Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation

Lauschke, Volker M.; Vorrink, Sabine U.; Moro, Sabrina; Rezayee, Fatemah; Nordling, Åsa; Hendriks, Delilah; Bell, Catherine; Sison-Young, Rowena; Park, B. Kevin; Goldring, Christopher E.; Ellis, Ewa; Johansson, Inger; Mkrtchian, Souren; Andersson, Tommy; Ingelman‐Sundberg, Magnus

doi:10.1002/hep.28780

Cited by 98 publications

(79 citation statements)

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“…1 E ). Whereas these genes were progressively downregulated in 2D monolayer culture as previously reported (14), their transcript levels were increased by 21-, 230- and 9-fold, respectively, in 3D culture and were indistinguishable from levels found in isolated cells. Thus, loss of key transcriptional regulators may provide an explanation for the observed differences in ADME gene expression between the culture systems.…”

Section: Resultssupporting

confidence: 83%

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long‐term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics

et al. 2017

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Adverse reactions or lack of response to medications are important concerns for drug development programs. However, faithful predictions of drug metabolism and toxicity are difficult because animal models show only limited translatability to humans. Furthermore, current in vitro systems, such as hepatic cell lines or primary human hepatocyte (PHH) 2-dimensional (2D) monolayer cultures, can be used only for acute toxicity tests because of their immature phenotypes and inherent instability. Therefore, the migration to novel phenotypically stable models is of prime importance for the pharmaceutical industry. Novel 3-dimensional (3D) culture systems have been shown to accurately mimic in vivo hepatic phenotypes on transcriptomic and proteomic level, but information about their metabolic stability is lacking. Using a combination of targeted and untargeted high-resolution mass spectrometry, we found that PHHs in 3D spheroid cultures remained metabolically stable for multiple weeks, whereas metabolic patterns of PHHs from the same donors cultured as conventional 2D monolayers rapidly deteriorated. Furthermore, pharmacokinetic differences between donors were maintained in 3D spheroid cultures, enabling studies of interindividual variability in drug metabolism and toxicity. We conclude that the 3D spheroid system is metabolically stable and constitutes a suitable model for in vitro studies of long-term drug metabolism and pharmacokinetics.—Vorrink, S. U., Ullah, S., Schmid, S., Nandania, J., Velagapudi, V., Beck, O., Ingelman-Sundberg, M., Lauschke, V. M. Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics.

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Section: Resultssupporting

confidence: 83%

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long‐term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics

et al. 2017

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“…When cultured in 2D monolayers, PHHs rapidly dedifferentiate within hours, at least in part due to wide-scale microRNA-mediated inhibition of drug-metabolizing enzymes, transporters, and other hepatic genes (Elaut et al, 2006; Lauschke et al, 2016b,c). In contrast, expression levels of most important phase I ( CYP2C8 , CYP2C9 , CYP3A4 , and CYP2D6 ) and phase II ( GSTT1 and UGT1A1 ) drug-metabolizing enzymes, drug and bile transporters ( ABCB11 and ABCC1 and SLCO1B1 ), ligand-activated nuclear receptors ( CAR , PXR , and PPARA ), and other genes with importance for hepatic functions ( ALB and HNF4A ) were preserved in 3D PHH spheroid cultures, approximating levels found in the corresponding freshly isolated cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…However, their rapid dedifferentiation in conventional two-dimensional (2D) monolayer cultures, paralleled by a loss of hepatic functionality, renders them unsuitable for long-term studies and significantly impairs their predictive power for DILI risk (Gerets et al, 2012; Lauschke et al, 2016c; Sison-Young et al, 2016; Heslop et al, 2017). To prevent dedifferentiation, an array of three-dimensional (3D) culture techniques has been developed in which hepatic phenotypes are maintained for extended periods of time (Lauschke et al, 2016a).…”

Section: Introductionmentioning

confidence: 99%

Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell–Derived Hepatocytes, HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for Drug-Induced Liver Injury

Bell¹,

Lauschke²,

Vorrink³

et al. 2017

Drug Metab Dispos

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Reliable and versatile hepatic in vitro systems for the prediction of drug pharmacokinetics and toxicity are essential constituents of preclinical safety assessment pipelines for new medicines. Here, we compared three emerging cell systems—hepatocytes derived from induced pluripotent stem cells, HepaRG cells, and three-dimensional primary human hepatocyte (PHH) spheroids—at transcriptional and functional levels in a multicenter study to evaluate their potential as predictive models for drug-induced hepatotoxicity. Transcriptomic analyses revealed widespread gene expression differences between the three cell models, with 8148 of 17,462 analyzed genes (47%) being differentially expressed. Expression levels of genes involved in the metabolism of endogenous as well as xenobiotic compounds were significantly elevated in PHH spheroids, whereas genes involved in cell division and endocytosis were significantly upregulated in HepaRG cells and hepatocytes derived from induced pluripotent stem cells, respectively. Consequently, PHH spheroids were more sensitive to a panel of drugs with distinctly different toxicity mechanisms, an effect that was amplified by long-term exposure using repeated treatments. Importantly, toxicogenomic analyses revealed that transcriptomic changes in PHH spheroids were in compliance with cholestatic, carcinogenic, or steatogenic in vivo toxicity mechanisms at clinically relevant drug concentrations. Combined, the data reveal important phenotypic differences between the three cell systems and suggest that PHH spheroids can be used for functional investigations of drug-induced liver injury in vivo in humans.

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“…However, their physiological phenotypes are lost in conventional 2D monolayer cultures due to the lack of necessary biochemical cues and cell–cell interactions as well as non-physiological biophysical properties of the culture substratum, e.g., with regards to stiffness [195,196]. As a consequence, PHH lose expression of genes characteristic for mature hepatocytes within hours of culture and acquire fetal-like phenotypes [197,198,199]. To prevent this dedifferentiation a variety of advanced 3D hepatocyte culture methodologies have been developed (extensively reviewed in reference [200]).…”

Section: Epigenetics and Inter-individual Differencesmentioning

confidence: 99%

The Importance of Patient-Specific Factors for Hepatic Drug Response and Toxicity

Lauschke

Ingelman‐Sundberg

2016

IJMS

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Responses to drugs and pharmacological treatments differ considerably between individuals. Importantly, only 50%–75% of patients have been shown to react adequately to pharmacological interventions, whereas the others experience either a lack of efficacy or suffer from adverse events. The liver is of central importance in the metabolism of most drugs. Because of this exposed status, hepatotoxicity is amongst the most common adverse drug reactions and hepatic liabilities are the most prevalent reason for the termination of development programs of novel drug candidates. In recent years, more and more factors were unveiled that shape hepatic drug responses and thus underlie the observed inter-individual variability. In this review, we provide a comprehensive overview of different principle mechanisms of drug hepatotoxicity and illustrate how patient-specific factors, such as genetic, physiological and environmental factors, can shape drug responses. Furthermore, we highlight other parameters, such as concomitantly prescribed medications or liver diseases and how they modulate drug toxicity, pharmacokinetics and dynamics. Finally, we discuss recent progress in the field of in vitro toxicity models and evaluate their utility in reflecting patient-specific factors to study inter-individual differences in drug response and toxicity, as this understanding is necessary to pave the way for a patient-adjusted medicine.

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Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation

Abstract: miRNAs are important drivers of hepatic dedifferentiation, and our results provide valuable information regarding the mechanisms behind liver regeneration and possibilities to inhibit dedifferentiation in vitro. (Hepatology 2016;64:1743-1756).

Cited by 98 publications

References 50 publications

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long‐term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics

Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long‐term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics

Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell–Derived Hepatocytes, HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for Drug-Induced Liver Injury

The Importance of Patient-Specific Factors for Hepatic Drug Response and Toxicity

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