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First published online 27 July 2004
doi: 10.1242/jcs.01279

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Small hepatocytes in culture develop polarized transporter expression and differentiation

¹ Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, 8091 Zurich, Switzerland
² Central Laboratory for Electron Microscopy, University of Zurich, P.O. Box, 8028 Zurich, Switzerland

View larger version (186K): [in a new window]   Fig. 1. Development and maturation of small-hepatocyte colonies in culture. (A) A small-hepatocyte colony after 1 week of culture. (B) A small-hepatocyte colony after 7 weeks of culture. Notice the hexagonal shape of most cells, indicating differentiation of small hepatocytes into mature hepatocytes. (C) Phase-contrast microscopy picture showing a clearly demarcated colony of cells appearing as mature hepatocytes. Cells were cultured for 9 weeks and then fixed with 4% paraformaldehyde in the presence of Triton X-100 and subsequently processed for immunofluorescence localization of Mrp2. (D) Immunofluorescence localization by laser-scanning microscopy indicated expression of Mrp2 in structures resembling bile canaliculi. Scale bars, 40 µm.

View larger version (17K): [in a new window]   Fig. 2. Time-dependent mRNA expression of Mrp1 (A) and AFP (B) in cultured rat small hepatocytes. Cells were cultured for up to 9 weeks. Cells were harvested at day 1 and weeks 1, 3, 5 and 9 with Trizol. mRNA of Mrp1 and AFP was quantitatively measured by real-time PCR and normalized to 18S rRNA and mRNA expression of 1-week cultures [Ct value for Mrp1=27.61±0.46; Ct value for 18S=13.9±0.97; the quotient (Ct value for Mrp1 ÷ Ct value for 18S)=13.71±0.8 (n=4, mean±standard deviation); Ct value for AFP=35.41±0.6; the quotient (Ct value for AFP/Ct value for 18S=21.5±1.0) (n=4, mean±SD)]. 0.0023% AFP mRNA was detected in 1-day cultures relative to 1-week cultures.

View larger version (69K): [in a new window]   Fig. 3. Coexpression of AFP and albumin in cultured rat small hepatocytes. Cells were cultured for 8 weeks, fixed with 4% paraformaldehyde in the presence of Triton X-100 and subsequently processed for immunofluorescence localization of AFP (a) and albumin (b). Typical cells coexpressing AFP and albumin are indicated by arrow heads. (c) Phase-contrast microscopy picture. Scale bar, 40 µm.

View larger version (11K): [in a new window]   Fig. 4. Time-dependent secretion of albumin in cultured rat small hepatocytes. Cells were cultured for 9 weeks and the culture medium was collected at day 1 and weeks 1, 3, 4, 5, 6, 7 and 9 of the culture period. Each value represents the albumin secretion into the culture medium in µg albumin per ml and 24 hours normalized to the 18S rRNA, which was quantitatively measured by real-time PCR. Each time point represents the mean±SD of five determinations. One of two independent experiments is given.

View larger version (162K): [in a new window]   Fig. 5. Time-dependent protein expression of the basolateral uptake systems Ntcp (A) and Oatp1b2 (B) in cultured rat small hepatocytes. Cells were cultured for up to 9 weeks. They were fixed at weeks 1 (a,b), 5 (c,d), 7 (e,f) and 9 (g,h) with 4% paraformaldehyde in the presence of Triton X-100 and subsequently processed for immunofluorescent transporter localization. (a,c,e,g) Phase-contrast microscopy pictures. (b,d,f,h) Confocal laser-scanning microscopy pictures. Scale bars, 40 µm.

View larger version (157K): [in a new window]   Fig. 6. Time-dependent protein expression of canalicular export systems Bsep (A) and Mrp2 (B) in cultured rat small hepatocytes. Cells were cultured for up to 9 weeks. They were fixed at weeks 1 (a,b), 5 (c,d), 7 (e,f) and 9 (g,h) with 4% paraformaldehyde in the presence of Triton X-100 and subsequently processed for immunofluorescent transporter localization. (a,c,e,g) Phase-contrast microscopy pictures. (b,d,f,h) Confocal laser-scanning microscopy pictures. Scale bars, 40 µm.

View larger version (135K): [in a new window]   Fig. 7. Double labeling of cultured rat small hepatocytes with canalicular transporters and plasma-membrane-domain-specific proteins. Cells were cultured for 7 weeks. They were fixed with 4% paraformaldehyde in the presence of Triton X-100 and subsequently processed for immunofluorescence localization of the various proteins. (A) Basolateral expression of Ntcp (a) and Oatp1b2 (d) did not overlap with expression of canalicular DPP IV (b,e). Arrows indicate basolateral membrane, which stained positive for Ntcp (a) and Oatp1b2 (d) but was negative for DPP IV (b,e). Arrowheads indicate bile canaliculi, which stained positive for DPP IV (b,e) but remained negative for Ntcp (a) and Oatp1b2 (d). (c,f) Corresponding phase-contrast microscopy pictures. (B) Canalicular expression of Bsep (a) and Mrp2 (d) did not overlap with basolateral expression of the 1-18 antigen (b,e). Arrows indicate the canalicular membrane, which stained positive for Bsep (a) and Mrp2 (d) but was negative for the 1-18 antigen (b,e). Arrowheads indicate the basolateral plasma membrane, which was positive for the 1-18 antigen (b,e) but remained negative for Bsep (a) and Mrp2 (d). (c,f) Corresponding phase-contrast pictures. Scale bar, 20 µm.

View larger version (39K): [in a new window]   Fig. 8. Three-dimensional reconstruction of bile canaliculi in long-term-cultured rat small hepatocytes. Cells were cultured for 7 weeks, fixed with 4% paraformaldehyde in the presence of Triton X-100 and subsequently processed for immunofluorescence localization of Mrp2. Confocal laser-scanning microscopy was used to record stacks of the cell colony that were processed for surface rendering. (Left) Immunofluorescence picture for Mrp2 of a representative stack in the middle of the z-axis. (Right) Three-dimensional view of the surface of the canalicular network after image analysis of stacked Mrp2 fluorescence. The canalicular space is shown in dark gray and the cell colony surface is shown in light gray. Scale bar, 20 µm.

View larger version (64K): [in a new window]   Fig. 9. Canalicular secretion of fluorescein-diacetate and cholylglycyl-fluorescein in long-term small-hepatocyte colonies. Cells were cultured for 7 weeks. They were then incubated with fluorescein-dicetate (A) or or cholylglycyl-fluorescein (B). After rinsing, fluorescence analysis demonstrated canalicular fluorescein (A) and cholylglycyl-fluorescein (B) secretion with some fluorescence remaining in hepatocytes. Scale bar, 50 µm.