Biology subjects

Verstegen, M.

Publications and source records attributed to Verstegen, M..

5 recordsLinked to original sources

bioRxiv2025

Tissue scarring provides a biomechanical framework to promote mammalian bile duct regeneration through the activation of integrin-SRC/FAK signalling.

Following chronic injury, the adult mammalian bile duct regenerates by forming new branches, essentially replumbing the ductular system to overcome blockages and breaks. To regenerate effectively, biliary epithelial cells (BECs) receive a range of pro-mitogenic signals from myofibroblasts, which concurrently deposit a collagen-rich scar around the duct as it regrows. Despite epithelial regeneration and scarring occurring side-by-side, whether the deposition of scar tissue regulates ductular regeneration per se remains unclear. By inducing ductular fibrosis and regeneration in vivo, we show that the formation of collagen-I-rich scars around regenerating ducts changes the local biomechanical properties of these tissues, promoting the growth of ducts. Critically, this changing structural landscape is perceived by a spatially restricted population of biliary epithelial cells which forms a "leading-tip" of integrin-2-high cells. This leading-tip undergoes partial-EMT-type reprogramming, allowing it to become migratory and coordinate ductular regeneration. We show that this process is directly driven through an integrin-2-SRC/FAK signalling axis; thereby connecting epithelial regeneration directly to the changing fibrotic environment in chronic ductular disease. HighlightsO_LIChronic liver disease results in the formation of stiff, collagen scars around ducts. C_LIO_LINew ducts acquire high levels of integrin-2 which is spatially localised to a "leading-tip", which loses epithelial features. C_LIO_LIIntegrin-2{beta}1-SRC/FAK signalling regulates ductular migration by linking changes in the bio-structural composition of the liver with ductular cells. C_LI

cell biology↗

bioRxiv2024

Novel hepatocyte-like liver organoids recapitulate crucial mature hepatic functions

Accurate liver disease modeling and drug toxicity testing still remain challenging as liver cells in vitro poorly resemble adult hepatocytes, as we previously demonstrated using whole transcriptome and cell identity analysis. To address this, we used our insights into hepatic modeling to develop hepatocyte-like liver organoids (HeLLOs), a novel human organoid model with mature hepatocyte functions superior to existing models. HeLLOs are easily established from (small) healthy or diseased liver tissues and rapidly expanded for an extended period in optimized culture conditions. Transcriptomic and functional analyses revealed that differentiated HeLLOs closely resemble fresh primary human hepatocytes (PHHs) and perform key hepatic functions such as gluconeogenesis, drug metabolism, and bile acid synthesis. We developed a HeLLO-based toxicity assay with higher sensitivity in predicting liver toxicity of known liver-toxic drugs compared to the gold-standard PHHs. By modeling disease-related mechanisms, such as bile acid transport, HeLLOs uncover transport-inhibition toxicity mechanisms of known liver toxic drugs. Single cell sequencing analysis of HeLLOs identified a heterogeneous cluster of cells with cholangiocyte-like and hepatocyte-like cells, overall resembling liver regenerative cells. As such, HeLLOs hold great promise for advancing liver disease modeling and drug testing. To our knowledge, HeLLOs are the best expandable liver model for predicting adverse drug reactions as well as modeling various liver disease mechanisms.

cell biology↗

bioRxiv2023

Integrative omics analysis reveals gene regulatory mechanisms distinguishing organoid-derived hepatocytes from primary human hepatocytes

Background and AimsHepatic organoid cultures are considered a powerful model system to study liver development and diseases in vitro. However, hepatocyte-like cells differentiated from such organoids remain immature compared to primary human hepatocytes. Therefore, a comprehensive understanding of differences in gene regulatory mechanisms between primary human hepatocytes and hepatic organoids is essential to obtain functional hepatocyte-like cells in vitro for fundamental and therapeutic applications. MethodsWe obtained primary human hepatocytes at high purity from all zones of the liver lobule using an optimized two-step perfusion protocol. We captured the single-cell transcriptome and chromatin accessibility landscape using scRNA-seq and ATAC-seq, respectively. We identified key transcription factors and compared the gene regulatory mechanisms in primary human hepatocytes and (un)differentiated intrahepatic cholangiocyte organoids. Using siRNA-mediated perturbations, we showed the functional relevance of an organoid-enriched transcription factor during in vitro differentiation of hepatocyte-like cells. ResultsOur integrative omics analysis revealed that Activator Protein 1 (AP-1) family members cooperate with hepatocyte-specific transcription factors, including HNF4A, in maintaining cellular functionality of mature human hepatocytes. Comparative analysis identified distinct transcription factor sets specifically active in human hepatocytes and organoids. Amongst these ELF3 is unique to intrahepatic cholangiocyte organoids and its expression level negatively correlate with expression of hepatic marker genes. Functional analysis of ELF3 furthermore revealed that ELF3 depletion optimizes the formation of hepatocyte-like cells from intrahepatic cholangiocyte organoids. ConclusionsCollectively, our integrative analysis provides insights into the transcriptional regulatory networks of human hepatocytes and hepatic organoids, thereby informing future strategies for better establishment of urgently-needed hepatic model systems in vitro.

molecular biology↗

bioRxiv2019

Mutational impact of chronic alcohol use on stem cells in cirrhotic liver

Excessive alcohol consumption increases the risk of developing liver cancer, but the mechanism through which alcohol drives carcinogenesis is as yet unknown. Here, we determined the mutational consequences of chronic alcohol use on the genome of human liver stem cells prior to cancer development. No change in base substitution rate or spectrum could be detected. Analysis of the trunk mutations in an alcohol-related liver tumor by multi-site whole-genome sequencing confirms the absence of specific alcohol-induced mutational signatures driving the development of liver cancer. However, we did identify an enrichment of nonsynonymous base substitutions in cancer genes in stem cells of the cirrhotic livers, such as recurrent nonsense mutations in PTPRK that disturb Epidermal Growth Factor (EGF)-signaling. Our results thus suggest that chronic alcohol use does not contribute to carcinogenesis through altered mutagenicity, but instead induces microenvironment changes which provide a fertile ground for selection of cells with oncogenic mutations.

cancer biology↗

bioRxiv2017

Locus-Specific Enhancer Hubs And Architectural Loop Collisions Uncovered From Single Allele DNA Topologies

Chromatin folding is increasingly recognized as a regulator of genomic processes such as gene activity. Chromosome conformation capture (3C) methods have been developed to unravel genome topology through the analysis of pair-wise chromatin contacts and have identified many genes and regulatory sequences that, in populations of cells, are engaged in multiple DNA interactions. However, pair-wise methods cannot discern whether contacts occur simultaneously or in competition on the individual chromosome. We present a novel 3C method, Multi-Contact 4C (MC-4C), that applies Nanopore sequencing to study multi-way DNA conformations of tens of thousands individual alleles for distinction between cooperative, random and competing interactions. MC-4C can uncover previously missed structures in sub-populations of cells. It reveals unanticipated cooperative clustering between regulatory chromatin loops, anchored by enhancers and gene promoters, and CTCF and cohesin-bound architectural loops. For example, we show that the constituents of the active b-globin super-enhancer cooperatively form an enhancer hub that can host two genes at a time. We also find cooperative interactions between further dispersed regulatory sequences of the active proto-cadherin locus. When applied to CTCF-bound domain boundaries, we find evidence that chromatin loops can collide, a process that is negatively regulated by the cohesin release factor WAPL. Loop collision is further pronounced in WAPL knockout cells, suggestive of a \"cohesin traffic jam\". In summary, single molecule multi-contact analysis methods can reveal how the myriad of regulatory sequences spatially coordinate their actions on individual chromosomes. Insight into these single allele higher-order topological features will facilitate interpreting the consequences of natural and induced genetic variation and help uncovering the mechanisms shaping our genome.

genetics↗