Fig. 5. Hepatic and cholangiocytic differentiation of HPPL. (A) Hepatic differentiation of HPPL was induced by OSM and EHS gel as shown by the expression of TAT and CPS (panel 1). The EHS gel treatment also induced the formation of cell clusters showing granulated cytosol and clear round nuclei (panel 3) when compared with the cells without OSM and EHS gel (panel 2). HPPL that became confluent were incubated with OSM for 5 days, and then overlaid with EHS gel for additional 5 days. OSM was not added to the medium during EHS-gel treatment. Numbers shown at the top of panel 1 represent days after addition of OSM. Bar, 25 µm. (B) Polysaccharide accumulation by HPPL. PAS staining showed that HPPL treated with OSM and EHS gel accumulated high levels of polysaccharide in their cytosol (red in panel 2) compared with HPPL before the induction of hepatic differentiation (panel 1). The nuclei were counter-stained with hematoxylin. Bar, 100 µm. (C) Clearance of ammonia from culture medium by HPPL. HPPL treated with OSM and EHS gel eliminated ammonium ions from the culture medium within 48 hours of incubation (open circles). By contrast, HPPL without hepatic differentiation failed to remove ammonium ions (filled circles). (D) Expression of cholangiocyte marker genes in HPPL before (lane 1) and after (lane 2) culture in collagen gel. HPPL were examined for the expression of CK7 and CK19, and integrin ß4 by RT-PCR after 1 week of culture in type I collagen gel. Mature hepatocytes and non-parenchymal cells (NPC) containing cholangiocytes isolated from adult liver were also examined for the expression of these genes. The thermal cycle was repeated 30 times for GAPDH and 35 times for CK7, CK19 and integrin ß4. (E) HPPL showed tube-like structures in collagen gel. After 5 days of incubation in type I collagen gel, the cells were stained with anti-CK19 antibody. Box in panel 1 was magnified and is shown in panel 2. Bar, 100 µm (panel 1); 20 µm (panel 2).