Effect of glucose and insulin supplementation on the isolation of primary human hepatocytes

Primary human hepatocytes (PHHs) remain the gold standard for in vitro investigations of xenobiotic metabolism and hepatotoxicity. However, scarcity of liver tissue and novel developments in liver surgery has limited the availability and quality of tissue samples. In particular, warm ischemia shifts the intracellular metabolism from aerobic to anaerobic conditions, which increases glycogenolysis, glucose depletion and energy deficiency. Therefore, the aim of the present study was to investigate whether supplementation with glucose and insulin during PHH isolation could reconstitute intracellular glycogen storage and beneficially affect viability and functionality. Furthermore, the study elucidated whether the susceptibility of the tissue’s energy status correlates with body mass index (BMI). PHHs from 12 donors were isolated from human liver tissue obtained from partial liver resections using a two-step EDTA/collagenase perfusion technique. For a direct comparison of the influence of glucose/insulin supplementation, we modified the setup, enabling the parallel isolation of two pieces of one tissue sample with varying perfusate. Independent of the BMI of the patient, the glycogen content in liver tissue was notably low in the majority of samples. Furthermore, supplementation with glucose and insulin had no beneficial effect on the glycogen concentration of isolated PHHs. However, an indirect improvement of the availability of energy was shown by increased viability, plating efficiency and partial cellular activity after supplementation. The plating efficiency showed a striking inverse correlation with increasing lipid content of PHHs. However, 60 h of cultivation time revealed no significant impact on the maintenance of albumin and urea synthesis or xenobiotic metabolism after supplementation. In conclusion, surgical procedures and tissue handling may decrease hepatic energy resources and lead to cell stress and death. Consequently, PHHs with low energy resources die during the isolation process without supplementation of glucose/insulin or early cell culture, while their survival rates are improved with glucose/insulin supplementation

Lipid metabolism in development and progression of hepatocellular carcinoma

Metabolic reprogramming is critically involved in the development and progression of cancer. In particular, lipid metabolism has been investigated as a source of energy, micro-environmental adaptation, and cell signalling in neoplastic cells. However, the specific role of lipid metabolism dysregulation in hepatocellular carcinoma (HCC) has not been widely described yet. Alterations in fatty acid synthesis, β-oxidation, and cellular lipidic composition contribute to initiation and progression of HCC. The aim of this review is to elucidate the mechanisms by which lipid metabolism is involved in hepatocarcinogenesis and tumour adaptation to different conditions, focusing on the transcriptional aberrations with new insights in lipidomics and lipid zonation. This will help detect new putative therapeutic approaches in the second most frequent cause of cancer-related death

Cross-species variability in lobular geometry and cytochrome P450 hepatic zonation: insights into CYP1A2, CYP2D6, CYP2E1 and CYP3A4

There is a lack of systematic research exploring cross-species variation in liver lobular geometry and zonation patterns of critical drug-metabolizing enzymes, a knowledge gap essential for translational studies. This study investigated the critical interplay between lobular geometry and key cytochrome P450 (CYP) zonation in four species: mouse, rat, pig, and human. We developed an automated pipeline based on whole slide images (WSI) of hematoxylin-eosin-stained liver sections and immunohistochemistry. This pipeline allows accurate quantification of both lobular geometry and zonation patterns of essential CYP proteins. Our analysis of CYP zonal expression shows that all CYP enzymes (besides CYP2D6 with panlobular expression) were observed in the pericentral region in all species, but with distinct differences. Comparison of normalized gradient intensity shows a high similarity between mice and humans, followed by rats. Specifically, CYP1A2 was expressed throughout the pericentral region in mice and humans, whereas it was restricted to a narrow pericentral rim in rats and showed a panlobular pattern in pigs. Similarly, CYP3A4 is present in the pericentral region, but its extent varies considerably in rats and appears panlobular in pigs. CYP2D6 zonal expression consistently shows a panlobular pattern in all species, although the intensity varies. CYP2E1 zonal expression covered the entire pericentral region with extension into the midzone in all four species, suggesting its potential for further cross-species analysis. Analysis of lobular geometry revealed an increase in lobular size with increasing species size, whereas lobular compactness was similar. Based on our results, zonated CYP expression in mice is most similar to humans. Therefore, mice appear to be the most appropriate species for drug metabolism studies unless larger species are required for other purposes, e.g., surgical reasons. CYP selection should be based on species, with CYP2E1 and CYP2D6 being the most preferable to compare four species. CYP1A2 could be considered as an additional CYP for rodent versus human comparisons, and CYP3A4 for mouse/human comparisons. In conclusion, our image analysis pipeline together with suggestions for species and CYP selection can serve to improve future cross-species and translational drug metabolism studies

Periportal steatosis in mice affects distinct parameters of pericentral drug metabolism

Little is known about the impact of morphological disorders in distinct zones on metabolic zonation. It was described recently that periportal fibrosis did affect the expression of CYP proteins, a set of pericentrally located drug-metabolizing enzymes. Here, we investigated whether periportal steatosis might have a similar effect. Periportal steatosis was induced in C57BL6/J mice by feeding a high-fat diet with low methionine/choline content for either two or four weeks. Steatosis severity was quantified using image analysis. Triglycerides and CYP activity were quantified in photometric or fluorometric assay. The distribution of CYP3A4, CYP1A2, CYP2D6, and CYP2E1 was visualized by immunohistochemistry. Pharmacokinetic parameters of test drugs were determined after injecting a drug cocktail (caffeine, codeine, and midazolam). The dietary model resulted in moderate to severe mixed steatosis confined to periportal and midzonal areas. Periportal steatosis did not affect the zonal distribution of CYP expression but the activity of selected CYPs was associated with steatosis severity. Caffeine elimination was accelerated by microvesicular steatosis, whereas midazolam elimination was delayed in macrovesicular steatosis. In summary, periportal steatosis affected parameters of pericentrally located drug metabolism. This observation calls for further investigations of the highly complex interrelationship between steatosis and drug metabolism and underlying signaling mechanisms

Cross-species variability in lobular geometry and cytochrome P450 hepatic zonation: insights into CYP1A2, CYP2D6, CYP2E1 and CYP3A4

There is a lack of systematic research exploring cross-species variation in liver lobular geometry and zonation patterns of critical drug-metabolizing enzymes, a knowledge gap essential for translational studies. This study investigated the critical interplay between lobular geometry and key cytochrome P450 (CYP) zonation in four species: mouse, rat, pig, and human. We developed an automated pipeline based on whole slide images (WSI) of hematoxylin-eosin-stained liver sections and immunohistochemistry. This pipeline allows accurate quantification of both lobular geometry and zonation patterns of essential CYP proteins. Our analysis of CYP zonal expression shows that all CYP enzymes (besides CYP2D6 with panlobular expression) were observed in the pericentral region in all species, but with distinct differences. Comparison of normalized gradient intensity shows a high similarity between mice and humans, followed by rats. Specifically, CYP1A2 was expressed throughout the pericentral region in mice and humans, whereas it was restricted to a narrow pericentral rim in rats and showed a panlobular pattern in pigs. Similarly, CYP3A4 is present in the pericentral region, but its extent varies considerably in rats and appears panlobular in pigs. CYP2D6 zonal expression consistently shows a panlobular pattern in all species, although the intensity varies. CYP2E1 zonal expression covered the entire pericentral region with extension into the midzone in all four species, suggesting its potential for further cross-species analysis. Analysis of lobular geometry revealed an increase in lobular size with increasing species size, whereas lobular compactness was similar. Based on our results, zonated CYP expression in mice is most similar to humans. Therefore, mice appear to be the most appropriate species for drug metabolism studies unless larger species are required for other purposes, e.g., surgical reasons. CYP selection should be based on species, with CYP2E1 and CYP2D6 being the most preferable to compare four species. CYP1A2 could be considered as an additional CYP for rodent versus human comparisons, and CYP3A4 for mouse/human comparisons. In conclusion, our image analysis pipeline together with suggestions for species and CYP selection can serve to improve future cross-species and translational drug metabolism studies

Computational Modeling in Liver Surgery

The need for extended liver resection is increasing due to the growing incidence of liver tumors in aging societies. Individualized surgical planning is the key for identifying the optimal resection strategy and to minimize the risk of postoperative liver failure and tumor recurrence. Current computational tools provide virtual planning of liver resection by taking into account the spatial relationship between the tumor and the hepatic vascular trees, as well as the size of the future liver remnant. However, size and function of the liver are not necessarily equivalent. Hence, determining the future liver volume might misestimate the future liver function, especially in cases of hepatic comorbidities such as hepatic steatosis. A systems medicine approach could be applied, including biological, medical, and surgical aspects, by integrating all available anatomical and functional information of the individual patient. Such an approach holds promise for better prediction of postoperative liver function and hence improved risk assessment. This review provides an overview of mathematical models related to the liver and its function and explores their potential relevance for computational liver surgery. We first summarize key facts of hepatic anatomy, physiology, and pathology relevant for hepatic surgery, followed by a description of the computational tools currently used in liver surgical planning. Then we present selected state-of-the-art computational liver models potentially useful to support liver surgery. Finally, we discuss the main challenges that will need to be addressed when developing advanced computational planning tools in the context of liver surgery.Peer Reviewe

Identification of a novel mechanism driving NAFLD progression and therapeutic strategies

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries with an increasing prevalence of approximately 25 %. NAFLD comprises several stages, starting as benign steatosis and progressing to non-alcoholic steatohepatitis (NASH), and in some cases to liver cirrhosis and hepatocellular carcinoma (HCC). Although several emerging therapies are currently in clinical trials, so far there are no approved drugs for treatment of NASH. The overarching goal of this thesis was to investigate the mechanisms of NAFLD stage transitions, and to establish preventive measures for the progression from benign steatosis to NASH. First, a mouse model of NAFLD progression was established by long-term feeding of male C57Bl/6N mice with western-style diet (WD) up to 54 weeks. The disease progression was evaluated time-dependently by biochemical, histopathological, and immunohistochemical analyses as well as by intravital two-photon-based imaging. This analyses revealed six stages in NAFLD progression: (1) benign steatosis, (2) macrophage crown-like structure formation, (3) macropinocytosis of bile, (4) ductular reaction, (5) dedifferentiation and functional shutdown, and (6) tumor nodule formation. The novel finding of this thesis was the identification of stage 3, where a retrograde vesicular uptake of bile from bile canaliculi to hepatocytes led to toxic accumulation of bile acids in the liver tissue, providing a link between NAFLD and cholestasis. The phenomenon was further identified as macropinocytosis by treating the mice with a macropinocytosis-specific inhibitor imipramine. As a result, a single application of imipramine efficiently blocked macropinocytosis in WD-fed mice. Interestingly, a long-term application of imipramine for 8 weeks decreased the bile acid concentrations in the liver tissue and led to significant NAFLD amelioration. Moreover, bile macropinocytosis was also found relevant in human NAFLD patients as detected by the presence of fragments of bile canaliculi within steatotic hepatocytes. In conclusion, an NAFLD mouse model recapitulating the different stages of human NAFLD progression to NASH and eventually to HCC was successfully established. Moreover, a novel mechanism possibly driving NALFD progression was identified as macropinocytosis of bile from bile canaliculi back to hepatocytes

Epigenetic characterization of human hepatocyte subpopulations in context of complex metabolic diseases and during in vitro differentiation of hepatocyte-like cells

The comprehensive transcriptional and epigenetic characterization of human hepatocyte subpopulations is necessary to achieve a better understanding of regulatory processes in health and complex metabolic diseases as well as during in vitro differentiation. Based on integrative analysis of genome-wide sequencing data, this thesis aims to unravel hepatocyte heterogeneity in different biological contexts. A deeper understanding of spatial organization of cells in human tissues is an important challenge. Using a unique experimental set-up based on laser capture microdissection coupled to next generation sequencing, which preserves spatial orientation and still provides genome-wide data of well defined subpopulations, the first combined spatial analysis of transcriptomes and methylomes across three micro-dissected zones of human liver provides a wealth of new positional insights, both in health and in context of fatty liver disease. In addition, these spatial maps serve as reference for projection of single cell data into hepatic pseudospace, which is still a major challenge. Hence, a novel pseudospace inference approach, which considerably improves spatial reconstruction of single cells into tissue context, is demonstrated for human liver. Finally, the identification of underlying regulatory networks by integrative epigenomic analysis of in vitro differentiated hepatocyte-like cells contributes to the development of reasonable cell culture interventions to improve differentiation.Die umfassende transkriptionelle und epigenetische Charakterisierung humaner Leberzellsubpopulationen ist notwendig für die Aufklärung regulatorischer Prozesse in gesundem Gewebe, sowie im Zusammenhang mit komplexen metabolischen Erkrankungen und während der in vitro Differenzierung. Ziel dieser Arbeit ist es, basierend auf der integrativen Analyse genomweiter Sequenzierungsdaten, die Heterogenität von Leberzellen besser zu verstehen. Die räumliche Organisation von Zellen in humanem Gewebe stellt eine große Herausforderung dar. Mit Hilfe von Lasermikrodissektion gekoppelt an Hochdurchsatzsequenzierung ist es möglich definierte Subpopulationen hinsichtlich ihres Gewebekontextes zu analysieren. Somit konnte die erste räumliche Analyse von Transkriptom und Methylom dreier Zonen der humanen Leber erstellt werden, die eine Vielzahl neuer Erkenntnisse sowohl in gesundem Lebergewebe als auch in Zusammenhang mit Fettlebererkrankungen liefert. Außerdem wurde auf Grundlage dieser räumlichen Karten ein neuer Ansatz zur Projektion von Einzelzelldaten in den räumlichen Gewebekontext etabliert. Schließlich konnte durch die integrative Analyse der ausschlaggebenden regulatorischen Netzwerke während der in vitro Differenzierung von Hepatozyten-ähnlichen Zellen neue Strategien zur Verbesserung der Differenzierung entwickelt werden