Biology subjects

Keren-Shaul, H.

Publications and source records attributed to Keren-Shaul, H..

14 recordsLinked to original sources

bioRxiv2026

Persistent stromal reprogramming defines incomplete mucosal healing and predicts therapeutic response in ulcerative colitis

Mucosal healing (MH) is the primary therapeutic endpoint in ulcerative colitis (UC), yet frequent relapses suggest it does not reflect complete tissue recovery. To define the basis of this vulnerability, we generated a multimodal atlas of UC integrating single-cell and bulk transcriptomics, Visium HD spatial profiling, and multiplexed imaging across 89 patients. We show that MH represents a distinct biological state marked by persistent stromal remodeling along three axes: emergence of inflammatory fibroblasts, sustained loss of OGN niche-supporting fibroblasts, and expansion of pericytes with matrix-remodeling features and reduced vascular association. Spatial analyses revealed persistent reorganization of mucosal tissue domains despite apparent clinical remission. Across independent cohorts, baseline inflammatory fibroblast and pericyte signatures robustly predicted non-response to anti-TNF therapy. These findings suggest that patients in MH remain in a biologically altered state linked to relapse risk and identify stromal reprogramming as a determinant of disease persistence and therapeutic response in UC.

immunology↗

bioRxiv2026

A stress-function tradeoff organizes epithelial heterogeneity across spatial scales in the human thyroid

Many organs are organized into repeating anatomical units, yet how cellular heterogeneity is structured within and between these units remains poorly understood. Here we use spatial transcriptomics to dissect multiscale heterogeneity in the human thyroid gland, a tissue composed of hormone-producing follicles. Across human thyroid samples spanning non-inflamed to inflamed states, we develop a follicle-aware analytical framework that separates intra-follicular from inter-follicular variability. We find that heterogeneity among thyrocytes is not dominated by differences in hormone synthesis but instead by two opposing transcriptional programs: an active hormone-producing state and a damage-response thyrocyte (DRT) state enriched for stress, immune, and damage-response pathways. DRTs are spatially clustered, associated with DNA damage markers, and are enriched near immune niches. Notably, the balance between active and damage-response programs constitutes a major axis of variability across cells, follicles, and patients. Our findings highlight a damage-response epithelial thyrocyte state that may be fundamental to follicular function in the human thyroid and provide a general framework for studying heterogeneity in tissues composed of repeating anatomical units.

systems biology↗

bioRxiv2026

Spatial analysis reveals the evolving organization of low-grade and high-grade IDH-mutant glioma

Adult diffuse gliomas are comprised of malignant cell states interwoven with the non-malignant brain microenvironment. Here we combine spatial transcriptomics and spatial proteomics of IDH-mutant gliomas to define organizational principles across histological grades. In low-grade tumors, spatial organization arises from underlying nonmalignant brain structures. For example, we classify low-grade tumor regions as embedded into white matter and identify a sharp white-grey matter junction that restricts cortical invasion and is associated with marked changes in tumor composition and cellular phenotypes. This junction is preferentially traversed by oligodendrocyte progenitor (OPC)-like malignant cells, which may drive tumor expansion. In contrast, intermediate-grade tumors are largely disorganized, with few recurring pairwise interactions between cancer cell states and TME cell types. In high-grade tumors, hypoxia/necrosis-associated global structure begins to emerge, reminiscent of IDH-wildtype glioblastoma. Together, these findings reveal two independent axes of glioma spatial organization--from brain anatomy-driven organization in low-grade tumors to hypoxia-associated structure in high-grade tumors--and establishes a framework that links tumor grade to recurrent spatial associations between cell states and cell types.

cancer biology↗

bioRxiv2026

Spatial Rewiring of Enterocyte Identity in Celiac Disease

Enterocytes in the human small intestine exhibit distinct functional states in different zones along the crypt-villus axis, a feature that is thought to convey optimal absorption. In celiac disease (CeD), autoimmune destruction of enterocytes leads to villus blunting, but how this altered tissue morphology affects enterocyte states is unclear. Using spatial and single-cell transcriptomics, we show that in patients with CeD, enterocytes acquire a novel identity characterized by co-expression of multiple zonal programs. This aberrant zonal co-expression results from reduced distances between BMP- and WNT-producing mesenchymal cells, leading to overlapping morphogen fields. In addition, we identify a subset of metaplastic cells that adopt gastric pit cell-like identities in discrete tissue patches. Our findings provide a detailed view of epithelial remodeling in CeD and establish a resource for understanding the cellular basis of malabsorption associated with villus blunting.

systems biology↗

bioRxiv2025

Single-cell quantification of senescence burden reveals cell type-specific ageing dynamics across organs

Cellular senescence, a hallmark of ageing, drives tissue dysfunction by promoting inflammation and fuelling disease. Yet, the dynamics of senescent cell accumulation across tissues and their cell type identity remain poorly understood. Here, we introduce the first, single-cell, protein-level approach, combining multiple senescence markers for the identification and quantification of senescent cells across multiple tissues in mice and in human PBMCs. Applying this method, we reveal widespread but heterogeneous changes in senescence marker expression across cell types and tissues. The cells we identify as senescent displayed transcriptomic senescence signatures, providing a direct molecular link between protein- and mRNA-level detection of senescence. Importantly, senescence accumulation was strongly coordinated within organs but showed little correlation across them, supporting the idea of a tissue specific progression of ageing. These findings refine our understanding of the tissue-specific dynamics of senescence accumulation with age, and provide a framework for evaluating diverse therapeutic interventions.

cell biology↗

bioRxiv2025

A spatial transcriptomics atlas of live donors reveals unique zonation patterns in the healthy human liver

Reconstructing gene expression atlases for human tissues is challenging due to limited access to healthy samples from live donors. Neurologically deceased donors often show ischemic changes, while tissues near diseased regions may have altered gene expression. The liver, with its unique regenerative capacity, allows analysis from live healthy donors (LHDs). Using spatial transcriptomics (Visum, Visium HD and MERFISH), we analyzed 16 liver samples: eight from young LHDs and eight from patients with liver pathology, sampling adjacent normal tissue. LHD livers displayed significant gene expression differences from adjacent normal tissues. Hepatocytes exhibited marked zonation along the porto-central axis of liver lobules, with key functions pericentrally shifted compared to other mammals. Our atlas identified dynamic programs in early steatotic hepatocytes, showing transitions from lipid uptake in low-lipid regions to insulin hypersensitivity in high-lipid regions. This study presents a spatial gene expression reference for the healthy human liver and insights into hepatocyte adaptations in steatosis.

cell biology↗

bioRxiv2024

Single-cell variations of circadian clock and immune gene expression in microglia and neurodegeneration

We investigated the diurnal rhythmicity of gene expression in microglia, the resident macrophages of the brain, in health and disease. Using RNA sequencing and single-cell analysis by RNAscope, we examined wild-type mice and the R6/2 transgenic mouse model of Huntingtons disease (HD). Our findings suggest context-dependent rhythmic gene expression in microglia, exhibiting substantial variability between individual cells and brain regions over 24 hours. Notably, we observed loss of rhythmic gene expression of key clock genes in microglia from symptomatic but not presymptomatic R6/2 mice. Moreover, we identified de novo 24-hour rhythmic gene expression and altered diurnal patterns of immune-related genes associated with neurodegenerative diseases in microglia from symptomatic R6/2 mice. Our findings suggest circadian reprogramming of microglia in the context of neurodegeneration.

neuroscience↗

bioRxiv2023

Integrative spatial analysis reveals a multi-layered organization of glioblastoma

Glioma contains malignant cells in diverse states. Hypoxic regions are associated with a unique histology of pseudopalisading cells, while other regions appear to have limited histological organization, reflecting the diffuse nature of glioma cells. Here, we combine spatial transcriptomics with spatial proteomics and novel computational approaches to define glioma cellular states at high granularity and uncover their organization. We find three prominent modes of cellular organization. First, cells in any given state tend to be spatially clustered, such that tumors are composed of small local environments that are each typically enriched with one major cellular state. Second, specific pairs of states preferentially reside in proximity across multiple scales. Despite the unique composition of each tumor, this pairing of states remains largely consistent across tumors. Third, the pairwise interactions that we detect collectively define a global architecture composed of five layers. Hypoxia appears to drive this 5-layered organization, as it is both associated with unique states of surrounding cells and with a long-range organization that extends from the hypoxic core to the infiltrative edge of the tumor. Accordingly, tumor regions distant from any hypoxic foci and tumors that lack hypoxia such as IDH-mutant glioma are less organized. In summary, we provide a conceptual framework for the organization of gliomas at the resolution of cellular states and highlight the role of hypoxia as a long-range tissue organizer.

cancer biology↗

bioRxiv2023

Transgene-Free Ex Utero Derivation of A Human Post-Implantation Embryo Model Solely from Genetically Unmodified Naive PSCs

Our ability to study early human post-implantation development remains highly limited due to the ethical and technical challenges associated with intrauterine development of the human embryo after implantation. Despite the great progress made on human gastruloids, axioloids and in vitro cultured blastoids, such elegant models do not constitute an integrated Stem cell-derived Embryo Models (SEMs) that includes all the key extra-embryonic tissues of the early post-implantation human conceptus (e.g., hypoblast, yolk-sac, trophoblasts, amnion, and extraembryonic mesoderm), and thus, do not recapitulate post-implantation epiblast development within the context of these extra-embryonic compartments. Mouse naive pluripotent stem cells (PSCs) have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation mouse SEMs, while bypassing the blastocyst-like stage, and eventually initiating organogenesis ex utero. Here, we implement critical adaptations to extend these finding to humans, while using only genetically unmodified human naive PSCs, thus circumventing the need for ectopic expression of lineage promoting transgenes. Such integrated human SEMs recapitulate the organization of all known compartments of early post-implantation stage human embryos, including epiblast, hypoblast, extra-embryonic mesoderm, and trophoblast surrounding the latter layers. The organized human SEMs recapitulate key hallmarks of post-implantation stage embryogenesis up to 13-14 days post-fertilization (dpf, Carnegie stage 6a), such as bilaminar disk formation, epiblast lumenogenesis, amniogenesis, anterior-posterior symmetry breaking, PGC specification, primary and secondary yolk sac formation, and extra-embryonic mesoderm expansion that defines a chorionic cavity and a connective stalk. This new platform constitutes a tractable stem cell-based model for experimentally interrogating previously inaccessible windows of human peri- and early post-implantation development.

developmental biology↗

bioRxiv2022

Acanthamoeba polyphaga de novo transcriptome and its dynamics during Mimivirus infection

Acanthamoeba polyphaga mimivirus (Mimivirus) is a giant virus that infects Acanthamoeba species - opportunistic human pathogens. We applied long- and short-read sequencing to generate a de novo transcriptome of the host and followed the dynamics of both host and virus transcriptomes over the course of infection. The assembled transcriptome of the host included 22,604 transcripts and 13,043 genes, with N50 = 2,372 nucleotides. Functional enrichment analysis revealed major changes in the host transcriptome, namely, enrichment in downregulated genes associated with cytoskeleton homeostasis and DNA replication, repair, and nucleotide synthesis. These modulations, together with those implicated by other enriched processes, indicate cell cycle arrest, an event we demonstrated experimentally. We also observed upregulation of host genes associated with transcription, secretory pathways and, as reported here for the first time, peroxisomes and the ubiquitin-proteasome system. In Mimivirus, the early stages of infections were marked by upregulated genes related to DNA replication, transcription, translation, and nucleotide metabolism, and the later stages, by enrichment in genes associated with lipids metabolism, carbohydrates, and proteases. Some of the changes observed in the amoebal transcriptome likely point to Mimivirus infection causing the dismantling of the host cytoskeleton, the translocation of endoplasmic reticulum membranes to viral factory areas, and cell cycle arrest.

microbiology↗

bioRxiv2021

Multi-resolution deconvolution of spatial transcriptomics data reveals continuous patterns of inflammation

The function of mammalian cells is largely influenced by their tissue microenvironment. Advances in spatial transcriptomics open the way for studying these important determinants of cellular function by enabling a transcriptome-wide evaluation of gene expression in situ. A critical limitation of the current technologies, however, is that their resolution is limited to niches (spots) of sizes well beyond that of a single cell, thus providing measurements for cell aggregates which may mask critical interactions between neighboring cells of different types. While joint analysis with single-cell RNA-sequencing (scRNA-seq) can be leveraged to alleviate this problem, current analyses are limited to a discrete view of cell type proportion inside every spot. This limitation becomes critical in the common case where, even within a cell type, there is a continuum of cell states that cannot be clearly demarcated but reflects important differences in the way cells function and interact with their surroundings. To address this, we developed Deconvolution of Spatial Transcriptomics profiles using Variational Inference (DestVI), a probabilistic method for multi-resolution analysis for spatial transcriptomics that explicitly models continuous variation within cell types. Using simulations, we demonstrate that DestVI is capable of providing higher resolution compared to the existing methods and that it can estimate gene expression by every cell type inside every spot. We then introduce an automated pipeline that uses DestVI for analysis of single tissue slices and comparison between tissues. We apply this pipeline to study the immune crosstalk within lymph nodes to infection and explore the spatial organization of a mouse tumor model. In both cases, we demonstrate that DestVI can provide a high resolution and accurate spatial characterization of the cellular organization of these tissues, and that it is capable of identifying important cell-type-specific changes in gene expression - between different tissue regions or between conditions. DestVI is available as an open-source software package in the scvi-tools codebase (https://scvi-tools.org).

bioinformatics↗

bioRxiv2020

Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors

The connection between different tissues is vital for the development and function of any organs and systems. In the musculoskeletal system, the attachment of elastic tendons to stiff bones poses a mechanical challenge that is solved by the formation of a transitional tissue, which allows the transfer of muscle forces to the skeleton without tearing. Here, we show that tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, which is regulated by sharing regulatory elements with these cells and by Kruppel-like factors transcription factors (KLF). To uncover the molecular identity of attachment cells, we first applied high-throughput RNA sequencing to murine humeral attachment cells. The results, which were validated by in situ hybridization and single-molecule in situ hybridization, reveal that attachment cells express hundreds of chondrogenic and tenogenic genes. In search for the underlying mechanism allowing these cells to express these genes, we performed ATAC sequencing and found that attachment cells share a significant fraction of accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis further revealed transcriptional enhancer signatures for the majority of these regions. We then examined a subset of these regions using transgenic mouse enhancer reporter. Results verified the shared activity of some of these enhancers, supporting the possibility that the transcriptome of attachment cells is regulated by enhancers with shared activities in tenocytes or chondrocytes. Finally, integrative chromatin and motif analyses, as well as the transcriptome data, indicated that KLFs are regulators of attachment cells. Indeed, blocking the expression of Klf2 and Klf4 in the developing limb mesenchyme led to abnormal differentiation of attachment cells, establishing these factors as key regulators of the fate of these cells. In summary, our findings show how the molecular identity of bi-fated attachment cells enables the formation of the unique transitional tissue that connect tendon to bone. More broadly, we show how mixing the transcriptomes of two cell types through shared enhancers and a dedicated set of transcription factors can lead to the formation of a new cell fate that connects them.

developmental biology↗

bioRxiv2019

Transneuronal interactions facilitate axonal compartment formation

The mechanisms controlling wiring of neuronal networks are largely unknown. The stereotypic architecture of the Drosophila mushroom-body (MB) offers a unique system to study circuit assembly. The adult medial MB {gamma}-lobe is comprised of a long bundle of axons that wires with specific modulatory and output neurons in a tiled manner defining five distinct zones. We found that the immunoglobulin superfamily protein Dpr12 is cell-autonomously required in {gamma}-neurons for their developmental regrowth into the distal {gamma}4/5 zones, where both Dpr12 and its interacting protein, DIP-{delta}, are enriched. DIP-{delta} functions in a subset of dopaminergic neurons that wire with {gamma}-neurons within the {gamma}4/5 zone. During metamorphosis, these dopaminergic projections arrive to the {gamma}4/5 zone prior to {gamma}-axons, suggesting that {gamma}-axons extend through a prepatterned region. Thus, Dpr12/DIP-{delta} transneuronal interaction is required for {gamma}4/5 zone formation. Our study sheds light onto molecular and cellular mechanisms underlying circuit formation within subcellular resolution.

neuroscience↗

bioRxiv2019

Pan-cancer single cell RNA-seq uncovers recurring programs of cellular heterogeneity

Cultured cell lines are the workhorse of cancer research, but it is unclear to what extent they recapitulate the cellular heterogeneity observed among malignant cells in tumors, given the absence of a native tumor microenvironment. Here, we used multiplexed single cell RNA-seq to profile ~200 cancer cell lines. We uncovered expression programs that are recurrently heterogeneous within many cancer cell lines and are largely independent of observed genetic diversity. These programs of heterogeneity are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-to-mesenchymal transition, and protein maturation and degradation. Notably, some of these recurrent programs recapitulate those seen in human tumors, suggesting a prominent role of intrinsic plasticity in generating intra-tumoral heterogeneity. Moreover, the data allowed us to prioritize specific cell lines as model systems of cellular plasticity. We used two such models to demonstrate the dynamics, regulation and drug sensitivities associated with a cancer senescence program also observed in human tumors. Our work describes the landscape of cellular heterogeneity in diverse cancer cell lines, and identifies recurrent patterns of expression heterogeneity that are shared between tumors and specific cell lines and can thus be further explored in follow up studies.

cancer biology↗