Biology subjects

Linnarsson, S.

Publications and source records attributed to Linnarsson, S..

At least 19 recordsLinked to original sources

bioRxiv2026

System-wide extraction of cis-regulatory rules from sequence-to-function models in human neural development

The genomic cis-regulatory code (CRC) underlies spatiotemporal specificity of gene expression. While sequence-to-function (S2F) models can accurately encode the CRC of transcriptional enhancers, decoding these models into human-interpretable rules remains a major challenge. Here we tackle this challenge in human neural development, for which we generate two new single-cell multiome atlases, one from a human embryo and one from neural tube organoids. We use this comparative framework to robustly extract combinations of transcription factor (TF) binding sites that are necessary and sufficient to design enhancers. As such we extract cis-regulatory rules for dorsal-ventral progenitors, neural crest, mesenchyme and neurons. To enable this, we develop a new strategy and computational package, called TF-MINDI, to embed, cluster, and annotate candidate TF binding sites, and to extract combinatorial rules for each cell type. We evaluate rule-based models in conjunction with blackbox S2F models through simulations, evolutionary comparisons with zebrafish, topic modeling, and enhancer reporter-assays. Our findings show robust and interpretable rule extraction and constitute a step forward in deciphering, explaining, and formalizing the CRC. TF-MINDI is available at: https://github.com/aertslab/TF-MINDI.

bioinformatics↗

bioRxiv2025

Organotypic Timelapse recording with Transcriptomic Readout (OTTR) links cell behaviour to cell identity in human tissues

Linking dynamic cellular behaviour to molecular states in intact human tissue remains challenging because during live imaging only limited molecular information can be captured while high-dimensional molecular measurements are destructive. Here we describe Organotypic Timelapse recording with Transcriptomic Readout (OTTR), which integrates week-long live imaging of sparsely labelled organotypic slice cultures with highly multiplexed in situ spatial transcriptomics. We applied OTTR to primary human glioblastoma and fetal cortical tissues. Using sparse labelling, we tracked the migration, proliferation, and lineage of tens of thousands of individual cells per sample. Following live imaging, precision resectioning and alignment allowed us to perform spatial transcriptomics on the very same tissue, thereby preserving the link between dynamic cell behaviours and transcriptomic states. We used OTTR to quantify cell-type specific migration patterns, lineage trees and the behaviour of cells near vasculature. OTTR provides a powerful, broadly applicable method for investigating the complex interplay between cell behaviour and molecular state in human tissues.

molecular biology↗

bioRxiv2025

Cell atlas of the developing human meninges reveals a dura origin of meningioma

The vertebrate central nervous system is enveloped by the meninges, consisting of the pia, arachnoid, and dura layers. The arachnoid is hypothesised to give rise to the most common primary intracranial tumours, meningiomas. However, molecular evidence supporting this hypothesis is lacking. There are no effective medical therapies to treat meningiomas that are resistant to local interventions, encumbered by our limited understanding of their cellular origin. To address this limitation in our understanding of meningioma biology, we generated a comprehensive reference single cell and spatial transcriptomic atlas of human fetal meninges at post-conceptional weeks 5-13. We found that the meningeal layers develop concurrently, and identified an inner CDH1-positive dura cell layer expressing tight junction genes consistent with barrier function. We show that transcriptionally, meningioma cells resemble dura-lineage cells, and that common meningioma driver genes were expressed preferentially in the dura lineage. Our findings suggest that meningiomas originate from dura lineage cells. HIGHLIGHTSO_LIscRNA-seq and spatial transcriptomics reveals architecture of human fetal meninges development C_LIO_LIMeningeal layers are formed concurrently, by a gradual refinement of cell states C_LIO_LIA CDH1+ inner dura sublayer expresses tight junction genes like the arachnoid barrier C_LIO_LIMeningioma tumours likely originate from inner dura lineage cells, not arachnoid C_LI

developmental biology↗

bioRxiv2024

Spatial Dynamics of the Developing Human Heart

Heart development relies on a topologically defined interplay between a diverse array of cardiac cells. We finely curated spatial and single-cell measurements with subcellular imaging-based transcriptomics validation to explore spatial dynamics during early human cardiogenesis. Analyzing almost 80,000 individual cells and 70,000 spatially barcoded tissue regions between the 5.5th and 14th postconceptional weeks, we identified 31 coarse- and 72 fine-grained cell states and mapped them to highly resolved cardiac cellular niches. We provide novel insight into the development of the cardiac pacemaker-conduction system, heart valves, and atrial septum, and decipher heterogeneity of the hitherto elusive cardiac fibroblast population. Furthermore, we describe the formation of cardiac autonomic innervation and present the first spatial account of chromaffin cells in the fetal human heart. We support independent exploration of our datasets by an open-access, spatially centric interactive viewer. In summary, our study delineates the cellular and molecular landscape of the developing hearts architecture, offering links to genetic causes of heart disease.

developmental biology↗

bioRxiv2024

Midbrain Dopaminergic Neuron Development is Regulated by Two Molecularly Distinct Subtypes of Radial Glia Cells

Understanding midbrain dopaminergic (mDA) neuron development is key to advancing cell replacement therapies for Parkinsons disease (PD). Recent single-cell RNA-sequencing (scRNA-seq) studies have identified different subtypes of transient radial glia (Rgl) cell types in the developing mouse and human ventral midbrain. However, their individual functions and their impact on mDA neuron development are unclear. Here we analyze the transcriptome of endogenous mouse and human ventral midbrain Rgl and assess the function of key midbrain floor plate Rgl factors in human stem cells during mDA neuron differentiation. We find that Rgl1 is defined by a neurogenic network centered on Arntl, and that this transcription factor regulates human mDA neurogenesis. Conversely, the transcriptome of Rgl3 is dominated by signaling and extracellular matrix molecules that control different aspects of human mDA neuron development. Thus, our results suggest a function of Rgl1 as a mDA progenitor, and of Rgl3 as a signaling mDA niche cell. Moreover, using human stem cells we demonstrate that new knowledge of cell-type specific intrinsic and extrinsic developmental factors can readily be applied to improve the generation of cells with therapeutic interest, such as human mDA neurons for PD.

neuroscience↗

bioRxiv2024

High-parametric protein maps reveal the spatial organization in early-developing human lung

The respiratory system, including the lungs, is essential for terrestrial life. While recent research has advanced our understanding of lung development, much still relies on animal models and transcriptome analyses. In this study conducted within the Human Developmental Cell Atlas (HDCA) initiative, we describe the protein-level spatiotemporal organization of the lung during the first trimester of human gestation. Using high-parametric tissue imaging with a 30-plex antibody panel, we analyzed human lung samples from 6 to 13 post-conception weeks, generating data from over 2 million cells across five developmental timepoints. We present a resource detailing spatially resolved cell type composition of the developing human lung, including proliferative states, immune cell patterns, spatial arrangement traits, and their temporal evolution. This represents an extensive single-cell resolved protein-level examination of the developing human lungs and provides a valuable resource for further research into the developmental roots of human respiratory health and disease.

developmental biology↗

bioRxiv2023

Glioblastoma is spatially organized by neurodevelopmental programs and a glial-like wound healing response

Glioblastoma is the deadliest brain cancer, characterized by large cellular diversity whose complexity and organizing principles are only starting to be uncovered. Both neurodevelopment-like and mesenchymal-like cell states have been described in glioblastoma1-8, with the latter being strongly implicated in malignancy and disease progression8-11. However, the nature of these mesenchymal-like cell states remains unresolved. Here, we performed deep single-cell RNA sequencing of rare glioblastoma cases where tissue could be sampled from tumor core to macroscopically normal cortex. We discovered that previously defined mesenchymal-like tumor cell states instead represented a wound response that was shared across both malignant and non-malignant cell types and was spatially confined to the tumor bulk. Using glioblastoma organoids, we showed that the wound response transcriptional state could be reversibly induced in vitro by hypoxia and human plasma. We used multiplex single-molecule spatial transcriptomics12 on a large patient cohort to show that the activation of wound response states was associated with hypoxia, and organized by distance to perivascular niches. Our findings help reconceptualize the cellular landscape of glioblastoma, wherein a reactive wound-response tissue state shared by all cells in the tumor bulk is superimposed on a fundamentally neurodevelopmental and glial tumor.

cancer biology↗

bioRxiv2023

Dynamics of chromatin accessibility during human first-trimester neurodevelopment

The human brain is capable of highly complex functions that develops through a tightly organized cascade of patterning events, expressed transcription factors and changes in chromatin accessibility. While extensive datasets exist describing gene expression across the developing brain with single-cell resolution, similar atlases of chromatin accessibility have been primarily focused on the forebrain. Here, we focus on the chromatin landscape and paired gene expression across the developing human brain to provide a comprehensive single cell atlas during the first trimester (6 - 13 post-conceptional weeks). We identified 135 clusters across half a million nuclei and using the multiomic measurements linked candidate cis-regulatory elements (cCREs) to gene expression. We found an increase in the number of accessible regions driven both by age and neuronal differentiation. Using a convolutional neural network we identified putative functional TF-binding sites in enhancers characterizing neuronal subtypes and we applied this model to cCREs upstream of ESRRB to elucidate its activation mechanism. Finally, by linking disease-associated SNPs to cCREs we validated putative pathogenic mechanisms in several diseases and identified midbrain-derived GABAergic neurons as being the most vulnerable to major depressive disorder related mutations. Together, our findings provide a higher degree of detail to some key gene regulatory mechanisms underlying the emergence of cell types during the first trimester. We anticipate this resource to be a valuable reference for future studies related to human neurodevelopment, such as identifying cell type specific enhancers that can be used for highly specific targeting in in vitro models.

developmental biology↗

bioRxiv2022

Epigenomic complexity of the human brain revealed by single-cell DNA methylomes and 3D genome structures

Delineating the gene regulatory programs underlying complex cell types is fundamental for understanding brain functions in health and disease. Here, we comprehensively examine human brain cell epigenomes by probing DNA methylation and chromatin conformation at single-cell resolution in over 500,000 cells from 46 brain regions. We identified 188 cell types and characterized their molecular signatures. Integrative analyses revealed concordant changes in DNA methylation, chromatin accessibility, chromatin organization, and gene expression across cell types, cortical areas, and basal ganglia structures. With these resources, we developed scMCodes that reliably predict brain cell types using their methylation status at select genomic sites. This multimodal epigenomic brain cell atlas provides new insights into the complexity of cell type-specific gene regulation in the adult human brain.

neuroscience↗

bioRxiv2022

A comparative atlas of single-cell chromatin accessibility in the human brain

The human brain contains an extraordinarily diverse set of neuronal and glial cell types. Recent advances in single cell transcriptomics have begun to delineate the cellular heterogeneity in different brain regions, but the transcriptional regulatory programs responsible for the identity and function of each brain cell type remain to be defined. Here, we carried out single nucleus ATAC-seq analysis to probe the open chromatin landscape from over 1.1 million cells in 42 brain regions of three neurotypical adult donors. Integrative analysis of the resulting data identified 107 distinct cell types and revealed the cell-type-specific usage of 544,735 candidate cis-regulatory DNA elements (cCREs) in the human genome. Nearly 1/3 of them displayed sequence conservation as well as chromatin accessibility in the mouse brain. On the other hand, nearly 40% cCREs were human specific, with chromatin accessibility associated with species-restricted gene expression. Interestingly, these human specific cCREs were enriched for distinct families of retrotransposable elements, which displayed cell-type-specific chromatin accessibility. We uncovered strong associations between specific brain cell types and neuropsychiatric disorders. We futher developed deep learning models to predict regulatory function of non-coding disease risk variants.

neuroscience↗

bioRxiv2022

Transcriptomic cytoarchitecture reveals principles of human neocortex organization

Variation in cortical cytoarchitecture is the basis for histology-based definition of cortical areas, such as Brodmann areas. Single cell transcriptomics enables higher-resolution characterization of cell types in human cortex, which we used to revisit the idea of the canonical cortical microcircuit and to understand functional areal specialization. Deeply sampled single nucleus RNA-sequencing of eight cortical areas spanning cortical structural variation showed highly consistent cellular makeup for 24 coarse cell subclasses. However, proportions of excitatory neuron subclasses varied strikingly, reflecting differences in intra- and extracortical connectivity across primary sensorimotor and association cortices. Astrocytes and oligodendrocytes also showed differences in laminar organization across areas. Primary visual cortex showed dramatically different organization, including major differences in the ratios of excitatory to inhibitory neurons, expansion of layer 4 excitatory neuron types and specialized inhibitory neurons. Finally, gene expression variation in conserved neuron subclasses predicts differences in synaptic function across areas. Together these results provide a refined cellular and molecular characterization of human cortical cytoarchitecture that reflects functional connectivity and predicts areal specialization.

neuroscience↗

bioRxiv2022

Comprehensive cell atlas of the first-trimester developing human brain

The adult human brain likely comprises more than a thousand kinds of neurons, and an unknown number of glial cell types, but how cellular diversity arises during early brain development is not known. Here, in order to reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics to uncover cell states and trajectories in human brains at 5 - 14 post-conceptional weeks (p.c.w.). We identified twelve major classes and over 600 distinct cell states, which mapped to precise spatial anatomical domains at 5 p.c.w. We uncovered detailed differentiation trajectories of the human forebrain, and a surprisingly large number of region-specific glioblasts maturing into distinct pre-astrocytes and pre-oligodendrocyte precursor cells (pre-OPCs). Our findings reveal the emergence of cell types during the critical first trimester of human brain development.

developmental biology↗

bioRxiv2022

Transcriptomic diversity of cell types across the adult human brain

The human brain directs a wide range of complex behaviors ranging from fine motor skills to abstract intelligence and emotion. However, the diversity of cell types that support these skills has not been fully described. Here we used high-throughput single-nucleus RNA sequencing to systematically survey cells across the entire adult human brain in three postmortem donors. We sampled over three million nuclei from approximately 100 dissections across the forebrain, midbrain, and hindbrain. Our analysis identified 461 clusters and 3313 subclusters organized largely according to developmental origins. We found area-specific cortical neurons, as well as an unexpectedly high diversity of midbrain and hindbrain neurons. Astrocytes also exhibited regional diversity at multiple scales, comprising subtypes specific to the telencephalon and to more precise anatomical locations. Oligodendrocyte precursors comprised two distinct major types specific to the telencephalon and to the rest of the brain. Together, these findings demonstrate the unique cellular composition of the telencephalon with respect to all major brain cell types. As the first single-cell transcriptomic census of the entire human brain, we provide a resource for understanding the molecular diversity of the human brain in health and disease.

neuroscience↗

bioRxiv2022

Comparative transcriptomics reveals human-specific cortical features

Humans have unique cognitive abilities among primates, including language, but their molecular, cellular, and circuit substrates are poorly understood. We used comparative single nucleus transcriptomics in adult humans, chimpanzees, gorillas, rhesus macaques, and common marmosets from the middle temporal gyrus (MTG) to understand human-specific features of cellular and molecular organization. Human, chimpanzee, and gorilla MTG showed highly similar cell type composition and laminar organization, and a large shift in proportions of deep layer intratelencephalic-projecting neurons compared to macaque and marmoset. Species differences in gene expression generally mirrored evolutionary distance and were seen in all cell types, although chimpanzees were more similar to gorillas than humans, consistent with faster divergence along the human lineage. Microglia, astrocytes, and oligodendrocytes showed accelerated gene expression changes compared to neurons or oligodendrocyte precursor cells, indicating either relaxed evolutionary constraints or positive selection in these cell types. Only a few hundred genes showed human-specific patterning in all or specific cell types, and were significantly enriched near human accelerated regions (HARs) and conserved deletions (hCONDELS) and in cell adhesion and intercellular signaling pathways. These results suggest that relatively few cellular and molecular changes uniquely define adult human cortical structure, particularly by affecting circuit connectivity and glial cell function.

neuroscience↗

bioRxiv2022

Single cell transcriptomics reveals correct developmental dynamics and high-quality midbrain cell types by improved hESC differentiation.

Stem cell technologies provide new opportunities for modeling cells in the healthy and diseased states and for regenerative medicine. In both cases developmental knowledge as well as the quality and molecular properties of the cells are essential for their future application. In this study we identify developmental factors important for the differentiation of human embryonic stem cells (hESCs) into midbrain dopaminergic (mDA) neurons. We found that Laminin-511, and dual canonical and non-canonical WNT activation followed by GSK3{beta} inhibition plus FGF8b, improved midbrain patterning. In addition, mDA neurogenesis and differentiation was enhanced by activation of liver X receptors and inhibition of fibroblast growth factor signaling. Moreover, single-cell RNA-sequencing analysis revealed a developmental dynamics similar to that of the endogenous human ventral midbrain and the emergence of high quality molecularly-defined midbrain cell types, including mDA neurons that become functional. Thus, our study identifies novel factors important for human midbrain development and opens the door for a future application of molecularly-defined hESC-derived midbrain cell types in Parkinsons disease.

neuroscience↗

bioRxiv2022

Decoding spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin

The human spinal cord contains diverse cell types, governed by a series of spatiotemporal events for tissue assembly and functions. However, the spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. Single-cell RNA sequencing and spatial transcriptomics techniques have advanced the understanding of human organ development considerably. By performing integrated analysis of single-cell and spatial multi-omics methods, we created a comprehensive developmental cell atlas of the first trimester human spinal cord. Our data revealed that the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. Beyond this resource, we unexpectedly discovered unique events in human spinal cord development compared to rodents, including earlier quiescence of active neural stem cells, different regulation of stem cell differentiation, and distinct spatiotemporal genetic regulations of cell fate choices. In addition, using our atlas we identified specific gene expression in cancer stem cells in ependymomas. Thus, we demonstrate spatiotemporal genetic regulation of human spinal cord development as well as its potential to understand novel disease mechanisms and to inspire new therapies.

developmental biology↗

bioRxiv2022

Scalable in situ single-cell profiling by electrophoretic capture of mRNA

Methods to spatially profile the transcriptome are dominated by a trade-off between resolution and throughput. Here, we developed a method named EEL FISH that can rapidly process large tissue samples without compromising spatial resolution. By electrophoretically transferring RNA from a tissue section onto a capture surface, EEL speeds up data acquisition by reducing the amount of imaging needed, while ensuring that RNA molecules move straight down towards the surface, preserving single-cell resolution. We applied EEL on eight entire sagittal sections of the mouse brain and measured the expression patterns of up to 440 genes to reveal complex tissue organisation. Moreover, EEL enabled the study of challenging human samples by removing autofluorescent lipofuscin, so that we could study the spatial transcriptome of the human visual cortex. We provide full hardware specification, all protocols and complete software for instrument control, image processing, data analysis and visualization.

neuroscience↗

bioRxiv2022

Developmental origins of cell heterogeneity in the human lung

The lung contains numerous specialized cell-types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a first comprehensive topographic atlas of early human lung development. We report 83 cell states, several spatially-resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated scRNA-Seq and spatial transcriptomics into a web-based, open platform for interactive exploration. To illustrate the utility of our approach we show distinct states of secretory and neuroendocrine cells, largely overlapping with the programs activated either during lung fibrosis or small cell lung cancer progression. We define the origin of uncharacterized airway fibroblasts associated with airway smooth muscle in bronchovascular bundles, and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.

developmental biology↗