Fig. 5

Characterization of CD146⁺ pericytes. A Flow cytometry analysis of a single cell suspension freshly isolated from human foreskin dermis. The hierarchical gating strategy involved the consecutive exclusion of debris, doublets, and dead cells using Zombie Aqua staining. The staining for CD146 and CD90 highlights the presence of two populations: pericytes (CD146⁺CD90⁺, 15,5 ± 2,1%) and fibroblasts (CD146⁻CD90⁺, 52,5 ± 2,6%). The staining with CD31 shows also the presence of a CD146⁺CD31⁺ cell population (3,1 ± 0,7%). n = 4 independent skin donors. B Representative immunofluorescence images of isolated CD146⁺ pericytes (n = 3 independent donors). The entire population of pericytes results positive for CD146, while HDMECs and FBs, used as control, displays a small population or no detection of CD146, respectively. Moreover, CD146⁺ pericytes results entirely positive for NG2, while only a small percentage is also positive for desmin. CD146⁺ pericytes are also positive for αSMA and CD90. No detection of these perivascular markers is observed on HDMECs, while FBs results positive for αSMA and CD90. Cell nuclei are stained with Hoechst (blue). Scale bar: 100 μm. C Western blot analysis of perivascular markers on CD146⁺ pericytes, HDMECs and FBs. CD146⁺pericytes show protein expression of CD146, NG2, desmin, αSMA and CD90. HDMECs display higher protein expression of CD146 compared to pericytes. αSMA and CD90 are also detected on FBs. Equal loading was assessed with anti-GAPDH antibody. (n = 3 independent donors)