18lineage tracing and genetic fate mapping of epicardial progenitor cells.Results:Relative copy number of Cre was assessed accurately, Tbx18-Cre integration site was detected specifically, CX40and CX43expression in Tbx18Cre-Cre knock-in mouse model were higher than wild type C57BL/6, Tbx18expression in Tbx18-Cre/Rosa26R-LacZ double-heterozygous mouse model was consistent with Tbx18cRNA probe in situ hybridization during early and middle gestations, genetic fate mapping showed Tbx18+epicardial progenitor cells migrated and differentiated into part of myocardium.Conclusions:High fidelity and temporal-spatial expression of Cre recombinase was demonstrated in the Tbx18-Cre knock-in mouse and no Cre leakage was found so far.Part Two Great potential of Tbx18+EPCs in differentiation into vascular smooth muscle cells in the mouseObjective:To reveal the differentiation potential of Tbxl8+EPCs into vascular smooth muscle cells in the mouse.Methods:Tbx18-Cre/Rosa26R-EYFP double-heterozygous mouse model was used in lineage tracing of EYFP+cells and immunofluorescence with Myh11was employed in smooth muscle cells detecting in vitro and in vivo. Tbx18-Cre/Rosa26R-LacZ double-heterozygous mouse model was established, LacZ protein in cells and tissues were observed following by X-gal staining.Results:EYFP+cells were confocal with Myh11to a certain extent in vitro and in vivo, cardiac tissues with X-gal staining had the same anatomy structure with smooth muscle.Conclusions:Tbx18+EPCs-derived cells had the great potential in differentiation into smooth muscle cells.