Abstract
Rat small hepatocytes have been shown to proliferate in culture and to form organoids with differentiated hepatocytes in vitro. To evaluate the degree of polarized transporter differentiation of rat small hepatocytes during 9 weeks of culturing, we studied the time-dependent expression and subcellular localization of the major bile salt and organic anion transport systems of hepatocytes [i.e. the basolateral sodium-taurocholate co-transporting protein (Ntcp), organic-anion-transporting polypeptide 1b2 (Oatp1b2), the canalicular bile-salt export pump (Bsep) and multidrug-resistance-associated protein 2 (Mrp2)]. Small hepatocytes proliferated and differentiated in culture and formed sharply demarcated colonies as assessed by morphology, alpha-fetoprotein, albumin and Mrp1 expression. Polarized surface transporter expression was evident after 5 weeks of culturing for Ntcp, Oatp1b2 and Mrp2, and after 7 weeks for Bsep. After 9 weeks in culture, the vast majority of matured hepatocytes expressed Ntcp/Oatp1b2 at the basolateral and Bsep/Mrp2 at the canalicular plasma-membrane domains. This polarized transporter expression was accompanied by canalicular secretion of fluorescein-diacetate and cholylglycyl-fluorescein. Furthermore, an anastomizing three-dimensional network of bile canaliculi developed within piling-up colonies. These data demonstrate that cultured rat small hepatocytes acquire a fully differentiated transporter expression phenotype during their development into hepatic 'organoid-like' clusters of mature hepatocytes. Thereby, the time-dependent sequence of transporter expression mirrored the ontogenesis of transporter expression in developing rat liver, supporting the concept that small hepatocytes correspond to the hepatocyte lineage derived from embryonic hepatoblasts and/or from a different pool of 'committed hepatocyte progenitor cells'.
Highlights
Transcellular bile secretion from sinusoidal blood plasma into bile canaliculi is an important function of differentiated hepatocytes in all mammalian species
Using the same cell isolation and culturing procedures as originally reported by Mitaka et al (Mitaka et al, 1999), colonies of 10-20 mononucleated small hepatocyte-like cells appeared after 1 week (Fig. 1A)
Thereafter, some colonies died and new colonies appeared, indicating continuous proliferation of small hepatocytes during the entire 9 weeks of culture time. This assumption was confirmed by measuring mRNA expression of the two hepatocyte proliferation markers Mrp1 (Roelofsen et al, 1997) and antibody to rat α-fetoprotein (AFP) (Mizejewski, 2001)
Summary
Transcellular bile secretion from sinusoidal blood plasma into bile canaliculi is an important function of differentiated hepatocytes in all mammalian species. At the canalicular membrane, conjugated bile salts and organic anions are actively secreted/excreted against the prevailing electrochemical gradient by the bile-salt export pump (Bsep; Abcb11) and the multidrug resistance protein 2 (Mrp; Abcc2), respectively (Keppler and Konig, 2000; Meier and Stieger, 2002). These members of the ATP-binding cassette (ABC) transporter superfamily provide the necessary driving force for the generation of bile-salt-dependent and -independent bile flow within the bile canaliculi
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