Fig. 3

Bipotential differentiation capabilities of single HpSC-derived clones. a qPCR analysis of hepatocyte markers, cholangiocyte markers, and stem cell-related markers. HpSC clones, FACS-sorted single HpSC-derived clones after culture for 14 days; hFetal liver, samples from human primary FLCs; hAdult liver, samples from human adult liver cells. Results shown as mean ± SD (n = 3 independent experiments). Mann–Whitney test, *P < 0.05, **P < 0.001, NS no significant difference. b Immunocytochemical staining of hepatocyte markers of ALB and AFP and cholangiocyte-specific markers CK7 and CK19 in single human HpSC-derived colonies after culture for 40 days. Nuclei counterstained with DAPI. Scale bars: 100 μm. c Hepatocyte differentiation capability of single HpSC-derived colonies characterized after long-term culture for up to day 90 in vitro by Periodic acid–Schiff (PAS) staining and by positive immunocytochemical staining with human ALB, human CYP3A4, human CYP2D6, and human CYP1A2. Nuclei counterstained with DAPI. Scale bars: 100 μm. d Cholangiocytic cyst formation of single HpSC-derived colonies. FACS-sorted single HpSC-derived colonies were replated in an extracellular matrix gel, and several epithelial cysts were formed after culture for 14 days. CK7 was positively detected in cysts. Cells stained with antibodies against ALB and CK7. Nuclei counterstained with DAPI. Scale bars: 50 μm. Experiments were performed at least three times, representative data are shown. HpSC hepatic stem cell, ALB Albumin, CK cytokeratin