Network Of Bile Canaliculi Research Articles

Hepatocyte differentiation requires anisotropic expansion of bile canaliculi.

During liver development, bipotential progenitor cells called hepatoblasts differentiate into hepatocytes or cholangiocytes. Hepatocyte differentiation is uniquely associated with multi-axial polarity, enabling the anisotropic expansion of apical lumina between adjacent cells and formation of a three-dimensional network of bile canaliculi (BC). Cholangiocytes, the cells forming the bile ducts, exhibit the vectorial polarity characteristic of epithelial cells. Whether cell polarization feeds back on the gene regulatory pathways governing hepatoblast differentiation is unknown. Here, we used primary hepatoblasts to investigate the contribution of anisotropic apical expansion to hepatocyte differentiation. Silencing of the small GTPase Rab35 caused isotropic lumen expansion and formation of multicellular cysts with the vectorial polarity of cholangiocytes. Gene expression profiling revealed that these cells express reduced levels of hepatocyte markers and upregulate genes associated with cholangiocyte identity. Time-course RNA sequencing demonstrated that loss of lumen anisotropy precedes these transcriptional changes. Independent alterations in apical lumen morphology induced either by modulation of the subapical actomyosin cortex or increased intraluminal pressure caused similar transcriptional changes. These findings suggest that cell polarity and lumen morphogenesis feedback to hepatoblast-to-hepatocyte differentiation.

Hepatocyte apical bulkheads provide a mechanical means to oppose bile pressure.

Hepatocytes grow their apical surfaces anisotropically to generate a 3D network of bile canaliculi (BC). BC elongation is ensured by apical bulkheads, membrane extensions that traverse the lumen and connect juxtaposed hepatocytes. We hypothesize that apical bulkheads are mechanical elements that shape the BC lumen in liver development but also counteract elevated biliary pressure. Here, by resolving their structure using STED microscopy, we found that they are sealed by tight junction loops, connected by adherens junctions, and contain contractile actomyosin, characteristics of mechanical function. Apical bulkheads persist at high pressure upon microinjection of fluid into the BC lumen, and laser ablation demonstrated that they are under tension. A mechanical model based on ablation results revealed that apical bulkheads double the pressure BC can hold. Apical bulkhead frequency anticorrelates with BC connectivity during mouse liver development, consistent with predicted changes in biliary pressure. Our findings demonstrate that apical bulkheads are load-bearing mechanical elements that could protect the BC network against elevated pressure.

Rab35 controls formation of luminal projections required for bile canalicular morphogenesis.

Hepatocytes display a unique biaxial polarity with shared apical luminal connections between adjacent hepatocytes that merge into a network of bile canaliculi. Belicova et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202103003) discovered that hepatocyte apical membranes generate Rab35-dependent extensions that traverse the lumen and are essential for bile canalicular formation and maintenance.

Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads.

Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis.

Modeling Alcoholic Liver Disease in a Human Liver-Chip

Fatty liver disease (FLD) is a major public health burden that affects up to 30% of people in Western countries and leads to progressive liver injury, comorbidities, and increased mortality. One risk factor for developing FLD is alcohol consumption, which is growing in prevalence worldwide. There is an urgent need for human-relevant preclinical models to improve our understanding of FLD progression to steatohepatitis in alcoholic liver disease (ALD) and to develop sensitive noninvasive diagnostics and therapies of FLD in general. Here, by leveraging a tri-culture human Liver-Chip with biomimetic hepatic sinusoids and bile canaliculi, we achieve the first in vitro modeling of ALD that uses human relevant blood alcohol concentrations (BAC) and affords multimodal profiling of clinically relevant endpoints. Our ALD Liver-Chip recapitulates established FLD markers in response to ethanol in a concentration-dependent manner, including lipid accumulation and oxidative stress. The ALD Liver-Chip also supports the study of secondary insults common in patients with advanced ALD, such as high blood endotoxin levels due to alcohol-associated increased intestinal permeability and barrier dysfunction. Moreover, the ALD Liver-Chip enables the measurement of structural changes of the hepatic bile canaliculi network as a novel in vitro quantitative readout of alcoholic liver toxicity, and the co-culture of human stellate cells to study their role in FLD progression and liver fibrosis. In summary, we report the development of a human ALD Liver-Chip as a new platform for modeling the progression of alcohol-induced liver injury and fatty liver disease with direct translation to clinical research.

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Covering the Cover

Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease

There are few invitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.

A Predictive 3D Multi-Scale Model of Biliary Fluid Dynamics in the Liver Lobule

Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow invivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury.

Bile canaliculi formation and biliary transport in 3D sandwich-cultured hepatocytes in dependence of the extracellular matrix composition

Primary human hepatocytes (PHH) are still considered as gold standard for investigation of in vitro metabolism and hepatotoxicity in pharmaceutical research. It has been shown that the three-dimensional (3D) cultivation of PHH in a sandwich configuration between two layers of extracellular matrix (ECM) enables the hepatocytes to adhere three dimensionally leading to formation of in vivo like cell-cell contacts and cell-matrix interactions. The aim of the present study was to investigate the influence of different ECM compositions on morphology, cellular arrangement and bile canaliculi formation as well as bile excretion processes in PHH sandwich cultures systematically. Freshly isolated PHH were cultured for 6days between two ECM layers made of collagen and/or Matrigel in four different combinations. The cultures were investigated by phase contrast microscopy and immunofluorescence analysis with respect to cell-cell connections, repolarization as well as bile canaliculi formation. The influence of the ECM composition on cell activity and viability was measured using the XTT assay and a fluorescent dead or alive assay. Finally, the bile canalicular transport was analyzed by live cell imaging to monitor the secretion and accumulation of the fluorescent substance CDF in bile canaliculi. Using collagen and Matrigel in different compositions in sandwich cultures of hepatocytes, we observed differences in morphology, cellular arrangement and cell activity of PHH in dependence of the ECM composition. Sandwich-cultured hepatocytes with an underlay of collagen seem to represent the best in vivo tissue architecture in terms of formation of trabecular cell arrangement. Cultures overlaid with collagen were characterized by the formation of abundant bile canaliculi, while the bile canaliculi network in hepatocytes cultured on a layer of Matrigel and overlaid with collagen showed the most branched and stable canalicular network. All cultures showed a time-dependent leakage of CDF from the bile canaliculi into the culture supernatant with variations in dependence on the used matrix combination. In conclusion, the results of this study show that the choice of ECM has an impact on the morphology, cell assembly and bile canaliculi formation in PHH sandwich cultures. The morphology and the multicellular arrangement were essentially influenced by the underlaying matrix, while bile excretion and leakage of sandwich-cultured hepatocytes were mainly influenced by the overlay matrix. Leaking and damaged bile canaliculi could be a limitation of the investigated sandwich culture models in long-term excretion studies.

Primary Human Hepatocytes from Metabolic-Disordered Children Recreate Highly Differentiated Liver-Tissue-Like Spheroids on Alginate Scaffolds

Human hepatocyte transplantation has not been routinely established as an alternative to liver transplantation in liver disease due to low cell engraftment rates. Preimplantation in vitro engineering of liver tissue using primary human hepatocytes on three-dimensional scaffolds could be an alternative model. Alginate bioscaffolds were seeded with 1×10(6) hepatocytes freshly isolated from the livers of three children suffering from different metabolic disorders. During a culture period of 14 days only a marginal loss of hepatocytes was observed via measurement of DNA content per scaffold. Formation of hepatocyte spheroids was detected from day 3 onward using transmission light microscopy. Biochemical assays for albumin, α1-antitrypsin, and urea revealed excellent metabolic function with its maximum at day 7. Low lactate dehydrogenase enzyme release demonstrated minor cellular membrane damage. Hematoxylin and eosin and periodic acid Schiff staining displayed high cell viability and well-preserved glycogen storage until day 7. Immunofluorescent staining of hepatocyte nuclear factor 4, zonula occludens protein 1, and cytokeratin 18 revealed highly differentiated hepatocytes in spheroids with a tissue-like structure on scaffolds. Fluorescent labeling of cytochrome P450 and bile canaliculi demonstrated detoxification ability as well as a well-shaped bile canaliculi network. Almost constant expression levels in most target genes were detected by quantitative real-time polymerase chain reaction. The results of TUNEL reaction implicated a safe scaffold-dissolving procedure. Our results indicate that alginate scaffolds provide a favorable microenvironment for liver neo-tissue recreation and regeneration. Further, we demonstrate that livers from children with inherited metabolic disorders could serve as an alternative cell source for in vitro experiments.

Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing

Primary cultures of human hepatocyte spheroids are a promising in vitro model for long-term studies of hepatic metabolism and cytotoxicity. The lack of robust methodologies to culture cell spheroids, as well as a poor characterization of human hepatocyte spheroid architecture and liver-specific functionality, have hampered a widespread adoption of this three-dimensional culture format. In this work, an automated perfusion bioreactor was used to obtain and maintain human hepatocyte spheroids. These spheroids were cultured for 3-4 weeks in serum-free conditions, sustaining their phase I enzyme expression and permitting repeated induction during long culture times; rate of albumin and urea synthesis, as well as phase I and II drug-metabolizing enzyme gene expression and activity of spheroid hepatocyte cultures, presented reproducible profiles, despite basal interdonor variability (n = 3 donors). Immunofluorescence microscopy of human hepatocyte spheroids after 3-4 weeks of long-term culture confirmed the presence of the liver-specific markers, hepatocyte nuclear factor 4α, albumin, cytokeratin 18, and cytochrome P450 3A. Moreover, immunostaining of the atypical protein kinase C apical marker, as well as the excretion of a fluorescent dye, evidenced that these spheroids spontaneously assemble a functional bile canaliculi network, extending from the surface to the interior of the spheroids, after 3-4 weeks of culture. Perfusion bioreactor cultures of primary human hepatocyte spheroids maintain a liver-specific activity and architecture and are thus suitable for drug testing in a long-term, repeated-dose format.

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'.

HETEROTOPICALLY TRANSPLANTED HEPATOCYTES PRODUCE LIVER NEO-TISSUE WITH PROLIFERATIVE ACTIVITY AND EXPRESSION OF A MATURE BILE CANALICULI NETWORK

P1218 Aims: As previously reported, we performed flow culture of hepatocytes on a biodegradable polymer matrix and observed formation of spheroids, which in vitro created a bile canaliculi network. We were now interested in their in vivo behavior. Therefore we designed a long term implantation study and compared hepatocyte transplantation without pre-culture with pre-cultured hepatocyte spheroid transplantation in a heterotopic matrix based transplantation model. Methods: Primary hepatocytes were isolated from male Lewis rats using a two step collagenase digestion method. Highly porous poly(L-lactic acid) (PLLA) sponges were seeded with 4 x 106 hepatocytes. They were transplanted onto the mesentery of Lewis rats either immediately after cell seeding, or after 3 days of culture. For hepatotrophic stimulation a porto-caval shunt operation was performed 2 weeks before hepatocyte transplantation. After 1 week, 1, 3, or 6 months the specimens were harvested and underwent histological, immunohistochemical, immunofluorescent and morphometric analysis. For statistical analysis the nonparametric Kruskal-Wallis test was used, assuming p< 0,1 as statistically significant. Results: Newly formed liver tissue could be found in all implants and was positive for PAS, Hepar 1, and Cytokeratin 18. Phaloidin immunofluorescent studies showed expression of a liver like bile canaliculi network in the spheroid group after 1 week in vivo. The non pre-cultured group expressed only some bile canaliculi at that time point. After 1 month no difference in expression between the two groups could be observed. The HE staining showed no sign of intracellular cholestasis throughout the observation period of 6 months after transplantation. Proliferation activity of the hepatocytes was demonstrated in both groups by PCNA stain and morphometric analysis, which showed an increase of the total heterotropic hepatocyte area. With p=0,023 (pre-cultured spheroids) and p=0,051 (non pre-cultured) for the monte-carlo-significance a statistically significant difference was shown with increasing rank order over the incubation time. After 6 months, the transplantation efficiency was twice as high in the spheroid group (90 ± 39%) compared to the non pre-cultured group (40 ± 25%). Conclusions: Matrix based heterotopic hepatocyte and hepatocyte spheroid transplantation led to liver neo-tissue with in vivo formation of a bile canaliculi network and long term proliferation activity. As there are no signs of cholestasis within the heterotopically formed liver neo-tissue even after 6 months in vivo, a removal of bile must be assumed. Transplantation of pre-cultured hepatocyte spheroids led to a higher efficiency and proliferation, compared to non pre-cultured hepatocyte transplantation. Pre-implantation tissue assembly, including the formation of a bile canaliculi network could possibly explain this advantage.

Effects of Fetal Calf Serum and Disruption of Cadherin Function on the Formation of Bile Canaliculi between Hepatocytes

The polarization of hepatocytes to form a connected network of bile canaliculi (BC) is necessary for the function of the liver. Hepatocyte polarization may be controlled by soluble factors and/or physical interactions between cells. Monolayer cultures of embryonic chicken hepatocytes in DMEM supplemented with ornithine, dexamethasone, and insulin express BC-specific antigens for at least 7 days. However, BC-specific antigen expression is lost within 3 days of culture initiation in DMEM containing 10% fetal calf serum. The dedifferentiating effects of fetal calf serum (FCS) can be reversed. Furthermore, cultures in medium containing ornithine, dexamethasone, insulin, and 10% FCS appear identical to cultures grown in 10% FCS alone. Thus FCS contains a soluble inhibitor of hepatocyte polarization. Aggregate cultures grown in suspension maintain hepatocyte polarization for 10-12 days. This may be due to the increased cell-cell contact between hepatocytes in aggregate culture or to more normal contact with the extracellular matrix. We have evaluated the role of cadherin-mediated interactions on hepatocyte polarization. Anti-E-cadherin Fab fragments disrupted the formation of long networks of BC in monolayer cultures but did not stop polarized expression of BC-specific antigens. The BC antigens in anti-E-cadherin-treated cells were concentrated in small areas between cells and were present at lower levels uniformly on the cell surface. These results indicate that E-cadherin is required for the formation of extended BC networks, but that other factors are responsible for maintaining the synthesis and localization of BC-specific antigens.

Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration.

Rat primary hepatocytes were cultured under different extracellular matrix configurations and evaluated for the acquisition and maintenance of structural and functional cell polarity. De novo repolarization of the plasma membrane was variable in rate and extent in hepatocyte cultures maintained on a conventional single layer of either gelled or ungelled collagen. However, cultures maintained in a collagen-sandwich configuration initiated uniform formation of a contiguous anastomosing network of bile canaliculi throughout the entire culture. Localization of apical membrane markers demonstrated normal distribution at the canalicular membrane. A marked rearrangement of the intracellular microfilaments to the cell periphery was observed and coincided with the development of the bile canaliculi. Acquisition of normal bile canalicular function and integrity was observed within 3-4 days postoverlay as indicated by the concentration and retention of carboxyfluorescein within the canalicular network. These results demonstrate that cultures of hepatocytes maintained in a sandwich configuration may serve as a more reliable and representative model in which to study the physiology of hepatic function as well as the morphogenesis of polarized membrane domains in vitro.

Development of bile canaliculi between chicken embryo liver cells in vivo and in vitro

The development of an organized network of bile canaliculi is essential for the normal functioning of the liver. We have characterized bile canaliculus development in situ from Days 3-19 and in vitro in cultured hepatocyte monolayers using electron microscopical and immunofluorescent staining with antibodies that specifically recognize antigens of the bile canaliculus. Although the liver first forms as a discrete epithelial bud of endodermal tissue at stage 12-14 (45-53 h after laying), canaliculi were first detected by our antibodies at low levels in 4-day embryos and at high levels in stage 27 (5 days after laying) and later embryos. During Days 4, 5, and 6 the canaliculi near the periphery of the rudiment do not stain while canaliculi in central areas, closer to the gut, are strongly stained. During this transition period the ultrastructure of the canaliculi in the peripheral regions is also less developed than the central canaliculi where the antigens appear. By 7 days post laying, canaliculi throughout the entire liver rudiment express the marker antigens equally and have the ultrastructural characteristics of mature, functional canaliculi. Cells prepared from liver of embryos of 11 days incubation and grown in monolayer culture reformed discernible canalicular specializations, as determined by immunofluorescent staining and electron microscopy, but only transiently (for 1 to 3 days after plating). Not all of the antigens were expressed or polarized in these cultures. The capacity of the embryonic parenchymal cells to develop and maintain polarity appears to depend on factors possibly including age-dependent changes in the cells themselves, interactions with other cell types or extracellular matrix, or the shape of the cells.