Small hepatocytes in culture develop polarized transporter expression and differentiation.
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'.
Division of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital, 8091 Zurich, Switzerland.
Marguerite-Anne
MA
Sidler Pfändler
Mathias
M
Höchli
Daniel
D
Inderbitzin
Peter J
PJ
Meier
Bruno
B
Stieger
D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000418:Albumins / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002449:Cell Aggregation / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology*; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D008297:Male; D026901:Membrane Transport Proteins / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent; D029362:Organic Anion Transporters, Sodium-Independent / Q000378:metabolism; D038081:Organogenesis / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D027981:Symporters; D000509:alpha-Fetoproteins / Q000378:metabolism
D000074020:ATP Binding Cassette Transporter, Subfamily B, Member 11; D018528:ATP-Binding Cassette Transporters; D000418:Albumins; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D029363:Organic Anion Transporters, Sodium-Dependent; D029362:Organic Anion Transporters, Sodium-Independent; D027981:Symporters; D000509:alpha-Fetoproteins; C490548:Abcb11 protein, rat; C423013:Abcc2 protein, rat; C421102:Slco1a1 protein, rat; C071908:sodium-bile acid cotransporter; C421109:multidrug resistance-associated protein 1