SEARCH · bioRxiv Science

Results for “Cell Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,513 records · Page 84Linked to original sources

bioRxiv2025

The Functional Epididymal Amyloid Cystatin-Related Epididymal Spermatogenic (CRES) is a Component of the Mammalian Brain Extracellular Matrix

CRES is the defining member of a reproductive subgroup of family 2 cystatins of cysteine protease inhibitors. We previously showed that CRES and other subgroup members are part of a highly plastic amyloid-containing extracellular matrix (ECM) with host defense functions in the mouse epididymal lumen. Based on parallels between the epididymis and the brain, we hypothesized that CRES and CRES amyloids might also function within the brain including the ECM. Here we show that CRES is produced by hippocampal neurons and astrocytes in the male and female mouse and human brain. Further, approximately 50% of hippocampal astrocytes from aged mice, like the aged human donor samples, had significantly reduced levels of CRES compared to younger mice, suggesting an age-related decline in CRES could contribute to altered brain function. Immunofluorescence experiments showed CRES colocalized with the ECM markers phosphacan and wisteria floribunda agglutinin indicating that CRES is part of the ECM. CRES monomer and high molecular weight SDS-resistant forms were found in insoluble fractions of the hippocampus, cortex, cerebellum, and midbrain and bound to the protein aggregation disease (PAD) ligand, which preferentially binds amyloids but not protein monomers, suggesting a population of CRES exists in the brain as an amyloid structure. Collectively, our studies demonstrate that CRES/CRES amyloid is present in the mammalian brain and may contribute to ECM structure and function. Significance StatementWe previously established that the cystatin-related epididymal spermatogenic (CRES) protein is part of an amyloid-containing extracellular matrix (ECM) that protects the male germline in the epididymal lumen. Here we demonstrate that CRES is present within the mouse and human brain. Using cell biological and biochemical approaches, we show that CRES is found in hippocampal astrocytes and specific neuronal populations, including those that possess perineuronal nets, and colocalized with ECM markers suggesting it is part of the ECM. Biochemical analyses suggested a population of CRES is present as an ordered amyloid structure. Our studies reveal CRES is present in the male and female mammalian brain and may contribute to brain structure and function as a biological amyloid. Keyword: hippocampus, mouse, human, plasticity

neuroscience↗

bioRxiv2025

Nuclease-NTPase systems use shared molecular features to controlbacterial anti-phage defense

Bacteria encode an enormous diversity of defense systems including restriction-modification and CRISPR-Cas that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate many recently identified anti-phage defense operons are comprised of a predicted nuclease and an accessory NTPase protein, suggesting additional classes of nucleic acid targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyze 16 nuclease-NTPase systems and define shared features that control anti-phage defense. Purification, biochemical characterization, and in vitro reconstitution of nucleic acid targeting for each system demonstrate protein-protein complex formation is a universal feature of nuclease-NTPase systems and explain patterns of phage targeting and susceptibility. We show that some nuclease-NTPase systems use highly degenerate recognition site preferences to enable exceptionally broad nucleic acid degradation. Our results uncover shared principles of anti-phage defense system function and provide a foundation to explain the widespread role of nuclease-NTPase systems in bacterial immunity.

microbiology↗

bioRxiv2025

Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

Improved methods are needed to gain insights in how proteins exert their myriad roles in cells and organs. Multiplex in situ proximity ligation assay (misPLA), described herein, can provide a window into the functional states of proteins in cells and tissues by applying pairs of antibody-oligonucleotide conjugates to generate amplifiable DNA circles upon proximal binding. The analysis reveals interactions and modifications among sets of proteins, read out by recording the identity and location of the resulting localized DNA amplification products. We applied misPLA to both primary and cultured cells and to formalin-fixated paraffin-embedded (FFPE) tissues, to map dynamic changes in protein localizations, phosphorylations and interactions across surface markers, MAPK, immune-checkpoints, T- and B-cell receptors, and adhesion panels. Comparisons of single-plex versus nine-plex assays confirmed that misPLA maintains sensitivity and specificity while increasing throughput and spatial context. Across breast cancer, lymphomas and chronic myeloid leukemia (CML) misPLA uncovered shared and disease-specific signaling patterns, underscoring convergence of oncogenic networks. By preserving tissue architecture and enabling high-content functional spatial proteomics at single-cell resolution, misPLA offers a versatile platform for dissecting signaling heterogeneity, pathway crosstalk, and therapeutic responses, with broad applications in cell biology, biomarker discovery and in precision oncology.

molecular biology↗

bioRxiv2025

FLEXTAG: A Small and Self-renewable Protein Labeling System for Anti-fading Multi-color Super-resolution Imaging

Super-resolution fluorescence imaging enables visualization of subcellular structures and molecular interactions at the nanoscale, but its broader application has been hindered by long-standing limitations in current protein tagging systems, including rapid photobleaching, tag-induced artifacts, poor post-fixation labeling efficiency, and restricted multiplexing capability. Here, we present FLEXTAG (Fluorescent Labeling for Exchangeable, X-resilient Tagging in Advanced Generic Nanoscopy), a comprehensive protein labeling system comprising three orthogonal, ultrasmall (12-18 kDa), and self-renewable protein tags that collectively overcome these major limitations of existing tagging systems, enabling optimized multi-color super-resolution imaging. Through continuous exchange of organic fluorophores, FLEXTAG supports unprecedented durations of high-resolution imaging in both live and fixed cells with minimal photobleaching. It is compatible with major super-resolution modalities, such as SIM, STED, STORM, and PAINT, and is applicable to a wide range of subcellular targets. To further address fixation-induced labeling inefficiency and background fluorescence, we developed a novel protection-based fixation method and chemical blocking strategies that significantly preserve tag accessibility and enhance signal-to-noise ratio, improvements that are broadly applicable to other protein tagging systems. Altogether, FLEXTAG enables long-term tracking of dynamic behaviors and interactions of subcellular targets, as well as mapping of nanoscale protein organizations and cellular architecture, advancing both basic research and translational applications in cell biology.

bioengineering↗

bioRxiv2025

Systematic Characterization of Optical Aberrations Reveals Cryo-FLM Localization Fidelity

Cryo-correlative light and electron microscopy (cryo-CLEM) facilitates in situ imaging and structural analysis by combining the molecular specificity of fluorescence microscopy with the ultrastructural resolution of cryo-electron microscopy. By further combining single molecule localization with cryo-CLEM, molecular positions of individual emitters can be revealed in the context of the electron density map of a cell, providing unique insights to profound questions in cell biology and virology. However, cryogenic fluorescence light microscopy (cryo-FLM) suffers from severe and spatially heterogeneous optical aberrations that distort the point spread function, limiting the accuracy of molecular localizations as well as downstream cryo-transmission electron microscopy workflows. Here, we present a systematic and quantitative analysis of optical aberrations in a commercial cryo-FLM system, uncovering the sources of significant distortions such as system imperfections, refractive index mismatches, and sample-induced heterogeneities. These system and sample induced aberrations lead to localization errors up to 90 nm laterally and over 300 nm axially, challenging the feasibility of precise molecular positioning within the vitrified specimen. We demonstrate that these errors are partially mitigated by spatially matched or adaptive point spread function models pushing the error rate down to ten nanometers or less, offering practical guidance for aberration-aware cryo-FLM and cryo-CLEM strategies. Our findings highlight the necessity of accurate, in situ point spread function modeling to achieve nanometer-scale localization in cryo-FLM. The experimental pipeline developed in this work establishes a novel tool to assess optical performance in cryo-CLEM and cryogenic focused ion beam milling workflows as the field strives toward accurate and precise molecular localization.

bioinformatics↗

bioRxiv2025

Defined human tri-lineage brain microtissues

Microglia are the immune cells of the central nervous system and are thought to be key players in both physiological and disease conditions. Several microglial features are poorly conserved between mice and human, such as the function of the neurodegeneration-associated immune receptor Trem2. Induced pluripotent stem cell (iPSC)-derived microglia offer a powerful opportunity to generate and study human microglia. However, human iPSC-derived microglia often exhibit activated phenotypes in vitro, and assessing their impact on other brain cell types remains challenging due to limitations in current co-culture systems. Here, we developed fully defined brain microtissues, composed of human iPSC-derived neurons, astrocytes, and microglia, co-cultured in 2D or 3D formats. Our microtissues are stable and self-sufficient over time, requiring no exogenous cytokines or growth factors. All three cell types exhibit morphologies characteristic of their in vivo environment and show functional properties. Co-cultured microglia develop more homeostatic phenotypes compared to microglia exposed to exogenous cytokines. Hence, these tri-cultures provide a unique approach to investigate cell-cell interactions between brain cell types. We found that astrocytes and not neurons are sufficient for microglial survival and maturation, and that astrocyte-derived M-CSF is essential for microglial survival. Single-cell and single-nucleus RNA sequencing analyses nominated a network of reciprocal communication between cell types. Brain microtissues faithfully recapitulated pathogenic -synuclein seeding and aggregation, suggesting their usefulness as human cell models to study not only normal but also pathological cell biological processes.

neuroscience↗

bioRxiv2025

Absolute Membrane Potential Recording with ASAP-Type Genetically Encoded Voltage Indicators Using Fluorescence Lifetime Imaging

The electrical membrane voltage (Vm) characterizes the functional state of biological cells, thus requiring precise, non-invasive Vm-sensing techniques. While voltage-dependent fluorescence intensity changes from genetically encoded voltage indicators (GEVIs) indicate Vm changes, variability in sensor expression confound determination of absolute Vm. Fluorescence lifetime imaging microscopy (FLIM) promises a solution to this problem, as fluorescence lifetime is expected to be unaffected by sensor expression and excitation intensity. By examining ASAP1, ASAP3, JEDI-1P, rEstus, and rEstus-NI (G138N:T141I) with one-photon excited FLIM measurements, we demonstrate that all sensors display a voltage-dependent lifetime. With the highest lifetime change in the Vm range of -100 to 50 mV of about 730 ps, ASAP3 and rEstus-NI are preferred for FLIM recordings. At a physiologically relevant Vm of -30 mV, the voltage sensitivity of rEstus-NI (6.6 ps/mV) is 3.6 and 1.4 times greater than that of ASAP1 and rEstus, respectively. As a proof of concept, we successfully used rEstus-NI to estimate absolute resting Vm in HEK293T, A375 melanoma, and MCF7 breast cancer cells and quantified spontaneous Vm fluctuations in A375 cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/669310v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1eaff8borg.highwire.dtl.DTLVardef@12f1a54org.highwire.dtl.DTLVardef@169ad83org.highwire.dtl.DTLVardef@fe9f44_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

A molecular glue: LecRKI.9 creates stiff plasma membrane cell wall connections

Water stress challenges plasma membrane - wall attachment. When plants are exposed to strong hyperosmotic stress, water exits the cell leading to plasmolysis, where the plasma membrane detaches partially from the cell wall and reveals discrete and persistent attachment sites in the form of Hechtian strands. Although these structures have been observed in several species for more than a century, the molecular bases behind plasma membrane - wall attachment remains elusive. Through a screen of candidate proteins, we reveal that the overexpression of two lectin receptor-like proteins (LecRK-I.9 and LecTM) increases the density of plasma membrane - wall connections in N. Benthamiana. The extracellular lectin domain of LecRK-I.9 was able to remain in the wall during plasmolysis. Conversely, deletion of the lectin domain in the LecRK-I.9 overexpressor restored the density of Hechtian strands to WT levels. In Arabidopsis, upon hyperosmotic stress, LecRK-I.9 formed largely immobile clusters, whereas lectin-deleted versions of LecRK-I.9 clusters were mobile. Cluster density correlated with predicted tensile stress levels and mechanical reinforcement in the wall before plasmolysis, consistent with a scenario in which attachment of the lectin domain also reflects the wall properties. Last, while overexpressing LecRK-I.9 conferred resistance to low water potential conditions, deletion of the lectin domain in the LecRK-I.9 overexpressor restored a WT response to water stress. Altogether, this demonstrates that the lectin domain of LecRK-I.9 creates persistent plasma membrane - wall attachment sites, with physiological relevance for plant resistance to water stress. SIGNIFICANCE STATEMENTAs observed since the mid-19th century, when placed in hyperosmotic conditions, plant cells undergo plasmolysis and form thin membraneous threads called Hechtian strands. These structures reflect the presence of persistent plasma membrane - cell wall attachment sites. Yet, the molecular components behind these sites are unknown. Through a candidate screen approach, we identify and demonstrate that the lectin domain of a receptor-like kinase provides such molecular glue. We also show that the clustering of the receptor is patterned and correlate with mechanically reinforced cell walls. Conversely, we find that the promotion of attachment increases resistance to water stress. This work revisits an old plant cell biology classic, and opens new avenues of research for plant adaptation to their environment.

plant biology↗

bioRxiv2025

Let there be multifunctionality: Uncovering the criticality zoo of the AC-DCgenetic circuit

Gene regulatory networks (GRNs) govern processes such as cell fate, patterning, and adaptation. While multistability and oscillations are both common GRN dynamics in cell biology, they are typically studied and engineered in isolation. Here, we challenge this separation using the AC-DC circuit, a minimal three- gene network that merges the classical toggle switch and repressilator. Using a thermodynamic formalism and Bayesian inference, we show that even a single-inducer version of the circuit can display diverse mul-tifunctional dynamics, including the coexistence of oscillations and multistability. In addition we explore robustness, classify emergent behaviours, and analyse critical slowing down and regime transitions. Re-markably, the AC-DC circuit can produce more than 30 topologically distinct bifurcation diagrams, chal-lenging the classical view that network topology rigidly constrains dynamical outcomes. This flexibility enables synthetic capabilities that couple hysteresis with oscillations, critical slowing down, and reversibility. By uncovering the hidden potential of minimal genetic circuits and outlining design principles for their implementation, this work opens new directions for harnessing emergent complexity using the basic building blocks of life.

systems biology↗

bioRxiv2025

In situ architecture of the endosymbiont Wolbachia pipientis

Hidden within host cells, the endosymbiont Wolbachia pipientis is the most prevalent bacterial infection in the animal kingdom. Scientific breakthroughs over the past century yielded fundamental mechanisms by which Wolbachia controls arthropod reproduction to shape dynamic ecological and evolutionary trajectories. However, the structure and spatial organization of symbiont machineries that underpin intracellular colonization and orchestrate maternal inheritance remain unknown. Here, we used cryo-electron tomography to directly image the nanoscale architecture of bacterial tools deployed for host manipulation and germline transmission. We discovered that Wolbachia assembles multiple structures at the host-endosymbiont interface including a filamentous ladder-like framework hypothesized to serve as a specialized motility mechanism that enables bacterial translocation to specific host cell compartments during embryogenesis and somatic tissue dissemination. In addition, we present the first in situ structure of the Rickettsiales vir homolog type IV secretion system (rvh T4SS). We provide evidence that the rvh T4SS nanomachine exhibits architectural similarities to the pED208-encoded T4SS apparatus including the biogenesis of rigid conjugative pili extending hundreds of nanometers beyond the bacterial cell surface. Coupled with integrative structural modeling, we demonstrate that in contrast to canonical T4SS architectures, the -proteobacterial T4SS outer membrane complex assembles a periplasmic baseplate structure predicted to comprise VirB9 oligomers complexed with cognate VirB10 subunits that form extended antennae projections surrounding the translocation channel pore. Collectively, these studies provide an unprecedented view into Wolbachia structural cell biology and unveil the molecular blueprints for architectural paradigms that reinforce ancient host-microbe symbioses.

microbiology↗

bioRxiv2025

Generative semantic multiplexing (SemaPlex) for accessible and scalable multiplexed fluorescence imaging

Multiplexed fluorescence imaging enhances spatially-resolved interrogation of complex, multi-molecular cell processes that are insufficiently sampled using standard 4-5 plex imaging. To improve accessibility and scalability for multiplexed imaging, we demonstrate generative Semantic Multiplexing (SemaPlex); a simple experimental and deep learning strategy for amplifying marker plexity several-fold by semantically unmixing multiple markers combined per imaging channel. We first characterise key determinants of SemaPlex performance, achieving precise computational multiplexing of 2-to-8 markers synthetically mixed in one channel, facilitating enhanced cell phenotype classification. We then demonstrate practical SemaPlex application, acquiring 10 markers over 4 channels (3*3-plex+1) to efficiently emulate real multiplexed labelling. This permitted accurate reconstruction of quantitative single-cell phenotypic manifolds delineating cell-cycle and mitotic dynamics, with internally validated error-detection. Finally, we exemplify use of semantic guides; additional input channels that significantly enhance multiplexing fidelity. SemaPlex makes multiple-fold increases in fluorescence imaging-plexity accessible, scalable and customisable; democratising multiplexed imaging-based interrogation of complex cell biology.

systems biology↗

bioRxiv2025

Structure of E. coli Twin-arginine translocase (Tat) complex with bound cargo

How the twin-arginine translocase (Tat) system transports fully folded substrate proteins across cellular membranes without disrupting membrane integrity has been a fundamental question in cell biology for decades. The Tat system recognizes cargo signal peptide via a conserved twin-arginine motif and is found in prokaryotes and plant organelles. Multi-subunit Tat complex facilitates proton motive force-dependent translocation process, yet its overall architecture remains unknown. Here, we present an atomic cryo-EM structure of a E. coli trimeric TatB3C3 complex bound to the substrate SufI. The complex adopts an unusual wide-open, bowl-shaped architecture with a polar inner cavity. Unexpectedly, the cargo is engaged in a dual-contact mode: while the signal peptide binds inside one TatBC unit, the folded domain docks tightly onto an adjacent unit. The structure offers a mechanistic framework for substrate engagement and translocation by the Tat system, suggesting a direct involvement of the entire Tat complex in substrate translocation.

biochemistry↗

bioRxiv2025

NUCGEN3D: A synthetic framework for large-scale 3D nuclear segmentation with open-source training data and models

AO_SCPLOWBSTRACTC_SCPLOWRobust nuclear segmentation in 3D microscopy images is a critical yet unresolved challenge in quantitative cell biology, hindered by the scarcity and variability of annotated volumetric datasets. Because such data are difficult to obtain, most state-of-the-art approaches, including Cellpose, segment individual 2D slices and then heuristically reconstruct 3D volumes, thereby losing critical spatial context. Our analysis of expert annotator performance confirms that ignoring 3D context introduces substantial variability in nuclear detection and annotation. While a few 3D models have been trained on small or toy datasets, no large-scale, openly available resource currently exists to enable robust training of high-capacity 3D segmentation networks. To address this, we present NucGen3D, a customizable simulation framework that generates large-scale, annotated 3D microscopy datasets from limited 2D input, specifically the 2018 Data Science Bowl dataset. NucGen3D produces realistic 3D volumes across diverse biological and imaging scenarios, including variations in nuclear morphology, spatial arrangement, acquisition artifacts, and imaging noise. Using this synthetic data, we trained two models from scratch: a 2D convolutional neural network under Cellpose-like conditions, and a fully 3D convolutional model that extends the 2D settings. We evaluated both on a challenging, independent real-world dataset with complex nuclear architectures. Both models, especially the 3D model, consistently outperformed state-of-the-art methods, including those trained on larger annotated datasets or based on more complex architectures. These results demonstrate that synthetic data can effectively substitute for real 3D annotations in training performing models at scale. To promote reproducibility and further research, we release both the NucGen3D framework and the fully trained 3D segmentation model as open source, making this the first end-to-end open resource for large-scale 3D nuclear segmentation.

bioinformatics↗

bioRxiv2025

AniMarkerDB: a comprehensive database for exploring cell types and marker genes in livestock and poultry at single-cell resolution

Single-cell RNA sequencing (scRNA-seq) has dramatically advanced the understanding of cellular heterogeneity. While numerous marker gene databases are available for humans and mice, a lack of systematic resources for livestock and poultry species remains, limiting progress in functional genomics, immunology, and breeding.. To address this challenge, we developed AniMarkerDB (https://animarkerdb.bio), a comprehensive and curated database dedicated to marker genes and immune-related epitopes in economically animals, including chicken, pig, and duck. AniMarkerDB integrates 7,010 marker gene across 37 tissues and 846 cell types, together with 71,442 immune epitope records from IEDB. All entries undergo rigorous literature curation, manual validation, and multi-level quality control, with standardized nomenclature and annotation to ensure data consistency and reusability. The platform supports flexible queries by species, tissue, cell type, or gene. It offers analytical tools for cross-species comparison model organisms such as human and mouse, interactive single-cell atlas visualization, and user-defined cell type annotation. Additionally, AniMarkerDB provides dynamic visualizations and export options, enabling researchers to efficiently obtain large-scale marker and epitope data for downstream applications such as infectious disease research, vaccine target design, and comparative immunology. Looking ahead, AniMarkerDB will expand species coverage and incorporate additional modalities, including single-cell atlases from healthy and disease models, establishing itself as a comprehensive and authoritative platform for animal cell biology, disease modeling, and translational research. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/682327v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@af7e7borg.highwire.dtl.DTLVardef@198d114org.highwire.dtl.DTLVardef@1c69ed2org.highwire.dtl.DTLVardef@e4fc43_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Systematic Discovery of Pathogen Effector Functions across Human Pathogens and Pathways

Pathogens deploy effector proteins to exploit host cell biology, and most pathogen open reading frames (ORFs) are rapidly evolving and lack functional annotation. We developed the eORFeome, a scalable functional genomics platform encompassing 3,835 effector ORFs from diverse viruses, bacteria, and parasites. High-throughput barcoded screens across NF{kappa}B, apoptosis, p53, cGAS-STING and MHC-I pathways revealed functions for hundreds of uncharacterized eORFs, unexpected new activities for known effectors, and distinct pathway-specific functions encoded by single ORFs. Illustrating the power of the approach, we identify HHV6A U14 as a p53 antagonist, HHV7 U21 as a dual-function STING antagonist and MHC-I antigen display inhibitor, and adenoviral 13.6K/i-leader protein as a de novo evolved TAP inhibitor that suppresses MHC-I display. These results establish a general framework for systematic effector annotation, uncover new mechanisms of host-pathogen interaction across kingdoms, and highlight pathogen effectors as a versatile toolkit for rewiring and probing human cellular pathways.

systems biology↗

bioRxiv2025

MANY PATHS TO DESTRUCTION: FAMILY-SPECIFIC TURNOVER AND STRESS RESPONSES FOR TRNA INTRONS

In organisms ranging from Archaea to humans, a subset of genes encoding tRNAs contain introns. Upon splicing, the tRNA exons are joined and the released free introns are rapidly degraded. Although tRNAs introns were previously considered to be "junk" sequences, we recently reported that free tRNA introns (fitRNAs) of S. cerevisiae serve as negative regulators of the cellular levels of mRNAs that bear long stretches of open reading frame sequence complementarity to tRNA introns. We also reported that 2 of the 10 families of tRNA introns, accumulate to elevated levels when cells suffer oxidative stress. The results led to the current investigations of the regulation of tRNA intron cellular levels. We document that tRNA intron turnover occurs by combinations of 5 RNA kinases, 5 to 3 and 3 to 5 exonucleases as well as by at least three endonucleases and, generally, the levels of each tRNA intron family are regulated by a unique combination of nucleases/kinases. Similarly, one family of excised intron can form circles whereas the other free tRNA intron families do not. Further, levels of individual tRNA introns differ in response to environmental conditions including type of media, stage in growth curves, and exposure to elevated temperature. Together, these findings highlight the many cellular pathways utilized to regulate tRNA intron levels and the specificity of these pathways for different tRNA families and varying cellular conditions. The results underscore the likely important roles of the newly discovered individual fitRNAs in regulation of cell biology and responses to environmental conditions.

molecular biology↗

bioRxiv2025

Derivation of primed sheep embryonic stem cells and conversion to an intermediate naive-like state

Embryonic stem cells (ESCs) derived from the inner cell mass of embryos possess unlimited self-renewal and pluripotency, offering a powerful system to study early development and enable genetic and biotechnological innovation. Although several livestock ESC lines have been reported in recent years, defining culture conditions that support stable long-term self-renewal and controlled transitions across pluripotent states remains challenging. Here, we report the de novo derivation of sheep embryonic stem cells (sESCs) from in vivo blastocysts using a chemically defined culture system. The derived cells exhibit morphological and molecular features of primed pluripotency and can be propagated under both feeder-dependent and feeder-free conditions without loss of identity or karyotypic stability. Building on this foundation, we developed enhancer-driven reporter lines that faithfully reflect OCT4 and SOX2 transcriptional activity, enabling dynamic visualization of pluripotency and differentiation in live cultures. These reporter systems revealed the responsiveness of sESCs to signaling modulation and provided a functional readout of pluripotency state transitions. When cultured in defined media previously shown to stabilize naive pluripotency in human ESCs, sESCs adopted dome-shaped colony morphology, maintained OCT4, SOX2, and NANOG expression, retained differentiation potential, and exhibited a transcriptomic profile consistent with resetting to an intermediate pluripotent state with naive-like morphological features. These findings establish stable sheep ESC lines and demonstrate their plasticity across the pluripotency spectrum, providing a valuable platform for investigating ruminant stem cell biology and advancing livestock biotechnology.

developmental biology↗

bioRxiv2017

A multi-center study on factors influencing the reproducibility of in vitro drug-response studies

Evidence that some influential biomedical results cannot be repeated has increased interest in practices that generate data meeting findable, accessible, interoperable and reproducible (FAIR) standards. Multiple papers have identified examples of irreproducibility, but practical steps for increasing reproducibility have not been widely studied. Here, seven research centers in the NIH LINCS Program Consortium investigate the reproducibility of a prototypical perturbational assay: quantifying the responsiveness of cultured cells to anti-cancer drugs. Such assays are important for drug development, studying cell biology, and patient stratification. While many experimental and computational factors have an impact on intra- and inter-center reproducibility, the factors most difficult to identify and correct are those with a strong dependency on biological context. These factors often vary in magnitude with the drug being analyzed and with growth conditions. We provide ways of identifying such context-sensitive factors, thereby advancing the conceptual and practical basis for greater experimental reproducibility.

cancer biology↗