Biology subjects

Marks, H.

Publications and source records attributed to Marks, H..

10 recordsLinked to original sources

bioRxiv2026

Correlative SHG-AFM imaging workflow for label-free quantitative analysis of collagen structure-function relationships

We present a user-friendly correlative second harmonic generation (SHG) and atomic force microscopy (AFM) imaging workflow for quantifying the nanomechanical properties of collagen in unfixed, unlabeled tissue sections. SHG Aligned Profiling for Elasticity and Segmentation or SHAPES utilizes SHG imaging to guide AFM force mapping, enabling label-free, anatomically specific selection of regions of interest, facilitating spatially resolved characterization of fibrous collagen morphology and local stiffness. Compared to brightfield or confocal contrast-guided AFM mapping, SHG improves anatomical specificity without fixation or staining, enabling downstream analysis on the same tissue section while preserving spatial correspondence between structural (SHG) and mechanical (AFM) data. Rapid identification of regions of interest also reduces the risk of costly AFM tip breakage, improving throughput and reducing operator burden. Utilizing only standard turn-key commercial systems common in many user facilities and clinical laboratories, multimodal image coregistration and automated identification of anatomical regions of interest are employed to integrate SHG and AFM datasets across complex sample topographies. Coregistration of SHG and AFM images substantially increases the number of usable datasets per measurement session, facilitating translation of these complementary modalities and bridging nanomechanical imaging with clinical research practice.

biophysics↗

bioRxiv2025

Immunotherapy with Pro-regenerative Macrophages from Embryonic Stem Cells Ameliorate Osteoarthritis via TNFAIP3-Mediated Chondroprotection

Immune cell-based therapies for osteoarthritis (OA) remains underexplored. Macrophages coordinate tissue repair, yet their functional heterogeneity and therapeutic applicability, particularly for designed stem cell-derived populations with tailored reparative properties are not fully delineated. Inspired by embryonic skeletal development as a paradigm of scarless regeneration, we identified a pro-regenerative macrophage population through a single-cell RNA atlas of developing skeletal tissues. We subsequently established a mechanically dynamic induction approach to efficiently generate pro-regenerative macrophages from human embryonic stem cells (hESCs), mimicking this developmental phenotype. In vitro, these macrophages mitigate cartilage degradation in OA explants and suppress pro-inflammatory, catabolic signaling in chondrocytes. Injection into murine OA joints robustly attenuates disease progression, reduces extracellular matrix breakdown, and resolves synovial inflammation, representing the first demonstration of immune cell-based therapy for OA. Mechanistically, these macrophages orchestrate chondroprotection not through broad immunosuppression, but by activating a TNFAIP3-dependent signaling hub that restrains inflammatory and catabolic responses in chondrocytes. This TNFAIP3 activation precisely reversed the OA-associated gene signature, suppressing catabolic mediators while enhancing reparative factors, thereby restoring the anabolic-catabolic balance. Our findings uncover a developmental blueprint for engineering pro-regenerative macrophages from stem cells and establish a pioneering targeted immune cell therapy that engages an intrinsic chondroprotective program, offering a transformative and translationally relevant strategy for OA treatment.

immunology↗

bioRxiv2025

Methylome profiling of SetDB1 deficient ESCs reveals diverse epigenetic cross-talk during pluripotency

SetDB1 is best known as a chromatin modifier catalyzing H3K9me3. However, recent studies show that SetDB1 can promote H3K27me3-deposition and CTCF-binding, and potentially recruit de novo DNA methyltransferases. Given the tight connection with these processes, we hypothesized that DNA methylation (DNAme) may integrate these combined features of SetDB1. Thereto, we conducted time-course whole-genome bisulfite sequencing following Setdb1 knockout (KO) in mouse embryonic stem cells (ESCs). In serum-cultured ESCs, nearly half of SetDB1 binding sites are DNA methylated, coinciding with H3K9me3, mainly silencing retrotransposons and imprinting control regions. Both H3K9me3 and DNAme are lost upon Setdb1 KO, but while TET2 rapidly removes DNAme at many of these sites, some retrotransposons are shielded from TET2 and lose DNAme slowly via passive dilution. SetDB1-mediated regulation via H3K27me3, CTCF, SMAD3, and histone acetylation are uncoupled from the DNAme-H3K9me3 axis. Hypomethylated naive ESCs show massive reactivation of retrotransposons upon Setdb1 KO, providing functional evidence that DNAme adds a protective layer against such activity. Altogether, our findings reveal how DNAme coordinates the multifaceted regulatory roles of SetDB1. HighlightsO_LIIn serum ESCs, SetDB1-dependent deposition of H3K9me3 and DNAme are tightly coupled, primarily silencing repeats and imprinted control regions; C_LIO_LILoss of SetDB1 causes demethylation of a large range of repeat types, the pace of which is dependent on TET pre-loading; C_LIO_LIThe regulatory modes of SetDB1 mediated by H3K27me3, CTCF, TGF-{beta} signalling and histone acetylation are uncoupled from the SetDB1 DNAme-H3K9me3 axis and/or from each other; C_LIO_LILoss of SetDB1-dependent DNAme in hypomethylated 2i ESCs reveals that DNAme serves as a buffering layer to repress SetDB1-mediated H3K9me3 targets. C_LI

molecular biology↗

bioRxiv2025

Double stranded RNA sensing drives interferon silencing in early development

In early mammalian development, the type I interferon (IFN) response is inactive, only becoming functional after gastrulation. As a result, the totipotent and pluripotent embryonic stages remain highly susceptible to pathogens, including viruses. Here, we show that pluripotent mouse embryonic stem cells (mESCs) suppress the RIG-I-like receptor sensing pathway by silencing the expression of the dsRNA sensor MDA5. We show that this silencing is required to avoid the recognition of dsRNAs from endogenous origin, which only accumulate in mESCs. Reintroducing MDA5 results in recognition of these endogenous dsRNAs, and activation of the IFN response through IRF3. The production of IFN alters the differentiation ability of mESCs, as well as the pluripotency gene expression program, as shown by epigenetic, transcriptomic and proteomic analyses. These findings are conserved in zebrafish, where MDA5 is also expressed at later stages of development. Similarly, zebrafish lack early-stage IFN activation and premature IFN signalling results in developmental defects. Altogether, we conclude that silencing the RIG-I-like receptor pathway during early development is the widely conserved and is required to prevent aberrant immune recognition of endogenous dsRNAs, safeguarding normal development.

cell biology↗

bioRxiv2024

The exit of naïve pluripotency contains a lipid metabolism-induced checkpoint for genome integrity.

Pluripotent progenitors undergo dramatic cellular and biochemical transformations during peri-implantation development. These large-scale reprogramming events are fundamental for subsequent differentiation, but how they are integrated and co-ordinated with the preservation of genome integrity remain unknown. Here, we uncover a metabolism-induced telomere checkpoint that takes place in pluripotent progenitors as they form rosette-like epithelial structures. We show that the glycolytic switch at the exit of naive pluripotency is preceded by an acceleration of mitochondrial respiration and de novo lipogenesis, fuelling the accumulation of lipid droplets required for morphogenesis. We find that downstream of these CIDEA-promoted metabolic events is the induction of ZSCAN4, a key pluripotency-associated regulator of telomere stability. Surprisingly, the build-up of lipid droplets corresponds to a transient shortening of telomeres, which triggers the activation of an elongation mechanism via ZSCAN4. Thus, telomere homeostasis can be safeguarded as essential lipid metabolic reprogramming unfolds to drive developmental progression.

developmental 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↗

bioRxiv2020

A plug and play microfluidic platform for standardized sensitive low-input Chromatin Immunoprecipitation

Epigenetic profiling by ChIP-Seq has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks and for screening for biomarkers. However, the ChIP-Seq protocol for low-input samples is laborious, time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput. Although prototypic microfluidic ChIP-Seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelisation. To enable standardized, automated ChIP-Seq profiling of low-input samples, we constructed PDMS-based plates containing microfluidic Integrated Fluidic Circuits capable of performing 24 sensitive ChIP reactions within 30 minutes hands-on time. These disposable plates can conveniently be loaded into a widely available controller for pneumatics and thermocycling, making the ChIP-Seq procedure Plug and Play (PnP). We demonstrate high-quality ChIP-seq on hundreds to few thousands of cells for multiple widely-profiled post-translational histone modifications, together allowing genome-wide identification of regulatory elements. As proof of principle, we managed to generate high-quality epigenetic profiles of rare totipotent subpopulations of mESCs using our platform. In light of the ready-to-go ChIP plates and the automated workflow, we named our procedure PnP-ChIP-Seq. PnP-ChIP-Seq allows non-expert labs worldwide to conveniently run robust, standardized ChIP-Seq, while its high-throughput, consistency and sensitivity paves the way towards large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies. Reviewer link to dataAll sequencing data has been submitted to the NCBI GEO database. Reviewer link: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=klwnocicrpaxrkv&acc=GSE120673

genetics↗

bioRxiv2019

Critical role for P53 in regulating the cell cycle of ground state embryonic stem cells

Mouse Embryonic Stem Cells (ESCs) grown in serum-supplemented conditions are characterized by an extremely short G1-phase due to the lack of G1-phase control. Concordantly, the G1-phase-specific P53-P21 pathway is compromised in serum ESCs. Here we provide evidence that P53 is activated upon transition of serum ESCs to their pluripotent ground state using serum-free 2i conditions and modulates G1-phase progression. Our data shows that the elongated G1-phase characteristic of ground state ESCs is dependent on P53. RNA-seq and ChIP-seq analyses reveal that P53 directly regulates the expression of the Retinoblastoma (RB) protein and that the hypo-phosphorylated, active RB protein plays a key role in G1-phase control. Our findings suggest that the P53-P21 pathway is active in ground state 2i ESCs and that its role in the G1-checkpoint is abolished in serum ESCs. Taken together, the data reveals a mechanism by which inactivation of P53 can lead to loss of RB and uncontrolled cell proliferation.

molecular biology↗

bioRxiv2019

Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

Polycomb Repressive Complex 2 (PRC2) plays an essential role in development by catalysing trimethylation of histone H3 lysine 27 (H3K27me3), resulting in gene repression. PRC2 consists of two sub-complexes, PRC2.1 and PRC2.2, in which the PRC2 core associates with distinct ancillary subunits such as MTF2 and JARID2, respectively. Both MTF2, present in PRC2.1, and JARID2, present in PRC2.2, play a role in core PRC2 recruitment to target genes in mouse embryonic stem cells (mESCs). However, it remains unclear how these distinct sub-complexes cooperate to establish Polycomb domains. Here, we combine a range of Polycomb mutant mESCs with chemical inhibition of PRC2 catalytic activity, to systematically dissect their relative contributions to PRC2 binding to target loci. We find that PRC2.1 and PRC2.2 mediate two distinct paths for recruitment, with mutually reinforced binding. Part of the cross-talk between PRC2.1 and PRC2.2 occurs via their catalytic product H3K27me3, which is bound by the PRC2 core-subunit EED, thereby mediating a positive feedback. Strikingly, removal of either JARID2 or H3K27me3 only has a minor effect on PRC2 recruitment, whereas their combined ablation largely attenuates PRC2 recruitment. This strongly suggests an unexpected redundancy between JARID2 and EED-H3K27me3-mediated recruitment of PRC2. Furthermore, we demonstrate that all core PRC2 recruitment occurs through the combined action of MTF2-mediated recruitment of PRC2.1 to DNA and PRC1-mediated recruitment of JARID2-containing PRC2.2. Both axes of binding are supported by EED-H3K27me3 positive feedback, but to a different degree. Finally, we provide evidence that PRC1 and PRC2 mutually reinforce reciprocal binding. Together, these data disentangle the interdependent and cooperative interactions between Polycomb complexes that are important to establish Polycomb repression at target sites.\n\nHighlightsO_LISystematic analysis of Polycomb complex binding to target loci in mESCs using null mutations and chemical inhibition.\nC_LIO_LIPRC1, PRC2.1 and PRC2.2 are all mutually dependent for binding to chromatin, mediated in part by H3K27me3.\nC_LIO_LIPRC2.1 recruitment is dependent on MTF2\nC_LIO_LIPRC2.2 recruitment by JARID2 is dependent on PRC1 and largely redundant with recruitment by H3K27me3\nC_LI

molecular biology↗

bioRxiv2016

Tracking the embryonic stem cell transition from ground state pluripotency

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition of ES cells. The population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naive pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state exhibit global transcriptome features consistent with features of early post-implantation epiblast and distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naive cells transition to a discrete formative phase of pluripotency preparatory to lineage priming.\n\nHighlightsO_LIThe Rex1 destabilized GFP reporter demarcates naive pluripotency.\nC_LIO_LIExit from the naive state is asynchronous in the population.\nC_LIO_LITransition is relatively acute in individual cells and precedes lineage priming.\nC_LIO_LITranscriptome and DNA methylome reflect events in the pre-gastrulation embryo.\nC_LI

developmental biology↗