Biology subjects

Heyn, H.

Publications and source records attributed to Heyn, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Macrophage and neutrophil heterogeneity at single-cell spatial resolution in inflammatory bowel disease

Ulcerative colitis (UC) and Crohns disease (CD) are chronic inflammatory intestinal diseases that show a perplexing heterogeneity in manifestations and response to treatment. The molecular basis for this heterogeneity remains uncharacterized. We applied single-cell RNA sequencing and CosMx Spatial Molecular Imaging to human colon and found the highest diversity in cellular composition in the myeloid compartment of UC and CD patients. Besides resident macrophage subsets (M0 and M2), patients showed a variety of activated macrophages including classical (M1 CXCL5 and M1 ACOD1) and new inflammation-dependent alternative (IDA) macrophages. In addition, we captured intestinal neutrophils in three transcriptional states. Subepithelial IDA macrophages expressed NRG1, which promotes epithelial differentiation. In contrast, NRG1low IDA macrophages were expanded within the submucosa and in granulomas, in proximity to abundant inflammatory fibroblasts, which we suggest may promote macrophage activation. We conclude that macrophages sense and respond to unique tissue microenvironments, potentially contributing to patient-to-patient heterogeneity.

immunology↗

bioRxiv2022

The effect of background noise and its removal on the analysis of single-cell expression data

BACKGROUNDIn droplet-based single-cell and single-nucleus RNA-seq experiments, not all reads associated with one cell barcode originate from the encapsulated cell. Such background noise is attributed to spillage from cell-free ambient RNA or barcode swapping events. Here, we characterize this background noise exemplified by three single-cell RNA-seq (scRNA-seq) and two single-nucleus RNA-seq (snRNA-seq) replicates of mouse kidney cells. For each experiment, kidney cells from two mouse subspecies were pooled, allowing to identify cross-genotype contaminating molecules and estimate the levels of background noise. RESULTSWe find that background noise is highly variable across replicates and individual cells, making up on average 3-35% of the total counts (UMIs) per cell and show that this has a considerable impact on the specificity and detectability of marker genes. In search of the source of background noise, we find that expression profiles of cell-free droplets are very similar to expression profiles of cross-genotype contamination and hence that the majority of background molecules originates from ambient RNA. Finally, we use our genotype-based estimates to evaluate the performance of three methods (CellBender, DecontX, SoupX) that are designed to quantify and remove background noise. We find that CellBender provides the most precise estimates of background noise levels and also yields the highest improvement for marker gene detection. By contrast, clustering and classification of cells are fairly robust towards background noise and only small improvements can be achieved by background removal that may come at the cost of distortions in fine structure. CONCLUSIONOur findings help to better understand the extent, sources and impact of background noise in single-cell experiments and provide guidance on how to deal with it.

bioinformatics↗

bioRxiv2022

β-catenin activity induces an RNA biosynthesis program promoting therapy resistance in T Acute Lymphoblastic Leukemia

Understanding the molecular mechanisms that contribute to the appearance of chemotherapy resistant cell populations is necessary to improve cancer treatment. We have now investigated the role of {beta}-catenin/CTNNB1 in the evolution of T-Acute Lymphoblastic Leukemia (T-ALL) patients and its involvement in therapy resistance. We have identified a specific gene signature that is directly regulated by {beta}-catenin, TCF/LEF factors and ZBTB33/Kaiso in T-ALL cell lines, which is highly and significantly represented in 5 out of 6 refractory patients from a cohort of 40 children with T-ALL. By subsequent refinement of this gene signature, we found that a subset of {beta}-catenin target genes involved with RNA-processing function are sufficient to segregate T-ALL refractory patients in three independent cohorts. We demonstrate the implication of {beta}-catenin in RNA and protein synthesis in T-ALL and provide experimental evidence that {beta}-catenin is crucial for the cellular response to chemotherapy, mainly in the cellular recovery phase after treatment. We propose that combination treatments involving chemotherapy plus {beta}-catenin inhibitors will enhance chemotherapy response and prevent disease relapse in T-ALL patients.

cancer biology↗

bioRxiv2022

An Atlas of Cells in the Human Tonsil

Palatine tonsils are secondary lymphoid organs representing the first line of immunological defense against inhaled or ingested pathogens. Here, we present a comprehensive census of cell types forming the human tonsil by applying single-cell transcriptome, epigenome, proteome and adaptive immune repertoire sequencing as well as spatial transcriptomics, resulting in an atlas of >357,000 cells. We provide a glossary of 121 annotated cell types and states, and disentangle gene regulatory mechanisms that drive cells through specialized lineage trajectories. Exemplarily, we stratify multiple tonsil-resident myeloid slancyte subtypes, establish a distant BCL6 superenhancer as locally active in both follicle-associated T and B cells, and describe SIX5 as a potentially novel transcriptional regulator of plasma cell maturation. Further, our atlas is a reference map to understand alterations observed in disease. Here, we discover immune-phenotype plasticity in tumoral cells and microenvironment shifts of mantle cell lymphomas (MCL). To facilitate such reference-based analysis, we develop HCATonsilData and SLOcatoR, a computational framework that provides programmatic and modular access to our dataset; and allows the straightforward annotation of future single-cell profiles from secondary lymphoid organs.

immunology↗

bioRxiv2022

A comprehensive single-cell expression atlas of human AML leukemia-initiating cells unravels the contribution of HIF pathway and its therapeutic potential

Relapse remains a major challenge in the clinical management of acute myeloid leukemia (AML), and is driven by rare therapy-resistant leukemia-initiating stem cells (LSCs) that reside in specific bone marrow niches. Hypoxia signaling keeps cells in a quiescent and metabolically relaxed state, desensitizing them to chemotherapy. This suggests the hypothesis that hypoxia contributes to AML-LSC function and chemoresistance and is a therapeutic target to sensitize AML-LSCs to chemotherapy. Here, we provide a comprehensive single-cell expression atlas (119,000 cells) of AML cells and AML-LSCs in paired diagnostic-relapse samples from risk-stratified patients with AML. The HIF/hypoxia pathway is attenuated in AML-LSCs compared with differentiated AML cells, but is enhanced when compared with healthy hematopoietic cells. Accordingly, chemical inhibition cooperates with standard-of-care chemotherapy to impair leukemogenesis, substantially eliminating AML-LSCs. These findings support the HIF pathway as a stem cell regulator in human AML, and reveal avenues for combinatorial targeted and chemotherapy-based approaches to specifically eliminate AML-LSCs.

cancer biology↗

bioRxiv2022

Local IL-17 orchestrates skin aging

Skin aging is characterized by structural and functional changes that lead to slower wound healing and higher rate of infections, which contribute to age-associated frailty. This likely depends on synergy between alterations in the local microenvironment and stem cell-intrinsic changes, underscored by pro-inflammatory microenvironments that drive pleotropic changes. To date, little is known about the precise nature and origin of the proposed age-associated inflammatory cues, or how they affect different tissue resident cell types. Based on deep single-cell RNA-sequencing of the entire dermal compartment, we now provide a comprehensive understanding of the age-associated changes in all skin cell types. We show a previously unreported skew towards an IL-17-expressing phenotype of Th cells, {gamma}{delta} T cells and innate lymphoid cells in aged skin. Aberrant IL-17 signaling is common to many autoimmune (e.g., rheumatoid arthritis and psoriasis) and chronic inflammatory diseases. Importantly, in vivo blockade of IL-17-triggered signaling during the aging process reduces the pro-inflammatory state by affecting immune and non-immune skin cells of both dermis and epidermis. Strikingly, IL-17 neutralization significantly delays the appearance of age-related traits, such as decreased epidermal thickness, increased cornified layer thickness and ameliorated hair follicle stem cell activation and hair shaft regeneration. Our results indicate that the aged skin shows chronic and persistent signs of inflammation, and that age-associated increased IL-17 signaling could be targeted as a strategy to prevent age-associated skin ailments in elderly.

cell biology↗

bioRxiv2021

Single-Cell Atlas of Common Variable Immunodeficiency reveals germinal center-associated epigenetic dysregulation in B cell responses

Common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, is characterized by impaired terminal B-cell differentiation and defective antibody responses. Incomplete genetic penetrance and a wide range of phenotypic expressivity in CVID suggest the participation of additional pathogenic mechanisms. Monozygotic (MZ) twins discordant for CVID are uniquely valuable for studying the contribution of epigenetics to the disease. We used single-cell epigenomics and transcriptomics to create a cell census of naive-to-memory B cell differentiation in a pair of CVID-discordant MZ twins. Our analysis identifies DNA methylation, chromatin accessibility and transcriptional defects in memory B cells that mirror defective cell-cell communication defects following activation. These findings were validated in a cohort of CVID patients and healthy donors. Our findings provide a comprehensive multi-omics map of alterations in naive-to-memory B-cell transition in CVID and reveal links between the epigenome and immune cell cross-talk. Our resource, publicly available at the Human Cell Atlas, paves the way for future diagnosis and treatments of CVID patients.

immunology↗

bioRxiv2021

A temporally controlled sequence of X-chromosomeinactivation and reactivation defines female mouse in vitro germ cells with meiotic potential

The early mammalian germ cell lineage is characterized by extensive epigenetic reprogramming, which is required for the maturation into functional eggs and sperm. In particular, the epigenome needs to be reset before parental marks can be established and then transmitted to the next generation. In the female germ line, reactivation of the inactive X-chromosome is one of the most prominent epigenetic reprogramming events, and despite its scale involving an entire chromosome affecting hundreds of genes, very little is known about its kinetics and biological function. Here we investigate X-chromosome inactivation and reactivation dynamics by employing a tailor-made in vitro system to visualize the X-status during differentiation of primordial germ cell-like cells (PGCLCs) from female mouse embryonic stem cells (ESCs). We find that the degree of X-inactivation in PGCLCs is moderate when compared to somatic cells and characterized by a large number of genes escaping full inactivation. Nevertheless, PGCLCs that fail to undergo X-inactivation show an abnormal gene expression signature and deficiencies in meiotic entry. Subsequent to X-inactivation we observe gradual step-wise X-reactivation, which is mostly completed by the end of meiotic prophase I. Cells deviating from these progressive kinetics and undergoing X-reactivation too rapidly fail to enter a meiotic trajectory. Our data reveals that a fine-tuned X-inactivation and -reactivation cycle is a critical feature of female germ cell developmental competence towards meiosis and oogenesis

developmental biology↗

bioRxiv2021

Glioblastoma cell fate is differentially regulated by the microenvironments of the tumour bulk and infiltrative margin

Glioblastoma recurrence originates from invasive cells at the tumour margin that escape surgical debulking, but their biology remains poorly understood. Here we generated three somatic mouse models recapitulating the main glioblastoma driver mutations to characterise margin cells. We find that, regardless of genetics, tumours converge on a common set of neural- like cellular states. However, bulk and margin display distinct neurogenic patterns and immune microenvironments. The margin is immune-cold and preferentially follows developmental-like trajectories to produce astrocyte-like cells. In contrast, injury-like programmes dominate in the bulk, are associated with immune infiltration and generate lowly-proliferative injured neural progenitor-like (iNPCs) cells. In vivo label-retention approaches further demonstrate that iNPCs account for a significant proportion of dormant glioblastoma cells and are induced by interferon signalling within T-cell niches. These findings indicate that tumour region is a major determinant of glioblastoma cell fate and therapeutic vulnerabilities identified in bulk may not extend to the margin residuum.

cancer biology↗

bioRxiv2021

Siglec-1 on dendritic cells mediates SARS-CoV-2 trans-infection of target cells while on macrophages triggers proinflammatory responses

COVID-19 pandemic is not yet under control by vaccination, and effective antivirals are critical for preparedness. Here we report that macrophages and dendritic cells, key antigen presenting myeloid cells (APCs), are largely resistant to SARS-CoV-2 infection. APCs effectively captured viruses within cellular compartments that lead to antigen degradation. Macrophages sense SARS-CoV-2 and released higher levels of cytokines, including those related to cytokine storm in severe COVID-19. The sialic acid-binding Ig-like lectin 1 (Siglec-1/CD169) present on APCs, which interacts with sialylated gangliosides on membranes of retroviruses or filoviruses, also binds SARS-CoV-2 via GM1. Blockage of Siglec-1 receptors by monoclonal antibodies reduces SARS-CoV-2 uptake and transfer to susceptible target cells. APCs expressing Siglec-1 and carrying SARS-CoV-2 are found in pulmonary tissues of non-human primates. Single cell analysis reveals the in vivo induction of cytokines in those macrophages. Targeting Siglec-1 could offer cross-protection against SARS-CoV-2 and other enveloped viruses that exploit APCs for viral dissemination, including those yet to come in future outbreaks.

microbiology↗

bioRxiv2021

HASTER is a transcriptional stabilizer of HNF1A

The biological purpose and disease relevance of long noncoding RNAs (lncRNAs) is poorly understood. We examined HASTER, a lncRNA antisense to HNF1A. Haploinsufficient mutations in HNF1A, encoding a homeodomain transcription factor, cause diabetes mellitus. Using mouse and human models, we show that HASTER maintains HNF1A at cell-specific physiological concentrations through positive and negative feedback loops. Haster mutant pancreatic {beta} cells thus showed variegated HNF1A overexpression or silencing, causing insulin-deficiency and diabetes. We demonstrate that the HASTER promoter acts in cis to prevent HNF1A overexpression and silencing, and link HASTER-dependent inhibition to local remodelling of 3D chromatin architecture. We further show that HASTER negative feedback ensures that HNF1A creates open chromatin at appropriate cell-specific genome regions. Our studies expose a cis-regulatory element that is unlike enhancers or silencers, and instead stabilizes expression levels of a pioneer transcription factor. They also show that disruption of a mammalian lncRNA can cause diabetes mellitus.

genomics↗

bioRxiv2020

A Single-Cell Tumor Immune Atlas for Precision Oncology

The tumor immune microenvironment is a main contributor to cancer progression and a promising therapeutic target for oncology. However, immune microenvironments vary profoundly between patients and biomarkers for prognosis and treatment response lack precision. A comprehensive compendium of tumor immune cells is required to pinpoint predictive cellular states and their spatial localization. We generated a single-cell tumor immune atlas, jointly analyzing >500,000 cells from 217 patients and 13 cancer types, providing the basis for a patient stratification based on immune cell compositions. Projecting immune cells from external tumors onto the atlas facilitated an automated cell annotation system for a harmonized interpretation. To enable in situ mapping of immune populations for digital pathology, we applied SPOTlight, combining single-cell and spatial transcriptomics data and identifying striking spatial immune cell patterns in tumor sections. We expect the tumor immune cell atlas, together with our versatile toolbox for precision oncology, to advance currently applied stratification approaches for prognosis and immuno-therapy.

cancer biology↗

bioRxiv2020

Single cell analysis reveals the impact of age and maturation stage on the human oocyte transcriptome

Study questionTo which degree does maternal age affect the transcriptome of human oocytes at the germinal vesicle (GV) stage or at metaphase II after maturation in vitro (IVM-MII)? Summary answerWhile the oocytes transcriptome is predominantly determined by maturation stage, transcript levels of genes related to chromosome segregation, mitochondria and RNA processing are affected by age after in vitro maturation of denuded oocytes. What is known alreadyFemale fertility is inversely correlated with maternal age due to both a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, and the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown GV and IVM-MII oocytes from women of varying reproductive age. Study design, size, durationIn this prospective molecular study, 37 women were recruited from May 2018 to June 2019. The mean age was 28.8 years (SD=7.7, range 18-43). A total of 72 oocytes were included in the study at GV stage after ovarian stimulation, and analyzed as GV (n=40) and in vitro matured oocytes (IVM-MII; n=32). Participants/materials, setting, methodsDenuded oocytes were included either as GV at the time of ovum pick-up or as IVM-MII after in vitro maturation for 30 hours in G2 medium, and processed for transcriptomic analysis by single-cell RNA-seq using the Smart-seq2 technology. Cluster and maturation stage marker analysis were performed using the Seurat R package. Genes with an average fold change greater than 2 and a p-value < 0.01 were considered maturation stage markers. A Pearson correlation test was used to identify genes whose expression levels changed progressively with age. Those genes presenting a correlation value (R) >= |0.3| and a p-value < 0.05 were considered significant. Main results and the role of chanceFirst, by exploration of the RNA-seq data using tSNE dimensionality reduction, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM-MII) with 4,445 and 324 putative marker genes, respectively. Next we identified genes, for which RNA levels either progressively increased or decreased with age. This analysis was performed independently for GV and IVM-MII oocytes. Our results indicate that the transcriptome is more affected by age in IVM-MII oocytes (1,219 genes) than in GV oocytes (596 genes). In particular, we found that genes involved in chromosome segregation and RNA splicing significantly increase in transcript levels with age, while genes related to mitochondrial activity present lower transcript levels with age. Gene regulatory network analysis revealed potential upstream master regulator functions for genes whose transcript levels present positive (GPBP1, RLF, SON, TTF1) or negative (BNC1, THRB) correlation with age. Limitations, reasons for cautionIVM-MII oocytes used in this study were obtained after in vitro maturation of denuded GV oocytes, therefore, their transcriptome might not be fully representative of in vivo matured MII oocytes. The Smart-seq2 methodology used in this study detects polyadenylated transcripts only and we could therefore not assess non-polyadenylated transcripts. Wider implications of the findingsOur analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM-MII stages. Nonetheless, hundreds of genes displayed altered transcript levels with age, particularly in IVM-MII oocytes. Especially affected by age were genes related to chromosome segregation and mitochondrial function, pathways known to be involved in oocyte ageing. Our study thereby suggests that misregulation of chromosome segregation and mitochondrial pathways also at the RNA-level might contribute to the age-related quality decline in human oocytes. Study funding/competing interest(s)This study was funded by the AXA research fund, the European commission, intramural funding of Clinica EUGIN, the Spanish Ministry of Science, Innovation and Universities, the Catalan Agencia de Gestio dAjuts Universitaris i de Recerca (AGAUR) and by contributions of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and to the "Centro de Excelencia Severo Ochoa". The authors have no conflict of interest to declare.

developmental biology↗

bioRxiv2020

SPOTlight:Seeded NMF regression to Deconvolute Spatial Transcriptomics Spots with Single-Cell Transcriptomes

The integration of orthogonal data modalities greatly supports the interpretation of transcriptomic landscapes in complex tissues. In particular, spatially resolved gene expression profiles are key to understand tissue organization and function. However, spatial transcriptomics (ST) profiling techniques lack single-cell resolution and require a combination with single-cell RNA sequencing (scRNA-seq) information to deconvolute the spatially indexed datasets. Leveraging the strengths of both data types, we developed SPOTlight, a computational tool that enables the integration of ST with scRNA-seq data to infer the location of cell types and states within a complex tissue. SPOTlight is centered around a seeded non-negative matrix factorization (NMF) regression, initialized using cell-type marker genes, and non-negative least squares (NNLS) to subsequently deconvolute ST capture locations (spots). Using synthetic spots, simulating varying reference quantities and qualities, we confirmed high prediction accuracy also with shallowly sequenced or small-sized scRNA-seq reference datasets. We trained the NMF regression model with sample-matched or external datasets, resulting in accurate and sensitive spatial predictions. SPOTlight deconvolution of the mouse brain correctly mapped subtle neuronal cell states of the cortical layers and the defined architecture of the hippocampus. In human pancreatic cancer, we successfully segmented patient sections into healthy and cancerous areas, and further fine-mapped normal and neoplastic cell states. Trained on an external pancreatic tumor immune reference, we charted the localization of clinical-relevant and tumor-specific immune cell states. Using SPOTlight to detect regional enrichment of immune cells and their co-localization with tumor and adjacent stroma provides an illustrative example in its flexible application spectrum and future potential in digital pathology.

bioinformatics↗

bioRxiv2020

Sampling artifacts in single-cell genomics cohort studies

Robust protocols and automation now enable large-scale single-cell RNA and ATAC sequencing experiments and their application on biobank and clinical cohorts. However, technical biases introduced during sample acquisition can hinder solid, reproducible results and a systematic benchmarking is required before entering large-scale data production. Here, we report the existence and extent of gene expression and chromatin accessibility artifacts introduced during sampling and identify experimental and computational solutions for their prevention.

genomics↗

bioRxiv2019

A TBX5 dosage-sensitive gene regulatory network for human congenital heart disease

Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD). However, underlying CHD gene regulatory network (GRN) imbalances are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD-linked transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5-dependent pathways-- including lineage decisions and genes associated with cardiomyocyte function and CHD genetics--in discrete subpopulations of cardiomyocytes. GRN analysis identified vulnerable nodes enriched for CHD genes, indicating that cardiac network stability is sensitive to TBX5 dosage. A GRN-predicted genetic interaction between Tbx5 and Mef2c was validated in mouse, manifesting as ventricular septation defects. These results demonstrate exquisite sensitivity to TBX5 dosage by diverse transcriptional responses in heterogeneous subsets of iPSC-derived cardiomyocytes. This predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.

developmental biology↗

bioRxiv2019

A Human Accelerated Region participates in early human forebrain patterning and expansion

The expansion of the mammalian brain is associated with specific developmental processes; however, not much is known about how evolutionary changes participated in the acquisition of human brain traits during early developmental stages. Here we investigated whether enhancers active during the phylotypic stage show human-specific genomic divergence which could contribute to the evolutionary expansion of the forebrain. Notably, we identified an active enhancer containing a human accelerated region (HAR) located in the Chromosome 14q12, a region enriched with neurodevelopmental genes, such as Foxg1, Nkx2.1 and Nova1. Reporter analysis revealed that the human variant is active in the forebrain in transgenic mice and that it has stronger enhancer activity than the mouse or chimpanzee versions. Humanization of the mouse enhancer variant in transgenic mice and in mouse organoids resulted in an expansion of Foxg1 expressing domains in the forebrain early neural progenitors with a bias towards dorsal identities. Overall, our results suggest that human-specific mutations in critical regulatory elements controlling early brain development impact the expansion and patterning of the forebrain.

developmental biology↗

bioRxiv2019

Robustness and applicability of functional genomics tools on scRNA-seq data

Many tools have been developed to extract functional and mechanistic insight from bulk transcriptome profiling data. With the advent of single-cell RNA sequencing (scRNA-seq), it is in principle possible to do such an analysis for single cells. However, scRNA-seq data has characteristics such as drop-out events, low library sizes and a comparatively large number of samples/cells. It is thus not clear if functional genomics tools established for bulk sequencing can be applied to scRNA-seq in a meaningful way. To address this question, we performed benchmark studies on in silico and in vitro single-cell RNA-seq data. We included the bulk-RNA tools PROGENy, GO enrichment and DoRothEA that estimate pathway and transcription factor (TF) activities, respectively, and compared them against the tools AUCell and metaVIPER, designed for scRNA-seq. For the in silico study we simulated single cells from TF/pathway perturbation bulk RNA-seq experiments. Our simulation strategy guarantees that the information of the original perturbation is preserved while resembling the characteristics of scRNA-seq data. We complemented the in silico data with in vitro scRNA-seq data upon CRISPR-mediated knock-out. Our benchmarks on both the simulated and real data revealed comparable performance to the original bulk data. Additionally, we showed that the TF and pathway activities preserve cell-type specific variability by analysing a mixture sample sequenced with 13 scRNA-seq different protocols. Our analyses suggest that bulk functional genomics tools can be applied to scRNA-seq data, outperforming dedicated single cell tools. Furthermore we provide a benchmark for further methods development by the community.

bioinformatics↗