Fig. 2

Isolation and validation of laryngeal tissue-resident clonal cells. a Clonal cells purified from laryngeal subsites of EM, LP and MF visualized by phase-contrast microscopy. Scale bars, 100 and 20 μm. Representative immunofluorescence staining images of laryngeal clonal cells expressing mesenchymal markers vimentin and α-SMA. b Expression of the genes Krt19 (epithelial cell marker) and Vwf (endothelial cell marker) was not detected, whereas that of Vim and Acta2 was detected in all laryngeal clonal cells. c MF stellate cells were validated to contain lipid droplets (arrow) by phase-contrast microscopy. Scale bars, 25 μm. These cells demonstrated vitamin A (retinoid) autofluorescence as assessed by d fluorescence microscopy (scale bars, 10 μm) and e retinoid-based FACS sorting. EM epiglottic mucosa, LP lamina propria, MF macula flava, α-SMA α-smooth muscle actin, FSC forward scatter