Microglia survey and regulate central nervous system myelination during embryonic development and adult homeostasis. However, whether microglia–myelin interactions are spatiotemporally regulated remains unexplored. Here, by examining spinal cord white matter tracts in mice, we determined that myelin degeneration was particularly prominent in the dorsal column (DC) during normal aging. This was accompanied by molecular and functional changes in DC microglia as well as an upregulation of transforming growth factor beta (TGF)β signaling. Disrupting TGFβ signaling in microglia led to unrestrained microglial responses and myelin loss in the DC, accompanied by neurological deficits exacerbated with aging. Single-nucleus RNA-sequencing analyses revealed the emergence of a TGFβ signaling-sensitive microglial subset and a disease-associated oligodendrocyte subset, both of which were spatially restricted to the DC. We further discovered that microglia rely on a TGFβ autocrine mechanism to prevent damage of myelin in the DC. These findings demonstrate that TGFβ signaling is crucial for maintaining microglial resilience to myelin degeneration in the DC during aging. This highlights a previously unresolved checkpoint mechanism of TGFβ signaling with regional specificity and spatially restricted microglia–oligodendrocyte interactions.

Integration of scRNA-seq data from millions of cells revealed a high diversity of cell types in the healthy and diseased human lung. In a large and complex organ, constantly exposed to external agents, it is crucial to understand the influence of lung tissue topography or external factors on gene expression variability within cell types. Here, we apply three spatial transcriptomics approaches, to: (i) localize the majority of lung cell types, including rare epithelial cells within the tissue topography, (ii) describe consistent anatomical and regional gene expression variability within and across cell types, and (iii) reveal distinct cellular neighborhoods in specific anatomical regions and examine gene expression variations in them. We thus provide a spatially resolved tissue reference atlas in three representative regions of the healthy human lung. We further demonstrate its utility by defining previously unknown imbalances of epithelial cell type compositions in chronic obstructive pulmonary disease lungs. Our topographic atlas enables a precise description of characteristic regional cellular responses upon experimental perturbations or during disease progression.

Introduction: RNA expression is modulated by tau. We used two mouse models, THY-Tau22 mice, which express pro-aggregation tau, and TauKO mice, which are null for tau, to improve our understanding of tau-altered mRNA expression in brain. Methods: Spatial transcriptomics on Tau22 and TauKO mice were used to interrogate regional mRNA expression changes. We focused on mRNA expression changes in the hippocampus and ventricles; two regions altered early in Alzheimer’s disease. Results: We identified the transthyretin mRNA, Ttr, as being dysregulated in a tau-dependent manner. Immunofluorescence (IF) revealed increased TTR protein expression in THY-Tau22 mice and lowered expression in TauKO mice in the choroid plexus epithelial cells. Conclusion: As TTR is involved in the clearance of Aβ and the prevention of Aβ aggregation, we evaluated endogenous mouse Aβ in TauKO mice and observed increased Aβ deposits. Our study reveals a hitherto unknown regulatory role of tau on Ttr mRNA and protein expression, which may participate in a feedback loop contributing to Aβ disease progression.

Heart development relies on topologically orchestrated cellular transitions and interactions, many of which remain poorly characterized in humans. Here, we combined unbiased spatial and single-cell transcriptomics with imaging-based validation across postconceptional weeks 5.5 to 14 to uncover the molecular landscape of human early cardiogenesis. We present a high-resolution transcriptomic map of the developing human heart, revealing the spatial arrangements of 31 coarse-grained and 72 fine-grained cell states organized into distinct functional niches. Our findings illuminate key insights into the formation of the cardiac pacemaker-conduction system, heart valves and atrial septum, and uncover unexpected diversity among cardiac mesenchymal cells. We also trace the emergence of autonomic innervation and provide the first spatial account of chromaffin cells in the fetal heart. Our study, supported by an open-access spatially centric interactive viewer, offers a unique resource to explore the cellular and molecular blueprint of human heart development, offering links to genetic causes of heart disease.

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 lung and provides a valuable resource for further research into the developmental roots of human respiratory health and disease.

Impairment of the central nervous system (CNS) poses a significant health risk for astronauts during long-duration space missions. In this study, we employed an innovative approach by integrating single-cell multiomics (transcriptomics and chromatin accessibility) with spatial transcriptomics to elucidate the impact of spaceflight on the mouse brain in female mice. Our comparative analysis between ground control and spaceflight-exposed animals revealed significant alterations in essential brain processes including neurogenesis, synaptogenesis and synaptic transmission, particularly affecting the cortex, hippocampus, striatum and neuroendocrine structures. Additionally, we observed astrocyte activation and signs of immune dysfunction. At the pathway level, some spaceflight-induced changes in the brain exhibit similarities with neurodegenerative disorders, marked by oxidative stress and protein misfolding. Our integrated spatial multiomics approach serves as a stepping stone towards understanding spaceflight-induced CNS impairments at the level of individual brain regions and cell types, and provides a basis for comparison in future spaceflight studies. For broader scientific impact, all datasets from this study are available through an interactive data portal, as well as the National Aeronautics and Space Administration (NASA) Open Science Data Repository (OSDR).

Sountoulidis et al. provide a spatial gene expression atlas of human embryonic lung during the first trimester of gestation and identify 83 cell identities corresponding to stable cell types or transitional states. 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 comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of 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.

The adult human brain comprises more than a thousand distinct neuronal and glial cell types, a diversity that emerges during early brain development. To reveal the precise sequence of events during early brain development, we used single-cell RNA sequencing and spatial transcriptomics and uncovered cell states and trajectories in human brains at 5 to 14 postconceptional weeks (pcw). We identified 12 major classes that are organized as ~600 distinct cell states, which map to precise spatial anatomical domains at 5 pcw. We described detailed differentiation trajectories of the human forebrain and midbrain and found a large number of region-specific glioblasts that mature into distinct pre-astrocytes and pre–oligodendrocyte precursor cells. Our findings reveal the establishment of cell types during the first trimester of human brain development.

Transcriptomics is one of the pivotal fields in molecular biology, enabling comprehensive analysis of gene expression patterns. Recent advancements in the biotechnology field have transformed the transcriptomics research, providing insights into the complexity of cellular processes in a greater detail. However, conventional transcriptomics methods such as bulk RNA sequencing or single-cell RNA sequencing rely on tissue dissociation and therefore lack spatial information, which limits our understanding of gene expression patterns within the tissue structures. The development of spatially resolved transcriptomics methods has revolutionized the study of transcriptomes, enabling analysis of gene expression patterns in the spatial context. The wide range of available transcriptomics technologies offer various levels of resolution and throughput, and combination of multiple techniques can be beneficial for studying biological systems and gain deeper understanding of their molecular processes. In this thesis, particular emphasis is given to the Visium spatial gene expression technology, which has gain widespread popularity in the research community over the recent years. 

In the article I, we expand the application of the Visium platform to fresh-frozen samples of lower RNA quality or otherwise challenging characteristics. To achieve this, we introduce specific modifications to the commercially available protocol and test its effectiveness across different tissue types of varying RNA quality, including pediatric brain tumors, human small intestine, and mouse bone and cartilage. By conducting comparative analysis, we demonstrate that the new protocol outperforms the standard Visium protocol when working with samples of moderate and lower RNA quality.

Article II introduces a novel method that enhances the resolution of the Visium gene expression method through tissue expansion. We showcase the implementation of this new protocol on two regions of mouse brain, olfactory bulb and hippocampus. We demonstrate the ability of this approach to study smaller tissue structures that were previously beyond the resolution capabilities of the Visium platform.

In the article III and IV, we demonstrate the practical application of the Visium approach and its combination with other methodologies in the field of developmental biology. We show how utilizing spatial transcriptomics methods help elucidate the spatial organization of cell types and cell states during organogenesis in the developing human spinal cord (article III) and developing lung tissue (article IV). By deploying single-cell RNA sequencing and spatial methods, we described the spatiotemporal gene expression profiles of various cell types as well as shared and unique events occurring during the spinal cord development in humans and rodents (article III). Applying this multimodal approach to lung tissue (article IV) allowed us to characterize novel cell states emerging during lung development and provided valuable insights into the structural organization of developing lungs. These studies highlight the findings and observations that can be gained by combining spatially resolved transcriptomics with other laboratory techniques to shed light on the spatial dynamics of cellular processes during organ development.

Transkriptomik är ett centralt område inom molekylärbiologi som möjliggör övergripande analys av genuttrycksmönster. De senaste framstegen inom bioteknik har revolutionerat transkriptomikforskningen vilket gett nya insikter i cellulära processers komplexitet på en mer detaljerad nivå. Konventionella transkriptomikmetoder som bulk-RNA-sekvensering eller single-cell RNA-sekvensering förlitar sig dock på dissociering av vävnad och saknar därför spatial information, vilket begränsar vår förståelse av genuttrycksmönster inom vävnaden. Utvecklingen av spatialt upplöst transkriptomik har revolutionerat studerandet av transkriptom genom att möjliggöra analys av genuttrycksmönster i sin spatiala kontext. Det breda utbudet av tillgängliga transkriptomiktekniker erbjuder olika nivåer av upplösning och kapacitet, och kombinationen av flera tekniker kan vara fördelaktig för studier av biologiska system för att uppnå en djupare förståelse av deras molekylära processer. I den här avhandlingen läggs särskild vikt på Visium-teknologin för spatialt genuttryck, som har blivit mycket populär inom forskarvärlden under de senaste åren.

I Artikel I expanderar vi tillämpningen av Visium-plattformen till färskfrusna prover med lägre RNA-kvalitet eller andra utmanande egenskaper. För att uppnå detta introducerar vi specifika modifieringar till den kommersiellt tillgängliga metoden och testar dess effektivitet på olika vävnadstyper med varierande RNA-kvalitet, inklusive barnhjärntumörer, mänsklig tunntarm och musbens- och broskvävnad. Våra jämförelse analyser visar att den nya metoden överträffar standard Visium-protokollet för prover med måttlig eller lägre RNA-kvalitet.

Artikel II introducerar en ny metod som förbättrar upplösningen av Visium-metoden genom utvidgning av vävnaden. Vi applicerar detta nya protokoll på två områden i mushjärnan, specifikt luktloben och hippocampus och demonstrerar dess förmågan att studera mindre vävnadsstrukturer som tidigare låg utanför Visium-plattformens upplösningsförmåga.

I Artikel III och IV demonstrerar vi den praktiska tillämpningen av Visium-metoden och dess kombination med andra metoder inom utvecklingsbiologi. Vi visar på hur användningen av spatiala transkriptomikmetoder hjälper till att belysa den rumsliga organisationen av celltyper och celltillstånd under organogenesen i den mänskliga ryggmärgs utvecklingen (artikel III) och i lungvävnads utvecklingen (artikel IV). Genom att använda single-cell RNA-sekvensering och spatiala metoder beskrev vi de spatiala genuttrycksmönstren hos olika celltyper samt delade och unika händelser som sker under ryggmärgsutvecklingen hos människor jämfört med gnagare (artikel III). Genom att tillämpa denna multimodala metod på lungvävnad (artikel IV) karaktäriserar vi även nya celltillstånd som uppstår under lungutvecklingen och erhåller värdefulla insikter i den strukturella organisationen av utvecklade lungor. Dessa studier framhäver de resultat och iakttagelser som kan erhållas genom att kombinera spatialt upplösta transkriptomik med andra laboratorietekniker för att klargöra den spatiala dynamiken hos cellulära processer under organs utveckling.

Capture array-based spatial transcriptomics methods have been widely used to resolve gene expression in tissues; however, their spatial resolution is limited by the density of the array. Here we present expansion spatial transcriptomics to overcome this limitation by clearing and expanding tissue prior to capturing the entire polyadenylated transcriptome with an enhanced protocol. This approach enables us to achieve higher spatial resolution while retaining high library quality, which we demonstrate using mouse brain samples.