Differentiation of CD45(−)/CD31(+) lung side population cells into endothelial and smooth muscle cells in vitro

Side population (SP) cells are a small subpopulation of cells found in many mammalian tissues and organs, identified by their capacity to efflux Hoechst 33342 dye. They are enriched for stem/progenitor cell activity. SP cells isolated from the adult mouse lung can be separated into a CD45(+) subset (bone marrow-derived) and a CD45(−) subset that can be subdivided into CD31(−) and CD31(+) subpopulations. CD45(−)/CD31(−) lung SP (LSP) cells are known to be mesenchymal stem cells. However, CD45(−)/CD31(+) LSP cells are not fully characterized. In the present study, it was found that CD45(−)/CD31(+) LSP cells were able to form colonies. Based on the expression of vascular endothelial growth factor receptor 2 (VEGFR2), these cells were separated into VEGFR2(−) and VEGFR2(+) cells. The CD45(−)/CD31(+)/VEGFR2(−) LSP cells expressed genes characteristic of smooth muscle and endothelial progenitors, and were able to differentiate into smooth muscle and endothelial cells in vitro. The CD45(−)/CD31(+)/VEGFR2(+) LSP cells expressed genes characteristic of endothelial progenitors and gave rise to endothelial cells, although not smooth muscle, in vitro. The data demonstrate that CD45(−)/CD31(+)/VEGFR2(−) LSP cells differentiated into CD45(−)/CD31(+)/VEGFR2(+) LSP cells and then endothelial cells, indicating that CD45(−)/CD31(+)/VEGFR2(+) LSP cells are likely to be derived from CD45(−)/CD31(+)/VEGFR2(−) LSP cells. Taken together, the results suggest that CD45(−)/CD31(+ )LSP cells can be separated into CD45(−)/CD31(+)/VEGFR2(−) LSP cells, which may be progenitors of endothelial and smooth muscle, whereas CD45(−)/CD31(+)/VEGFR2(+) LSP cells may serve as late commitment endothelial progenitors in the adult mouse lung.