Volume 126, Issue 4, Pages 1147-1156 (April 2004) Origin and characterization of a human bipotent liver progenitor cell line  Romain Parent, Marie-Jeanne Marion, Laetitia Furio, Christian Trépo, Marie-Anne Petit  Gastroenterology  Volume 126, Issue 4, Pages 1147-1156 (April 2004) DOI: 10.1053/j.gastro.2004.01.002

Figure 1 Growth properties of HepaRG cells. (A ) BrdU incorporation index. The number of positively stained cells was represented as the percentage of the total cell number. The labeling and detection were performed with the BrdU Kit I from Roche. (B) Methyltetrazolium (MTS) reducing activity. The CellTiter Aqueous One Solution cell proliferation assay (Promega) was used. Optical density (OD) was directly proportional to the number of metabolically active cells. Determinations were performed at different times after seeding, from day 2 to day 28. Gastroenterology 2004 126, 1147-1156DOI: (10.1053/j.gastro.2004.01.002)

Figure 2 Kinetic morphological studies of HepaRG cells. The cells were examined with phase-contrast microscopy at different phases of the culture: early proliferative (A ) (day 2), late proliferative (B) (days 4–5), intermediate or stationary (C ) (days 7–14), and differentiated (D) (days 28–35). From day 14, the culture medium was supplemented with 2% dimethyl sulfoxide. H, hepatocyte-like areas; B, biliary-like areas (magnification, 100×). Gastroenterology 2004 126, 1147-1156DOI: (10.1053/j.gastro.2004.01.002)

Figure 3 Morphological properties of HepaRG cells. Cells were analyzed at day 28 after seeding. (A ) Phase-contrast microscopy examination. H, hepatocyte-like areas; B, biliary-like areas (magnification, 300×). (B) Indirect immunofluorescence with a primary antibody against the ZO-1 antigen, a tight junction-specific protein. Arrows indicate the location of bile canaliculi (magnification, 400×). (C ) Effect of EGF. Commitment toward the hepatocytic lineage (H) after 4 weeks of culture in the presence of EGF 20 ng/mL (magnification, 200×). (D) Single-cell cloning experiment. After 10 weeks of culture in a 96-well plate, the cells were grown for 14 days in a 12-well plate and observed at day 28, i.e., 2 weeks after induction of the differentiation process (magnification, 150×). Gastroenterology 2004 126, 1147-1156DOI: (10.1053/j.gastro.2004.01.002)

Figure 4 Expression kinetics of hepatocytic-specific markers on HepaRG cells. Detection of albumin and cytokeratin 18 was performed by Western blotting (A ); albumin HP-1 and cytokeratin 18 were detected by indirect immunofluorescence (B); cytokeratin 18 was analyzed by flow cytometry (C ). 1, proliferative phase; 2, intermediate phase; 3, differentiated phase; kDa, kilodaltons. Gastroenterology 2004 126, 1147-1156DOI: (10.1053/j.gastro.2004.01.002)

Figure 5 Expression kinetics of oval cell-specific markers on HepaRG cells. Detection was performed by Western blotting (A ), indirect immunofluorescence (B), or flow cytometry (C ). 1, proliferative phase; 2, intermediate phase; 3, differentiated phase; kDa, kilodaltons. The markers used were CK19, M2-PK, OV-1, OV-6 (CK14 and CK19), and CD34. The FCM analysis presented here was performed at day 28 (differentiated phase), but the same results were obtained at the earlier phases (proliferative or intermediate). Gastroenterology 2004 126, 1147-1156DOI: (10.1053/j.gastro.2004.01.002)

Figure 6 Immunohistochemistry on tissue sections from the patient’s liver from which HepaRG cells originated. (A ) Hematoxylin/eosin/safranin staining: (a) magnification 100×; (b) magnification 400×. (B) Immunoperoxidase staining: (a) CK19, (b) CK18, (c) M2-PK, and (d ) OV-1. Arrows indicate neoductules within the fibrotic area. An EnVision system (Dako) was used. Gastroenterology 2004 126, 1147-1156DOI: (10.1053/j.gastro.2004.01.002)