Biology subjects

Meehan, R. R.

Publications and source records attributed to Meehan, R. R..

6 recordsLinked to original sources

bioRxiv2026

Development and characterisation of an optimised in vitro differentiation protocol for deriving hepatocyte-like cells from mouse embryonic stem cells

IntroductionReliable generation of hepatocyte-like cells (HLCs) from pluripotent stem cells remains limited by heterogeneity and incomplete maturation of the cells. Derivation of induced pluripotent- and embryonic stem cells into hepatocytes typically relies on complex, and costly reagent-intensive protocols, with inconsistent reporting of differentiation efficiencies and functional maturation criteria. Variability in protocol designs highlights the need for optimisation, particularly in mouse embryonic stem cells (mESCs) systems that can be more comparable with mouse models for underpinning translational and toxicological studies. Here, we developed and evaluated two cytokine-based strategies: an advanced hepatic-inducing cocktail (A-HIC) and a simplified hepatic-inducing cocktail (HIC), both designed to reduce complexity while increasing functional maturation. MethodsHepatic differentiation and maturation were assessed by morphology, immunofluorescence, flow cytometry, and qRT-PCR. Functional competence was evaluated via urea production, glutathione synthesis, indocyanine green handling, cytochrome P450 inducibility, and impedance-based cell layer integrity monitoring. ResultsMorphological, molecular and phenotypic analyses confirmed that both protocols supported hepatic lineage progression, generating heterogeneous populations of hepatoblast-like and more mature HLCs. Gene expression confirmed the loss of pluripotency, transient endoderm induction, and subsequent hepatic specification. Functionally, cells exhibited glycogen storage, inducible urea production, glutathione depletion, and active ICG uptake and clearance, with stable monolayer formation by day 21. A-HIC-derived HLCs demonstrated enhanced maturation, with higher ASGR1 expression and stronger Cyp1a1 induction. DiscussionThese findings suggest that both protocols generate functional HLCs; however, A-HIC yields a higher proportion of functionally mature cells with reduced variability. This approach enables a simple, cost-effective, and time-efficient generation of HLCs, supported by improved functional characterisation with potential applicability to more complex pluripotent systems, including human iPSC-based models for disease modelling and toxicology.

cell biology↗

bioRxiv2025

Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

Pericentromeric heterochromatin (PCH) is delineated by the enrichment of repressive epigenetic modifications, specifically trimethylated histone H3 at lysine 9 (H3K9me3) and DNA methylation (5-methylcytosine), which establish and maintain a condensed, transcriptionally silenced chromatin state. Depletion of either H3K9me3 or DNA methylation in mouse embryonic stem cells (mESCs) induces a permissive chromatin configuration that permits de novo recruitment and deposition of normally excluded Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2), characterized by H2AK119ub1 and H3K27me3 modifications, respectively, at PCH. Here, we demonstrate that H2AK119ub1 and H3K27me3 are independently recruited to hypomethylated PCH using a doxycycline-inducible mESC model allowing modulation of Dnmt1 expression levels and catalytic activity. We further investigate the roles of proposed mediators of PRC1/2 targeting, including SCML2, BEND3, KDM2b, and TET enzymes, in this context, our findings indicate that neither PRC1 nor PRC2 recruitment at hypomethylated PCH depends on these factors. Additionally, our data suggest that the permissive chromatin environment resulting from DNA hypomethylation is the principal facilitator of Polycomb complex spreading, offering novel insights into the mechanisms governing epigenetic modifier dynamics and interactions during periods of DNA methylation reprogramming.

molecular biology↗

bioRxiv2019

DNA methylation directs polycomb-dependent 3D genome re- organisation in naive pluripotency

The DNA hypomethylation that occurs when embryonic stem cells (ESCs) are directed to the ground state of naive pluripotency by culturing in 2i conditions results in redistribution of polycomb (H3K27me3) away from its target loci. Here we demonstrate that 3D genome organisation is also altered in 2i. We found chromatin decompaction at polycomb target loci as well as loss of long-range polycomb interactions. By preventing DNA hypomethylation during the transition to the ground-state, we are able to restore the H3K27me3 distribution, and polycomb-mediated 3D genome organisation that is characteristic of primed ESCs grown in serum, to ESCs in 2i. However, these cells retain the functional characteristics of 2i ground state ESCs. Our findings demonstrate the central role of DNA methylation in shaping major aspects of 3D genome organisation but caution against assuming causal roles for the epigenome and 3D genome in gene regulation and function in ESCs.

cell biology↗

bioRxiv2017

A reassessment of DNA immunoprecipitation-based genomic profiling

DNA immunoprecipitation sequencing (DIP-seq) is a common enrichment method for profiling DNA modifications in mammalian genomes. However, DIP-seq profiles often exhibit significant variation between independent studies of the same genome and from profiles obtained by alternative methods. Here we show that these differences are primarily due to intrinsic affinity of IgG for short unmodified DNA repeats. This pervasive experimental error accounts for 50 - 99% of regions identified as enriched for DNA modifications in DIP-seq data. Correction of this error profoundly alters DNA modification profiles for numerous cell types, including mouse embryonic stem cells, and subsequently reveals novel associations between DNA modifications, chromatin modifications and biological processes. We propose new methodological guidelines that minimize the impact of these errors on future DIP-seq experiments and allow new insights to be made from the wealth of existing DIP-seq data.

genetics↗

bioRxiv2017

Methyl donor deficient diets cause distinct alterations in lipid metabolism but are poorly representative of human NAFLD

Non-alcoholic fatty liver disease (NAFLD) is a global health issue. Dietary methyl donor restriction is used to induce a NAFLD/non-alcoholic steatohepatitis (NASH) phenotype in rodents, however the extent to which this model reflects human NAFLD remains incompletely understood. To address this, we undertook hepatic transcriptional profiling of methyl donor restricted rodents and compared these to published human NAFLD datasets.\n\nAdult C57BL/6J mice were maintained on control, choline deficient (CDD) or methionine/choline deficient (MCDD) diets for four weeks; the effects on methyl donor and lipid biology were investigated by bioinformatic analysis of hepatic gene expression profiles followed by a cross-species comparison with human expression data of all stages of NAFLD.\n\nCompared to controls, expression of the very low density lipoprotein (VLDL) packaging carboxylesterases (Ces1d, Ces1f, Ces3b) and the NAFLD risk allele Pnpla3 were suppressed in MCDD; with Pnpla3 and the liver predominant Ces isoform, Ces3b, also suppressed in CDD. With respect to 1-carbon metabolism, down-regulation of Chka, Chkb, Pcty1a, Gnmt and Ahcy with concurrent upregulation of Mat2a suggests a drive to maintain S-adenosylmethionine levels. There was minimal similarity between global gene expression patterns in either dietary intervention and any stage of human NAFLD, however some common transcriptomic changes in inflammatory, fibrotic and proliferative mediators were identified in MCDD, NASH and HCC.\n\nIn conclusion, this study suggests suppression of VLDL assembly machinery may contribute to hepatic lipid accumulation in these models, but that CDD and MCDD rodent diets are minimally representative of human NAFLD at the transcriptional level.\n\nSummary statementWe used transcriptional profiling of methyl donor restricted rodents to examine effects on methyl donor and lipid biology. We report novel mechanisms for lipid accumulation in this model and describe significant disparity between both dietary interventions and human disease.

molecular biology↗

bioRxiv2017

Tex19.1 Restricts LINE-1 Mobilisation in Mouse Embryonic Stem Cells

Mobilisation of retrotransposons to new genomic locations is a significant driver of mammalian genome evolution. In humans, retrotransposon mobilisation is mediated primarily by proteins encoded by LINE-1 (L1) retrotransposons, which mobilise in pluripotent cells early in development. Here we show that TEX19.1, which is induced by developmentally programmed DNA hypomethylation, can directly interact with the L1-encoded protein L1-ORF1p, stimulate its polyubiquitylation and degradation, and restrict L1 mobilisation. We also show that TEX19.1 likely acts, at least in part, through promoting the activity of the E3 ubiquitin ligase UBR2 towards L1-ORF1p. Moreover, we show that loss of Tex19.1 increases L1-ORF1p levels and mobilisation of L1 reporters in pluripotent mouse embryonic stem cells implying that Tex19.1 prevents new retrotransposition-mediated mutations from arising in the germline genome. These data show that post-translational regulation of L1 retrotransposons plays a key role in maintaining trans-generational genome stability in the epigenetically dynamic developing mammalian germline.

genetics↗