| 1. |
- Andrusivova, Zaneta
(författare)
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Development and application of spatial transcriptomics methods
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Asp, Michaela, et al.
(författare)
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Spatial Isoform Profiling within Individual Tissue Sections
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Spatial Transcriptomics has been shown to be a persuasive RNA sequencingtechnology for analyzing cellular heterogeneity within tissue sections. Thetechnology efficiently captures and barcodes 3’ tags of all polyadenylatedtranscripts from a tissue section, and thus provides a powerful platform whenperforming quantitative spatial gene expression studies. However, the currentprotocol does not recover the full-length information of transcripts, andconsequently lack information regarding alternative splice variants. Here, weintroduce a novel protocol for spatial isoform profiling, using SpatialTranscriptomics barcoded arrays.
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| 3. |
- Braun, Emelie, et al.
(författare)
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Comprehensive cell atlas of the first-trimester developing human brain
- 2023
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 382:6667, s. 172-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Fan, Yuhang, et al.
(författare)
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Expansion spatial transcriptomics
- 2023
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Ingår i: Nature Methods. - : Springer Nature. - 1548-7091 .- 1548-7105. ; 20:8, s. 1179-1182
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Fernandez Navarro, Jose, 1982-, et al.
(författare)
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Spatial Transcriptomics characterization of Alzheimer’s disease in the adult mouse brain
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease (AD) is a devastating neurological disease associated with progressive loss of mental skills, cognitive and physical functions. Here, our goal was to uncover novel and known molecular targets in the structured layers of the hippocampus and olfactory bulbs that may contribute to hippocampal synaptic dysfunction and smelling defects in AD mice. Spatial Transcriptomics was used to identify high confidence genes that were differentially regulated in AD mice relative to controls. A discussion of how these genes may contribute to AD pathology is provided.
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| 6. |
- Fernandez Navarro, Jose, et al.
(författare)
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Spatial Transcriptomics Reveals Genes Associated with Dysregulated Mitochondrial Functions and Stress Signaling in Alzheimer Disease
- 2020
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Ingår i: iScience. - : Elsevier Inc.. - 2589-0042. ; 23:10
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Tidskriftsartikel (refereegranskat)abstract
- Cellular Neuroscience; Omics; Transcriptomics Alzheimer disease (AD) is a devastating neurological disease associated with progressive loss of mental skills and cognitive and physical functions whose etiology is not completely understood. Here, our goal was to simultaneously uncover novel and known molecular targets in the structured layers of the hippocampus and olfactory bulbs that may contribute to early hippocampal synaptic deficits and olfactory dysfunction in AD mice. Spatially resolved transcriptomics was used to identify high-confidence genes that were differentially regulated in AD mice relative to controls. A diverse set of genes that modulate stress responses and transcription were predominant in both hippocampi and olfactory bulbs. Notably, we identify Bok, implicated in mitochondrial physiology and cell death, as a spatially downregulated gene in the hippocampus of mouse and human AD brains. In summary, we provide a rich resource of spatially differentially expressed genes, which may contribute to understanding AD pathology.
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| 7. |
- Firsova, Alexandra, et al.
(författare)
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Topographic atlas of cell states identifies regional gene expression in the adult human lung
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Single cell mRNA sequencing of the whole organ has become a popular technique to reveal rare types and subtypes of previously characterized cells as well as to distinguish and characterize gene expression of previously unknown cell types. Unsupervised clustering can reveal tens or even hundreds of variable genes that characterize cell types. Variation in gene expression is often observed within one cell type, and sometimes cannot be biologically explained without mapping of mRNA on tissue. In this study we aim to (i) map the majority of cell types of human lung, (ii) describe variability in their gene expression and (iii) relate this gene expression to cellular location and neighborhoods. Using three different spatial transcriptomics approaches, we mapped epithelial cell states of airways and submucosal gland, and defined cell type-unrelated gene expression variability along proximo-distal axis, including potential regulators and co-regulators of such cell states in the mesenchymal and immune cell niches. In addition, we mapped rare cell types, such as subtypes of neuroendocrine cells, ionocytes and tuft (brush) cells, revealing tracheal preference for ionocytes, and distal airways for GHRL-positive neuroendocrine cells. Finally, we used the created map as a reference for the diseased tissue from patients with stage II COPD and revealed perturbed cell states and COPD-specific imbalance of cell types, affecting immune and AT0 clusters.
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| 8. |
- Larsson, Ludvig, et al.
(författare)
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Spatial transcriptomics
- 2022
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Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 185:15, s. 2840-2840.e1
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Spatially resolved transcriptomics methodologies using RNA sequencing principles have and will continue to contribute to decode the molecular landscape of tissues. Linking quantitative sequencing data with tissue morphology empowers profiling of cellular morphology and transcription over time and space in health and disease. To view this SnapShot, open or download the PDF.
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| 9. |
- Lázár, Enikő, et al.
(författare)
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Spatial Dynamics of the Developing Human Heart
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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. In summary, our study delineates the cellular and molecular landscape of the developing heart’s architecture, offering links to genetic causes of heart disease.
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| 10. |
- Li, Xiaofei, et al.
(författare)
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Profiling spatiotemporal gene expression of the developing human spinal cord and implications for ependymoma origin
- 2023
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Ingår i: Nature Neuroscience. - : Springer Nature. - 1097-6256 .- 1546-1726. ; 26:5, s. 891-901
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Tidskriftsartikel (refereegranskat)abstract
- The authors created a comprehensive developmental cell atlas for spatiotemporal gene expression of the human spinal cord, revealed species-specific regulation during development and used the atlas to infer novel markers for pediatric ependymomas. The spatiotemporal regulation of cell fate specification in the human developing spinal cord remains largely unknown. In this study, by performing integrated analysis of single-cell and spatial multi-omics data, we used 16 prenatal human samples to create a comprehensive developmental cell atlas of the spinal cord during post-conceptional weeks 5-12. This revealed how the cell fate commitment of neural progenitor cells and their spatial positioning are spatiotemporally regulated by specific gene sets. We identified unique events in human spinal cord development relative to rodents, including earlier quiescence of active neural stem cells, differential regulation of cell differentiation and distinct spatiotemporal genetic regulation of cell fate choices. In addition, by integrating our atlas with pediatric ependymomas data, we identified specific molecular signatures and lineage-specific genes of cancer stem cells during progression. Thus, we delineate spatiotemporal genetic regulation of human spinal cord development and leverage these data to gain disease insight.
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| 11. |
- Maniatis, Silas, et al.
(författare)
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Spatiotemporal dynamics of molecular pathology in amyotrophic lateral sclerosis
- 2019
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Ingår i: Science. - : AMER ASSOC ADVANCEMENT SCIENCE. - 0036-8075 .- 1095-9203. ; 364:6435, s. 89-
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Tidskriftsartikel (refereegranskat)abstract
- Paralysis occurring in amyotrophic lateral sclerosis (ALS) results from denervation of skeletal muscle as a consequence of motor neuron degeneration. Interactions between motor neurons and glia contribute to motor neuron loss, but the spatiotemporal ordering of molecular events that drive these processes in intact spinal tissue remains poorly understood. Here, we use spatial transcriptomics to obtain gene expression measurements of mouse spinal cords over the course of disease, as well as of postmortem tissue from ALS patients, to characterize the underlying molecular mechanisms in ALS. We identify pathway dynamics, distinguish regional differences between microglia and astrocyte populations at early time points, and discern perturbations in several transcriptional pathways shared between murine models of ALS and human postmortem spinal cords.
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| 12. |
- Masarapu, Yuvarani, et al.
(författare)
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Spatially resolved multiomics on the neuronal effects induced by spaceflight in mice
- 2024
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- 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).
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| 13. |
- Mirzazadeh, Reza, et al.
(författare)
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Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of methods targeting the polyA tails of mRNA relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available spatial resolved transcriptomics assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), a workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we test the RRST protocol on tissue sections collected from five challenging tissue types, including human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyze 52 tissue sections and demonstrate that RRST is a versatile, powerful, and reproducible protocol for fresh frozen specimens of different qualities and origins.
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| 14. |
- Muus, Christoph, et al.
(författare)
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Single-cell meta-analysis of SARS-CoV-2 entry genes across tissues and demographics
- 2021
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Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 27:3, s. 546-559
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Tidskriftsartikel (refereegranskat)abstract
- Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2(+)TMPRSS2(+) cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention. An integrated analysis of over 100 single-cell and single-nucleus transcriptomics studies illustrates severe acute respiratory syndrome coronavirus 2 viral entry gene coexpression patterns across different human tissues, and shows association of age, smoking status and sex with viral entry gene expression in respiratory cell populations.
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| 15. |
- Overbey, Eliah G., et al.
(författare)
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Challenges and considerations for single-cell and spatially resolved transcriptomics sample collection during spaceflight
- 2022
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Ingår i: CELL REPORTS METHODS. - : Elsevier BV. - 2667-2375. ; 2:11
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Forskningsöversikt (refereegranskat)abstract
- Single-cell RNA sequencing (scRNA-seq) and spatially resolved transcriptomics (SRT) have experienced rapid development in recent years. The findings of spaceflight-based scRNA-seq and SRT investigations are likely to improve our understanding of life in space and our comprehension of gene expression in various cell systems and tissue dynamics. However, compared to their Earth-based counterparts, gene expression experiments conducted in spaceflight have not experienced the same pace of development. Out of the hundreds of spaceflight gene expression datasets available, only a few used scRNA-seq and SRT. In this perspective piece, we explore the growing importance of scRNA-seq and SRT in space biology and discuss the challenges and considerations relevant to robust experimental design to enable growth of these methods in the field.
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| 16. |
- Sariyar, Sanem, et al.
(författare)
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High-parametric protein maps reveal the spatial organization in early-developing human lung
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The respiratory system, encompassing the lungs, trachea, and vasculature, is essential for terrestrial life. Although recent research has illuminated aspects of lung development, such as cell lineage origins and their molecular drivers, much of our knowledge is still based on animal models, or is deduced from transcriptome analyses. In this study, conducted within the Human Developmental Cell Atlas (HDCA) initiative, we describe the spatiotemporal organization of lung during the first trimester of human gestation in situ and at protein level. We used high-parametric tissue imaging on human lung samples, aged 6 to 13 post-conception weeks, using a 30-plex antibody panel. Our approach yielded over 2 million individual lung cells across five developmental timepoints, with an in-depth analysis of nearly 1 million cells. We present a spatially resolved cell type composition of the developing human lung, with a particular emphasis on their proliferative states, spatial arrangement traits, and their temporal evolution throughout lung development. We also offer new insights into the emerging patterns of immune cells during lung development. To the best of our knowledge, this study is the most extensive protein-level examination of the developing human lung. The generated dataset is a valuable resource for further research into the developmental roots of human respiratory health and disease.
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| 17. |
- Sountoulidis, Alexandros, et al.
(författare)
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A topographic atlas defines developmental origins of cell heterogeneity in the human embryonic lung
- 2023
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Ingår i: Nature Cell Biology. - : Springer Nature. - 1465-7392 .- 1476-4679.
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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