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Cell polarity and formation of bile canaliculi can be achieved in hepatocytes which are generated from patient-derived induced pluripotent stem cells. This allows for the study of endogenous mutant proteins, patient-specific pathogenesis, and drug responses for diseases where hepatocyte polarity and bile canaliculi play a key role. Here, we describe a step-by-step protocol for the generation of bile canaliculi-forming hepatocytes from induced pluripotent stem cells and their evaluation.

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.

Peroxisome proliferator-activated receptor alpha (PPARα) controls lipid homeostasis through regulation of lipid transport and catabolism. PPARα activators are clinically used for hyperlipidemia treatment. The role of PPARα in bile acid (BA) homeostasis is beginning to emerge. Herein, Ppara-null and hepatocyte-specific Ppara-null (Ppara(ΔHep)) as well as the respective wild-type mice were treated with the potent PPARα agonist Wy-14,643 (Wy) and global metabolomics performed to clarify the role of hepatocyte PPARα in the regulation of BA homeostasis. Levels of all serum BAs were markedly elevated in Wy-treated wild-type mice but not in Ppara-null and Ppara(ΔHep) mice. Gene expression analysis showed that PPARα activation (1) down-regulated the expression of sodium-taurocholate acid transporting polypeptide and organic ion transporting polypeptide 1 and 4, responsible for the uptake of BAs into the liver; (2) decreased the expression of bile salt export pump transporting BA from hepatocytes into the bile canaliculus; (3) upregulated the expression of multidrug resistance-associated protein 3 and 4 transporting BA from hepatocytes into the portal vein. Moreover, there was a notable increase in the compositions of serum, hepatic and biliary cholic acid and taurocholic acid following Wy treatment, which correlated with the upregulated expression of the Cyp8b1 gene encoding sterol 12α-hydroxylase. The effects of Wy were identical between the Ppara(ΔHep) and Ppara-null mice. Hepatocyte PPARα controlled BA synthesis and transport not only via direct transcriptional regulation but also via crosstalk with hepatic farnesoid X receptor signaling. These findings underscore a key role for hepatocyte PPARα in the control of BA homeostasis.

Background: Neonates on long-term parenteral nutrition (PN) may develop parenteral nutrition-associated liver disease (PNALD). Aluminum (Al) is a known contaminant of infant PN, and we hypothesize that it substantially contributes to PNALD. In this study, we aim to assess the impact of Al on hepatocytes in a piglet model. Methods: We conducted a randomized control trial using a Yucatan piglet PN model. Piglets, aged 3-6 days, were placed into two groups. The high Al group (n=8) received PN with 63 µg/kg/day of Al, while the low Al group (n=7) received PN with 24 µg/kg/day of Al. Serum samples for total bile acids (TBA) were collected over two weeks, and liver tissue was obtained at the end of the experiment. Bile canaliculus morphometry was studied by transmission electron microscopy (TEM) and Image-J software analysis. In December 2019, the American Academy of Pediatrics issued a key technical report on Al effects in infants and children. This report was revised and commented on in this chapter. Results: The canalicular space was smaller, and the microvilli were shorter in the high Al group than in the low Al group. There was no difference in the TBA between the groups. Conclusions: Al causes structural changes in the hepatocytes despite unaltered serum bile acids. High Al in PN is associated with short microvilli, which could decrease the functional excretion area of the hepatocytes and impair bile flow. Long-term toxicity studies from the Al content in PN components supported the US Food and Drug Administration (FDA) in the decision to implement specific rules. It has been required that large-volume ingredients reduce the Al concentration and all small volume components be specifically labeled with the Al concentration. However, both US FDA and we consider that despite these rules, the total Al concentration from some components remains above the recommended final concentration. US FDA and our concerns about toxicity from the Al present in infant PN formulas are grounded, requiring more research on this topic.

The hepatic barrier is indispensable for the physiological functions of the liver and is impaired under various pathological conditions. Tight junctions reportedly play a central role in hepatic barrier regulation; however, there is limited direct evidence supporting this observation, with few in vivo models or confirmations of the implicated molecular mechanisms presented to date. We inactivated the tight junction component gene, Tjp2/ZO-2, and the related molecule, Tjp1/ZO-1, in mouse livers. In humans, TJP2/ZO-2 mutations have been implicated in the development of human progressive familial intrahepatic cholestasis 4 (PFIC4). The mice deficient in either ZO-1 or ZO-2 in the liver did not exhibit major abnormalities. However, the ablation of both molecules impaired the molecular architecture as well as the structure and function of hepatocyte tight junctions, which disrupted the hepatic barrier and was lethal to the mice by 6 weeks of age. In mutant mice, bile canaliculus formation and cellular polarity were compromised; also, transporter expression and localization were deregulated. Moreover, typical hepatic zonation and bile duct formation were inhibited, and sinusoidal vessels were disorganized. These findings clarify the role of tight junctions and polarity in the hepatic barrier as well as the effect that their disruption has on liver tissue. The observations also suggest that liver-specific ZO-1−/− and ZO-2−/− mice could be used as models for PFIC4, and this will provide new insights into liver pathophysiology and clinical applications.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

The effects of each type of machine perfusion preservation (MP) of liver grafts donated after cardiac death on the bile canaliculi of hepatocytes remain unclear. We analyzed the intracellular three-dimensional ultrastructure of the bile canaliculi and hepatocyte endomembrane systems in porcine liver grafts after warm ischemia followed by successive MP with modified University of Wisconsin gluconate solution. Transmission and osmium-maceration scanning electron microscopy revealed that lumen volume of the bile canaliculi decreased after warm ischemia. In liver grafts preserved by hypothermic MP condition, bile canaliculi tended to recover in terms of lumen volume, while their microvilli regressed. In contrast, midthermic MP condition preserved the functional form of the microvilli of the bile canaliculi. Machine perfusion preservation potentially restored the bile canaliculus lumen and alleviated the cessation of cellular endocrine processes due to warm ischemia. In addition, midthermic MP condition prevented the retraction of the microvilli of bile canaliculi, suggesting further mitigation of the damage of the bile canaliculi.

Exosomes are extracellular vesicles with a size of 40–150 nm that are released from a multitude of cell types and are present in biological fluids, which are particularly rich in membrane proteins. These vesicles are present in the bile, where they function as a special regulator of the digestive system; however, their source and characteristics in normal gallbladders have never been discovered. Bile, liver, and gallbladder were obtained from healthy turtles after starvation treatment. Biliary exosomes were extracted and their morphology, particle sizes, and specific proteins were characterized by transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) staining, and Western blotting. Furthermore, immunohistochemistry and TEM were used to investigate the distribution of exosomes in both liver and gallbladder. Biliary exosomes had a round or oval shape with apparent bilayer membranes. Biliary exosomes were uniform in size, with a range from 40 to 180 nm in diameter. Immunohistochemistry showed that the CD63 exosome marker was positive and primarily distributed in the hepatocyte junction, while the free surface of gallbladder tested negative. In addition, exosomes were present in bile but absent on the apical free surface of gallbladder. This study provides direct evidences that biliary exosomes are secreted by hepatocytes into bile canaliculus and flow with bile into the gallbladder. The source and the isolated protocol of biliary exosomes may provide technical support and a theoretical basis for the exploration of potential antiviral or anti-inflammatory properties of biliary exosomes.

Liver kinase B1 (LKB1) and its downstream effector AMP‐activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich‐cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver‐specific (albumin‐Cre) LKB1 knockout mice (LKB1−/−) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1−/− livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6‐carboxyfluorescein diacetate (6‐CFDA), which is processed in hepatocytes into its fluorescent derivative 6‐carboxyfluorescein (6‐CF) and secreted into the canaliculi. In LKB1−/− mice, 6‐CF remained largely in hepatocytes, canalicular secretion was delayed, and 6‐CF appeared in the blood. To test whether 6‐CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6‐CFDA. In wild‐type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1−/− mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. Conclusion: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317‐1329)

Background: Neonates on long-term parenteral nutrition (PN) may develop parenteral nutrition-associated liver disease (PNALD). Aluminum (Al) is a known contaminant of infant PN, and we hypothesize that it substantially contributes to PNALD. In this study, we aim to assess the impact of Al on hepatocytes in a piglet model. Methods: We conducted a randomized control trial using a Yucatan piglet PN model. Piglets, aged 3&ndash, 6 days, were placed into two groups. The high Al group (n = 8) received PN with 63 &micro, g/kg/day of Al, while the low Al group (n = 7) received PN with 24 &micro, g/kg/day of Al. Serum samples for total bile acids (TBA) were collected over two weeks, and liver tissue was obtained at the end of the experiment. Bile canaliculus morphometry were studied by transmission electron microscopy (TEM) and ImageJ software analysis. Results: The canalicular space was smaller and the microvilli were shorter in the high Al group than in the low Al group. There was no difference in the TBA between the groups. Conclusions: Al causes structural changes in the hepatocytes despite unaltered serum bile acids. High Al in PN is associated with short microvilli, which could decrease the functional excretion area of the hepatocytes and impair bile flow.

Introduction: Hepatopulmonary syndrome (HPS) is a serious, progressive disease. Its pathophysiology resides in a hypoxic intrapulmonary shunt and severe clinical deterioration. Liver transplantation (LT) is the only effective treatment in appropriately selected patients. Objective: To acknowledge the importance of early diagnosis of HPS. Patients and methods: Observational, descriptive, retrospective trial including 8 patients with HPS that received LT between April 2006 and August 2014. The clinical data prior to transplantation and follow-up after the procedure were reviewed. A multivariate analysis (stepwise forward logistic regression analysis) was used to identify the variable that could potentially increase the risk of death. Results: Of the 8 patients, death could only be significantly predicted based on the pre-LT arterial blood partial oxygen pressure (PaO2) (p = 0.002). The average pre-LT PaO2 of the patients that died was 51.5 ±2.49 SD, with a statistically significant difference (p = 0.002). None of the variables was statistically significant for HPS reversibility. The survival rate of patients diagnosed with HPS following the LT was 62.5%. Conclusions: The level of pre-LT hypoxemia is an important predictor for immediate postoperative mortality. Early detection of the condition is critical to reduce the post LT morbidity and mortality so that the indication for transplant is made at the right time, regardless of the stage of liver disease. The most efficient clinical strategy could be the use of appropriate early detection protocols for HPS through screening of hypoxemia in patients with portal hypertension. Introducción: El síndrome hepatopulmonar (SHP) es una enfermedad grave y progresiva cuya fisiopatología reside en un shunt intrapulmonar con hipoxia y deterioro clínico severo. Como único tratamiento efectivo se ha postulado el trasplante hepático (TH), en pacientes adecuadamente seleccionados. Objetivo: Reconocer la importancia del diagnostico temprano del SHP. Pacientes y métodos: Mediante un estudio observacional, descriptivo, con carácter retrospectivo de 8 pacientes con SHP, a los que se les realizó TH en el período entre Abril 2006-Agosto 2014. Se han revisado los datos clínicos previos al trasplante y el seguimiento tras este. Se empleó un estudio multivariante (stepwise forward logistic regression analisis), para determinar cual variable podría incrementar el riesgo de muerte. Resultados: De los 8 pacientes, el resultado de muerte sólo pudo ser predicho significativamente por el factor presión parcial de oxígeno en sangre arterial (PaO2) pre-TH (p=0,002). La PaO2 pre-TH promedio de los pacientes que fallecieron era de 51,5 +/- 2,49 DS, siendo la diferencia estadísticamente significativa (p=0,002). Ninguna variable resultó estadísticamente significativa para reversibilidad del SHP. La supervivencia de los pacientes con criterio de SHP posterior al TH fue de 62,5%. Conclusiones: El grado de hipoxemia pre-TH es un factor predictor importante de mortalidad en el postoperatorio inmediato. La precocidad en la detección de la entidad es fundamental tanto para disminuir la morbimortalidad post TH como para indicar éste en el momento óptimo independientemente del estadío de la enfermedad hepática. Protocolos adecuados de detección precoz del SHP mediante screening de hipoxemias en pacientes con hipertensión portal, puede ser la estrategia clínica más eficiente.

Proliferation of adult rat hepatocytes is observed in serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 mmol/L nicotinamide and 10 ng/mL epidermal growth factor (EGF). The proliferating cells are mainly mononucleate and form small cell colonies surrounded by mature hepatocytes. Although these cells in focal colonies have a less-differentiated appearance, immunocytochemically and ultrastructurally they possess hepatic characteristics. The size of small hepatocytes is one-third to half that of mature hepatocytes. Therefore, we call the cells forming a colony, small hepatocytes. The small hepatocytes can be subcultured for several passages. Furthermore, the cells are rich in the supernatant following 50 g centrifugation for 1 min after collagenase liver perfusion. When the cells are cultured in DMEM supplemented with 10% foetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL EGF and 1% dimethyl sulphoxide, each small hepatocyte can clonally proliferate for more than 3 months. A small hepatocyte divides to form a colony and the number of cells reaches more than 100 within 20 days. With time in culture, cells with a large cytoplasm appear within a colony. They have many mitochondria and large peroxisomes with crystalline nucleoids and are typical, mature hepatocytes. Immunoreactivity to connexin 32 and well-developed bile canaliculus structures are often observed in the cell-cell borders. Thus, we suggest that small hepatocytes may be considered to be 'committed progenitor cells' that can further differentiate into mature hepatocytes.