Fig. 4.

Long-term expansion of iHEPs is associated with phenotype loss by EMT. Phase-contrast microscopy images obtained from iHEP cultures at p30, showing the presence of spindle-shaped cells (red dashed circle and arrowheads) surrounding epithelial cells (a). Confocal microscopy image showing ɑSMA staining (red) in spindle-shaped cells (b). Quantification of fibroblast-like cells based on ɑSMA staining and high-content image morphology analysis, comparing early- and late-passage iHEPs, using HepG2 hepatoma line as control (c). Sequential time-lapse stills showing a fibroblast-like cell emerging from iHEP colony (d). Images of late iHEPs stimulated with osteogenic differentiation medium evaluated on D1 and D7 (phase-contrast microscopy) and stained for calcium-rich matrix with Azilarin red, at D15 (e). Quantification of Alizarin red staining in early and late iHEPs, using HepG2 hepatoma cell line as control. Bars 100 μm. *p < 0.05; **p < 0.01