Biology subjects

Hagemann-Jensen, M.

Publications and source records attributed to Hagemann-Jensen, M..

18 recordsLinked to original sources

bioRxiv2026

Single-cell proteomics reveals cell-type-specific functional coordination in PBMCs

The coordination of molecular networks defines cellular functions. This is reflected in molecular covariation within a cell type, which is more subtle than the differences separating cell types and has therefore been difficult to quantify. To achieve the depth, consistency and accuracy required to resolve such covariation, we leveraged single-cell proteomics (plexDIA) and transcriptomics (Smart-seq3xpress) to analyze the proteomes and transcriptomes of thousands of single peripheral blood mononuclear cells (PBMCs). plexDIA quantified more protein-coding gene products per cell with higher data completeness while Smart-seq3xpress quantified more gene products across all cells. The protein measurements revealed cell-type-specific proteome architecture -- shaped in part by protein stability, and complex coordination -- that was undetectable in our mRNA data. Specifically, protein covariation within a cell type suggests cell-type-specific protein-protein interactions and functional rewiring of biological pathways independent of previously characterized abundance differences. Covariation analysis within cell types resolves a B cell-specific axis of translational states inversely covarying with GDF6 cytokine abundance. Our framework captures cell-type-specific functional coordination representing a distinct information layer accessible via single-cell proteomics.

systems biology↗

bioRxiv2026

Lipid-ASO therapeutics exhibit differential tissue targeted delivery upon systemic or local CNS administration

Antisense oligonucleotides (ASOs) are a powerful therapeutic modality, but their full potential is hindered by pharmacokinetic properties that affect tissue and cellular delivery. Lipid conjugation is increasingly used to modulate ASO's biodistribution and promote extrahepatic activity, yet lipid dependent effects on in vivo functional delivery, particularly in the central nervous system (CNS), remain less explored. Here, we performed a side by side in vivo comparison of cholesterol, palmitic acid (C16:0), docosanoic acid (C22:0), and eicosapentaenoic acid (C20:5) conjugated to a fully phosphorothioated 3 10 3 LNA gapmer ASO targeting the Malat1 long non coding RNA. Lipid-ASO conjugates were administered systemically or locally in the brain of mice and evaluated for tissue level and cellular level distribution by imaging, qPCR and single-cell RNA sequencing, simultaneously annotating cell origin and global transcriptional changes within the cell. Following systemic administration in mice, lipid conjugation improved overall multi organ efficacy compared to unconjugated ASO, but with pronounced tissue specific differences. Single cell sequencing of liver and heart transcriptomes revealed lipid dependent cellular uptake patterns and transcriptional responses distinct from administration of unconjugated ASO. After intracerebroventricular administration, selected fatty acid conjugates enhanced silencing in deep brain regions such as the striatum, whereas cholesterol conjugation impaired functional delivery despite increased CNS retention. Light-sheet microscopy showed restricted parenchymal penetration of cholesterol ASOs compared with broader but heterogeneous distribution of palmitic acid conjugate. Together, these findings demonstrate that lipid identity critically determines ASO efficacy, productive cellular uptake, and regional CNS engagement, emphasizing the need for context specific lipid design in ASO therapeutic development.

pharmacology and toxicology↗

bioRxiv2026

Immediate transcriptional changes initiated by direct cell-cell contact between cytotoxic T cells and cancer cells

Many biological processes are regulated by the direct interaction between two or more cell types. However, our understanding of the immediate dynamic changes in gene transcription upon physical interaction between two cells has remained limited due to technical limitations. Here we address these limitations in a model system of cancer-specific T cell receptor (TCR)-modified CD8 T cells where single and cancer-interacting T cells were isolated by image-enabled cell sorting and transcripts from heterotypic cancer cell and T cell pairs were in silico assigned. This approach uncovers immediate, dynamic changes in gene expression following the specific interaction between TCR-modified CD8 T cells and cancer cells. In addition to dissecting transcriptional cascades dependent on the peptide sensitivity of the TCR, we for the first time directly compare these gene expression changes between single T cells and T cells in direct physical contact with cancer cells. Modeling of the observed transcriptomic activation signature identifies phenotypically distinct tumor infiltrating CD8 T cell subsets associated with reduced TCR diversity in in vivo datasets. Taken together, the paradigm developed here allows for future clonal identification of T cell receptors mediating ongoing effective cytotoxic responses in vivo.

immunology↗

bioRxiv2025

RNA BaseCode: Long-Read RNA Sequences with Short-Read Accuracy

RNA sequencing has revolutionized the life sciences by enabling comprehensive profiling of gene expression across tissues and individual cells. Most mammalian genes produce multiple isoforms with distinct functional consequences, yet their accurate detection and quantification remain challenging. Here we present RNA BaseCode, a synthetic long-read RNA sequencing method that combines the high accuracy and quantitative precision of short-read sequencing with internal random barcodes to reconstruct full-length RNA sequences. We applied RNA BaseCode to purified RNA to benchmark its performance against short-read and long-read RNA sequencing methods. RNA BaseCode demonstrated superior basecalling accuracy, higher quantitative precision, and an improved ability to reconstruct full-length transcripts compared to long-read sequencing. This method enables comprehensive characterization of RNA isoform expression at scale, providing a powerful and broadly applicable tool for investigating isoform regulation across biological contexts as well as the roles of these isoforms in health and disease.

genomics↗

bioRxiv2025

A novel method to sort and enrich sensory neurons

Peripheral sensory neurons, residing in the dorsal root ganglia (DRG), relay sensory information from the periphery to the central nervous system. Although single-cell transcriptomic studies have identified over 20 distinct sensory neuron subtypes, functional analysis and assessment of subtype-specific pathological changes remain difficult. Effective isolation and enrichment of sensory neurons are challenging yet essential for functional studies. Therefore, we used single-cell transcriptomic data from DRG to identify a panel of neuronal surface markers, including Nrxn2 and Pirt. Using these markers, we developed a fluorescence-activated cell sorting (FACS) panel for neuronal enrichment and analysis that does not rely on transgenic mouse strains and can be broadly applied. The panel was validated by microscopy and single-cell RNA (scRNA) sequencing, which also revealed broad representation of neuronal subtypes. Expression of these markers in human DRG underscores the translational value of this isolation method for sensory and pain studies. Overall, this study provides a valuable tool for isolating DRG neurons, advancing research on sensory neuron function and pain biology, and facilitating neuroimmune studies.

neuroscience↗

bioRxiv2025

Plasmodium falciparum subverts neutrophil function via host miR-451a loaded extracellular vesicles driving bacterial superinfection susceptibility

Malaria caused by Plasmodium falciparum (Pf) compromise innate immunity, yet the underlying mechanisms remain elusive. The immune dysregulation caused by the parasite may lead to bacterial superinfections and increase mortality. We reveal that Pf exploits extracellular vesicles (EVs) secreted by infected red blood cells (iRBC-EVs) to deliver host-derived miR451a to human neutrophils, impairing their antimicrobial defences. Neutrophil phagocytosis of iRBC-EVs suppresses reactive oxygen species (ROS) production and compromised microbicidal activity against Salmonella typhimurium. Microfluidic assays show that miR451a transfer significantly disrupts neutrophil chemotaxis and swarming upon microbial challenge. Transcriptomic profiling indicates that EVs and miR451a reprogram neutrophil gene expression, notably upregulating ferroptosis-related genes, suggesting a role in further impairing immune responses. We have uncovered a novel mechanism of iRBC-EVs-induced neutrophil immune suppression and provide insights into increased susceptibility to bacterial superinfections in malaria. These findings have implications for therapeutic strategies aimed at mitigating bacterial superinfections and sepsis in malaria-endemic regions.

immunology↗

bioRxiv2024

Spontaneously regenerative corticospinal neurons in mice

The spinal cord receives inputs from the cortex via corticospinal neurons (CSNs). While predominantly a contralateral projection, a less-investigated minority of its axons terminate in the ipsilateral spinal cord. We analyzed the spatial and molecular properties of these ipsilateral axons and their post-synaptic targets in mice and found they project primarily to the ventral horn, including directly to motor neurons. Barcode-based reconstruction of the ipsilateral axons revealed a class of primarily bilaterally-projecting CSNs with a distinct cortical distribution. The molecular properties of these ipsilaterally-projecting CSNs (IP-CSNs) are strikingly similar to the previously described molecular signature of embryonic-like regenerating CSNs. Finally, we show that IP-CSNs are spontaneously regenerative after spinal cord injury. The discovery of a class of spontaneously regenerative CSNs may prove valuable to the study of spinal cord injury. Additionally, this work suggests that the retention of juvenile-like characteristics may be a widespread phenomenon in adult nervous systems.

neuroscience↗

bioRxiv2024

Multi-layered dosage compensation of the avian Z chromosome

Sex-chromosome dosage represents a challenge for heterogametic species to maintain correct proportion of gene products across chromosomes in each sex. While therian mammals (XX/XY system) achieve near-perfect balance of X-chromosome mRNAs through X-upregulation and X-inactivation, birds (ZW/ZZ system) have been found to lack efficient compensation at RNA level, challenging the necessity of resolving major gene-dosage discrepancies in avian cells. Through allele-resolved multiome analyses, we comprehensively examined dosage compensation in female (ZW), male (ZZ), and rare intersex (ZZW) chicken. Remarkably, this revealed that females exhibit upregulation of their single Z through increased transcriptional burst frequency similar to mammalian X-upregulation, and that Z-protein levels are further balanced via enhanced translation efficiency in females. Global analyses of transcriptional kinetics elements in birds demonstrate remarkable conservation of the genomic encoding of burst kinetics between mammals and birds. Our study uncovers new mechanisms for achieving sex-chromosome dosage compensation and highlights the importance of gene-dosage balance across diverse species.

molecular biology↗

bioRxiv2024

Regionalized cell and gene signatures govern oesophageal epithelial homeostasis

Regionalized disease prevalence is a common feature of the gastrointestinal tract. Herein, we employed regionally resolved Smart-seq3 single-cell sequencing, generating a comprehensive cell atlas of the adult mouse oesophagus. Characterizing the oesophageal axis, we unveil non-uniform distribution of epithelial basal cells, fibroblasts and immune cells. In addition, we reveal a position-dependent, but cell subpopulation-independent, transcriptional signature, collectively generating a regionalized oesophageal landscape. Combining in vivo models with organoid co-cultures, we demonstrate that proximal and distal basal progenitor cell states are functionally distinct. We find that proximal fibroblasts are more permissive for organoid growth compared to distal fibroblasts and that the immune cell profile is regionalized in two dimensions, where proximal-distal and epithelial-stromal gradients impact epithelial maintenance. Finally, we predict and verify how WNT-, BMP-, IGF-and NRG-signalling are differentially engaged along the oesophageal axis. We establish a cellular and transcriptional framework for understanding oesophageal regionalization, providing a functional basis for epithelial disease susceptibility.

cell biology↗

bioRxiv2023

Erythroid differentiation intensifies RNA mis-splicing in SF3B1-mutant myelodysplastic syndromes with ring sideroblasts

Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly originate from mutations in the splicing factor SF3B1 (SF3B1mt). SF3B1mt cause RNA mis-splicing, mechanistically established as the major driver of RS development. However, little is known about RS fate and biology after their initial formation in the human bone marrow. We here achieve isolation of viable RS from patient samples, enabling the first complete investigation of SF3B1mt development from stem cell to RS. We show that RS skew MACS-isolated CD34+ data towards erythroid features not recapitulated in single-cell RNAseq. We demonstrate that RS divide, differentiate, enucleate and actively respond to mis-splicing/oxidative stress, decreasing wildtype stem cell fitness via GDF15 overproduction. We identify circulating RS as a uniform clinical feature associated with disease burden. Finally, we establish that SF3B1mt mis-splicing intensifies during erythroid differentiation and demonstrate through combined transcriptomics/proteomics an uncoupling of RNA/protein biology in RS encompassing severe and dysfunctional mis-splicing of proapoptotic genes. Statement of significanceWe here combine a novel method for RS isolation with state-of-the-art multiomics to perform the first complete investigation of SF3B1mt MDS-RS hematopoiesis from stem cell to RS. We identify the survival mechanisms underlying SF3B1mt erythropoiesis and establish an active role for erythroid differentiation and RS themselves in SF3B1mt MDS-RS pathogenesis.

cancer biology↗

bioRxiv2022

Spatial transcriptomics of T and B cell receptors uncovers lymphocyte clonal dynamics in human tissue.

The spatial distribution of lymphocyte clones within tissues is critical to their development, selection, and expansion. We have developed Spatial Transcriptomics of VDJ sequences (Spatial VDJ), which maps immunoglobulin and TR antigen receptors in human tissue sections. Spatial VDJ captures lymphocyte clones matching canonical T, B, and plasma cell distributions in tissues and amplifies clonal sequences confirmed by orthogonal methods. We confirm spatial congruency between paired receptor chains, develop a computational framework to predict receptor pairs, and link the expansion of distinct B cell clones to different tumor-associated gene expression programs. Spatial VDJ delineates B cell clonal diversity, class switch recombination, and lineage trajectories within their spatial context. Taken together, Spatial VDJ captures lymphocyte spatial clonal architecture across tissues, which could have important therapeutic implications. One-Sentence SummarySpatial transcriptomics-based technology co-captures T and B cell receptors within their anatomical niche in human tissue.

immunology↗

bioRxiv2022

Clonally heritable gene expression imparts a layer of diversity within cell types

Cell types can be classified based on shared patterns of transcription. Variability in gene expression between individual cells of the same type has been ascribed to stochastic transcriptional bursting and transient cell states. We asked whether long-term, heritable differences in transcription can impart diversity within a cell type. Studying clonal human lymphocytes and mouse brain cells, we uncover a vast diversity of heritable transcriptional states among different clones of cells of the same type in vivo. In lymphocytes we show that this diversity is coupled to clone specific chromatin accessibility, resulting in distinct expression of genes by different clones. Our findings identify a source of cellular diversity, which may have important implications for how cellular populations are shaped by selective processes in development, aging and disease.

systems biology↗

bioRxiv2021

Molecular spikes: a gold standard for single-cell RNA counting

Molecule counting is central to single-cell sequencing, yet no experimental strategy to evaluate counting performance exists. Here, we introduce molecular spikes, novel RNA spike-ins containing inbuilt unique molecular identifiers that we use to identify critical experimental and computational conditions for accurate RNA counting across single-cell RNA-sequencing methods. The molecular spikes are a new gold standard that can be widely used to validate RNA counting in single cells.

genomics↗

bioRxiv2021

Scalable full-transcript coverage single cell RNA sequencing with Smart-seq3xpress

Plate-based single-cell RNA-sequencing methods with full-transcript coverage typically excel at sensitivity but are more costly and time-consuming. Here, we miniaturized and streamlined the Smart-seq3 protocol for drastically reduced cost and increased throughput. Applying Smart-seq3xpress to 16,349 human peripheral blood mononuclear cells revealed a highly granular atlas complete with both common and rare cell types whose identification previously relied on additional protein measurements or the integration with a reference atlas.

genomics↗

bioRxiv2021

PhylEx: Accurate reconstruction of clonal structure via integrated analysis of bulk DNA-seq and single cell RNA-seq data

We propose PhylEx: a clonal-tree reconstruction method that integrates bulk genomics and single-cell transcriptomics data. In addition to the clonal-tree, PhylEx also assigns single-cells to clones, which effectively produce clonal expression profiles, and generates clonal genotypes. By analyzing scRNA-seq integrated with bulk DNA-seq, PhylEx can take advantage of co-occurrences of the mutations found in the cells. In the probabilistic model underlying PhylEx, the raw read counts from scRNA-seq follow a mixture of Beta-Binomial distributions, which accounts for the sparse nature of single-cell gene expression data; the mixture lessens the penalty caused by mutations not observed due to mono-allelic expression. We rigorously evaluated PhylEx on simulated datasets as well as a biological dataset consisting of a previously well-characterized high-grade serous ovarian cancer (HGSOC) cell line. PhylEx outperformed the state-of-the-art methods by a wide margin both when comparing capacity for clonal-tree reconstruction and capacity for correctly clustering mutations. By analyzing HGSOC and HER2+ breast cancer data, we also show that PhylEx clears the way for phylo-phenotypic analysis of cancer, i.e., that the clonal expression profiles, induced by the cell-to-clone assignments, can be exploited in a manner beyond what is possible with only expression-based clustering.

bioinformatics↗

bioRxiv2020

Profiling of small non-coding RNAs across cellular and biofluid compartments: implications for multiple sclerosis immunopathology

Multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS), is associated with dysregulation of microRNAs (miRNA). We here analyzed all classes of small non-coding RNAs (sncRNAs) in matching peripheral blood mononuclear cells (PBMCs), plasma, cerebrospinal fluid (CSF) cells and cell-free CSF from relapsing-remitting (RRMS, n=12 in relapse, n=11 in remission), secondary progressive (SPMS, n=6) MS patients and non-inflammatory and inflammatory neurological disease controls (NINDC, n=11; INDC, n=5). We show widespread changes in small nuclear, nucleolar, transfer RNAs and miRNAs. In CSF cells, 133/133 and 115/117 differentially expressed sncRNAs are increased in RRMS relapse compared to remission and RRMS compared to NINDC, respectively. In contrast, 65/67 differentially expressed PBMC sncRNAs are decreased in RRMS compared to NINDC. The striking contrast between periphery and CNS suggests that sncRNA-mediated mechanisms, including alternative splicing, RNA degradation and mRNA translation, regulate the transcriptome of pathogenic cells primarily in the target organ.

molecular biology↗

bioRxiv2020

Deducing transcriptional kinetics and molecular functions of long non-coding RNAs using allele-sensitive single-cell RNA-sequencing

An increasing number of studies have demonstrated the regulatory importance of long non-coding RNAs (lncRNAs), yet little is known about their transcriptional dynamics and it remains challenging to determine their regulatory functions. Here, we used allele-sensitive single-cell RNA-seq (scRNA-seq) to demonstrate that lncRNAs have lower burst frequencies with twice as long duration between bursts, compared to mRNAs. Additionally, we observed an increased cell-to-cell variability in lncRNA expression that was due to more sporadic bursting (lower frequency) with larger numbers of RNA molecules being produced. Exploiting heterogeneity in asynchronously growing cells, we identified and experimentally validated lncRNAs with cell-state specific functions involved in cell cycle progression and apoptosis. Finally, utilizing allele-resolved RNA expression, we identified cis functioning lncRNAs and observed that knockdown of these lncRNAs modulated either transcriptional burst frequency or size of the nearby protein-coding gene. Collectively, our results identify distinct transcriptional regulation of lncRNAs and we demonstrate a role for lncRNAs in the regulation of transcriptional bursting of mRNAs.

genomics↗

bioRxiv2019

Single-cell RNA counting at allele- and isoform-resolution using Smart-seq3

Large-scale sequencing of RNAs from individual cells can reveal patterns of gene, isoform and allelic expression across cell types and states1. However, current single-cell RNA-sequencing (scRNA-seq) methods have limited ability to count RNAs at allele- and isoform resolution, and long-read sequencing techniques lack the depth required for large-scale applications across cells2,3. Here, we introduce Smart-seq3 that combines full-length transcriptome coverage with a 5 unique molecular identifier (UMI) RNA counting strategy that enabled in silico reconstruction of thousands of RNA molecules per cell. Importantly, a large portion of counted and reconstructed RNA molecules could be directly assigned to specific isoforms and allelic origin, and we identified significant transcript isoform regulation in mouse strains and human cell types. Moreover, Smart-seq3 showed a dramatic increase in sensitivity and typically detected thousands more genes per cell than Smart-seq2. Altogether, we developed a short-read sequencing strategy for single-cell RNA counting at isoform and allele-resolution applicable to large-scale characterization of cell types and states across tissues and organisms.

genomics↗