Figure 7.

In vitro smooth muscle differentiation potential of CD45⁻/CD31⁺/VEGFR2⁻ and CD45⁻/CD31⁺/VEGFR2⁺ LSP cells. Images (scale bar, 20 µm) show the representative microscopic fields of CD45⁻/CD31⁺/VEGFR2⁻ LSP cells double-labeled for αSMA and αSMT after 14 days in culture under smooth muscle differentiation-inducing conditions; numerous cells stained positively with (A) anti-αSMA antibody (red) and (B) anti-αSMT antibody (green). (C) Nuclei were counterstained with DAPI (blue). (D) Merge of the optical channels. Single immunofluorescence labeling with (E) anti-αSMA (red) and (F) anti-αSMT (green) is shown (scale bar, 10 µm). (G) Reverse transcription-quantitative polymerase chain reaction analysis was performed to compare gene expression by freshly isolated CD45⁻/CD31⁺/VEGFR2⁻ LSP cells (grey bar), CD45⁻/CD31⁺/VEGFR2⁻ LSP cells after 14 days culture (white bar), freshly isolated CD45⁻/CD31⁺/VEGFR2⁺ LSP cells (light blue bar) and CD45⁻/CD31⁺/VEGFR2⁻ LSP cells after 14 days culture (red bar). Freshly isolated CD45⁻ LMP cells are included as a control (black bar). Relative expression levels of the ABCG2, CD133, αSMA, αSMT and MHC genes were compared between the three types of cells. Data are presented as the mean ± standard deviation (n=3 experiments). ^*P<0.05; LSP, lung side population; LMP, lung main population; UD, undetectable; ABCG2, ATP-binding cassette super-family G member 2; VEGFR2, vascular endothelial growth factor receptor 2; vWF, von Willebrand factor; αSMT, α-smooth muscle tropomyosin; αSMA, α-smooth muscle actin; MHC, myosin heavy chain; DAPI, 4-6-diamidino-2-phenylindol-dihydrochloride.