scholarly journals Multiplexed single-cell transcriptomic analysis of normal and impaired lung development in the mouse

2019 ◽  
Author(s):  
K. M. Hurskainen ◽  
I. Mižíková ◽  
D. P. Cook ◽  
C. Cyr-Depauw ◽  
F. Lesage ◽  
...  
Keyword(s):  
Single Cell ◽  
Lung Development ◽  
Alveolar Epithelium ◽  
Cell Types ◽  
Capillary Endothelium ◽  
Hyperoxia Exposure ◽  
Capillary Endothelial Cells ◽  
Macrophage Populations ◽  
Cellular Compartments ◽  
Dynamic Populations

ABSTRACTDuring late lung development alveolar and microvascular development is finalized to enable sufficient gas exchange. Impaired late lung development manifests as bronchopulmonary dysplasia (BPD) in preterm infants. Single-cell RNA sequencing (scRNA-seq) allows for assessment of complex cellular dynamics during biological processes, such as development. Here, we use MULTI-seq to generate scRNA-seq profiles of over 66,000 cells from 36 mice during normal or impaired lung development secondary to hyperoxia. We observed dynamic populations of cells, including several rare cell types and putative progenitors. Hyperoxia exposure, which mimics the BPD phenotype, alters the composition of all cellular compartments, particularly alveolar epithelium, capillary endothelium and macrophage populations. We identified several BPD-associated signatures, including Pdgfra in fibroblasts, Activin A in capillary endothelial cells, and Csf1-Csf1r and Ccl2-Ccr2 signaling in macrophages and neutrophils. Our data provides a novel single-cell view of cellular changes associated with late lung development in health and in disease.

scholarly journals Single cell transcriptomic analysis of murine lung development on hyperoxia-induced damage

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maria Hurskainen ◽  
Ivana Mižíková ◽  
David P. Cook ◽  
Noora Andersson ◽  
Chanèle Cyr-Depauw ◽  
...  
Keyword(s):  
Single Cell ◽  
Lung Development ◽  
Alveolar Epithelium ◽  
Cell Types ◽  
Capillary Endothelium ◽  
Stromal Fibroblasts ◽  
Main Driver ◽  
Macrophage Populations ◽  
Induced Changes ◽  
Cellular Compartments

AbstractDuring late lung development, alveolar and microvascular development is finalized to enable sufficient gas exchange. Impaired late lung development manifests as bronchopulmonary dysplasia (BPD) in preterm infants. Single-cell RNA sequencing (scRNA-seq) allows for assessment of complex cellular dynamics during biological processes, such as development. Here, we use MULTI-seq to generate scRNA-seq profiles of over 66,000 cells from 36 mice during normal or impaired lung development secondary to hyperoxia with validation of some of the findings in lungs from BPD patients. We observe dynamic populations of cells, including several rare cell types and putative progenitors. Hyperoxia exposure, which mimics the BPD phenotype, alters the composition of all cellular compartments, particularly alveolar epithelium, stromal fibroblasts, capillary endothelium and macrophage populations. Pathway analysis and predicted dynamic cellular crosstalk suggest inflammatory signaling as the main driver of hyperoxia-induced changes. Our data provides a single-cell view of cellular changes associated with late lung development in health and disease.

Lung tissue volume changes induced by hypertonic NaCl: morphometric evaluation

1981 ◽  
Vol 51 (6) ◽  
pp. 1443-1450 ◽  
Author(s):  
D. Wangensteen ◽  
H. Bachofen ◽  
E. R. Weibel
Keyword(s):  
Lung Tissue ◽  
Alveolar Epithelium ◽  
Cell Types ◽  
Ringer Solution ◽  
Capillary Endothelium ◽  
Vascular Perfusion ◽  
Tissue Samples ◽  
Membrane Area ◽  
Capillary Lumen ◽  
Osmotic Effect

Lung tissue was examined to determine how the volumes of alveolar septum components change when NaCl is added to the vascular perfusate, increasing the osmolarity by 70 mosM. Isolated rabbit lungs were perfused with Ringer solution containing dextran, either with or without added NaCl, and fixed by vascular perfusion. Tissue samples from both “control” and “hypertonic” lungs, prepared for electron microscopy, were examined using established morphometric procedures. Volumes of septal cells, interstitial space, capillary lumen, surface-lining layer, and endothelial and epithelial areas were measured, all normalized against the endothelium basement-membrane area. Results showed that hypertonic NaCl caused a reduction in total cell and surface-lining layer volumes but no change in interstitial or capillary lumen volumes. This supports the hypothesis that small molecules have no osmotic effect across the pulmonary capillary endothelium but do cause a fluid flux from cells and across the alveolar epithelium. Areas and volume measurements for different septal cell types suggest a heterogeneous response: epithelial cells showed significant decreases and endothelial cells changed little, if at all.

scholarly journals Antibodies to mouse lung capillary endothelium.

1988 ◽  
Vol 36 (7) ◽  
pp. 741-749 ◽  
Author(s):  
M C Rorvik ◽  
D P Allison ◽  
J A Hotchkiss ◽  
H P Witschi ◽  
S J Kennel
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Interstitial Cells ◽  
Mouse Lung ◽  
Capillary Endothelium ◽  
Specific Cell ◽  
Alveolar Wall ◽  
Gold Particles ◽  
Capillary Endothelial Cells ◽  
Quantitative Analyses

We are interested in developing monoclonal antibodies (MoAbs) that recognize specific cell types in the lung of BALB/c mice. Normal mouse lung homogenate was used to immunize F344 rats and hybridomas were produced by fusion of rat spleen cells with mouse myeloma SP 2/0. Two hybridomas were selected which produced MoAbs active in immunohistochemistry of lung cells. MoAb 273-34A and 411-201B both show extensive peroxidase staining of capillary endothelial cells within alveolar walls of lungs at the light microscopic level. To demonstrate cell specificity, immunoelectron microscopy with gold-labeled antibody was performed. Lightly fixed lungs were frozen and thin-sectioned before staining with MoAb and 5-nm gold particles coupled to secondary antibody. Quantitative analyses of these cryosections show that both antibodies, used at optimal concentrations, are specific for binding to capillary endothelial cells. More than 95% of the gold particles are associated with capillary endothelial cells on the thin side of the alveolar wall. When capillaries adjoined thick septa containing interstitial cells, about two thirds of the gold particles were associated with endothelial cells and about one quarter with interstitial cells. These MoAbs should be useful in studying the role of endothelial cells in toxic lung injury.

scholarly journals Machine and deep learning single-cell segmentation and quantification of multi-dimensional tissue images

2019 ◽  
Author(s):  
Eliot T McKinley ◽  
Joseph T Roland ◽  
Jeffrey L Franklin ◽  
Mary Catherine Macedonia ◽  
Paige N Vega ◽  
...  
Keyword(s):  
Deep Learning ◽  
Single Cell ◽  
Shape Descriptor ◽  
Cell Types ◽  
Colorectal Adenomas ◽  
Cell Segmentation ◽  
Tissue Imaging ◽  
Antibody Staining ◽  
Multiple Cell ◽  
Cellular Compartments

AbstractIncreasingly, highly multiplexed in situ tissue imaging methods are used to profile protein expression at the single-cell level. However, a critical limitation is a lack of robust cell segmentation tools applicable for sections of tissues with a complex architecture and multiple cell types. Using human colorectal adenomas, we present a pipeline for cell segmentation and quantification that utilizes machine learning-based pixel classification to define cellular compartments, a novel method for extending incomplete cell membranes, quantification of antibody staining, and a deep learning-based cell shape descriptor. We envision that this method can be broadly applied to different imaging platforms and tissue types.

scholarly journals A single-cell atlas of human fetal lung development between 14 and 19 weeks of gestation

2021 ◽  
Author(s):  
Laurent Renesme ◽  
Flore Lesage ◽  
David Cook ◽  
Shumei Zhong ◽  
Satu M Hanninen ◽  
...  
Keyword(s):  
Single Cell ◽  
Lung Development ◽  
Molecular Mechanisms ◽  
Human Lung ◽  
Cell Communication ◽  
Cell Types ◽  
Developmental Trajectory ◽  
Marker Genes ◽  
Different Cell Types ◽  
Pseudoglandular Stage

Rationale: Human lung development has been mainly described in morphologic studies and the potential underlying molecular mechanisms were extrapolated from animal models. Therefore, there is a need to gather knowledge from native human lung tissue. In this study we describe changes at a single-cell level in human fetal lungs during the pseudoglandular stage. Methods: We report the cellular composition, cell trajectories and cell-to-cell communication in developing human lungs with single-nuclei RNA sequencing (snRNA-seq) on 23,251 nuclei isolated from nine human fetuses with gestational ages between 14 to 19 weeks of gestation. Results: We identified nine different cell types, including a rare pulmonary neuroendocrine cells population. For each cell type, marker genes are reported, and selected marker genes are used for spatial validation with fluorescent RNA in situ hybridization. Enrichment and developmental trajectory analysis provide insight into molecular mechanisms and signaling pathways within individual cell clusters according to gestational age. Lastly, ligand-receptor analysis highlights determinants of cell-to-cell communication among the different cell types through the pseudoglandular stage, including general developmental pathways (NOTCH and TGFB), as well as more specific pathways involved in vasculogenesis, neurogenesis, and immune system regulation. Conclusion: These findings provide a clinically relevant background for research hypotheses generation in projects studying normal or impaired lung development and help to develop and validate surrogate models to study human lung development, such as human lung organoids.

scholarly journals Single-cell transcriptomics reveal cell type-specific molecular changes and altered intercellular communications in chronic obstructive pulmonary disease

2021 ◽  
Author(s):  
Qiqing Huang ◽  
Jingshen Wang ◽  
Shaoran Shen ◽  
Yuanyuan Wang ◽  
Yan Chen ◽  
...  
Keyword(s):  
Chronic Obstructive Pulmonary Disease ◽  
Single Cell ◽  
Pulmonary Disease ◽  
Alveolar Epithelium ◽  
Cell Types ◽  
Chronic Obstructive ◽  
Cell Type ◽  
Obstructive Pulmonary Disease ◽  
Cell Type Specific ◽  
Intercellular Communications

AbstractChronic obstructive pulmonary disease (COPD) is a common and heterogeneous respiratory disease, the molecular complexity of which remains poorly understood, as well as the mechanisms by which aging and smoking facilitate COPD development. Here, using single-cell RNA sequencing of more than 65,000 cells from COPD and age-stratified control lung tissues of donors with different smoking histories, we identified monocytes, club cells, and macrophages as the most disease-, aging-, and smoking-relevant cell types, respectively. Notably, we found these highly cell-type specific changes under different conditions converged on cellular dysfunction of the alveolar epithelium. Deeper investigations revealed that the alveolar epithelium damage could be attributed to the abnormally activated monocytes in COPD lungs, which could be amplified via exhaustion of club cell stemness as ages. Moreover, the enhanced intercellular communications in COPD lungs as well as the pro-inflammatory interaction between macrophages and endothelial cells indued by smoking could facilitate signaling between monocyte and the alveolar epithelium. Our findings complement the existing model of COPD pathogenesis by emphasizing the contributions of the previously less appreciated cell types, highlighting their candidacy as potential therapeutic targets for COPD.

scholarly journals The spatio-temporal program of liver zonal regeneration

2021 ◽  
Author(s):  
Shani Ben-Moshe ◽  
Tamar Veg ◽  
Rita Manco ◽  
Stav Dan ◽  
Aleksandra A. Kolodziejczyk ◽  
...  
Keyword(s):  
Liver Regeneration ◽  
Single Cell ◽  
Stellate Cell ◽  
Cell Types ◽  
Matrix Remodeling ◽  
Spatially Resolved ◽  
Single Cell Rna Sequencing ◽  
Ability To Regenerate ◽  
Spatio Temporal ◽  
Macrophage Populations

The liver carries a remarkable ability to regenerate rapidly after acute zonal damage. Single-cell approaches are necessary to study this process, given the spatial heterogeneity of multiple liver cell types. Here, we use spatially-resolved single cell RNA sequencing (scRNAseq) to study the dynamics of mouse liver regeneration after acute acetaminophen (APAP) intoxication. We find that hepatocytes proliferate throughout the liver lobule, creating the mitotic pressure required to repopulate the necrotic pericentral zone rapidly. A subset of hepatocytes located at the regenerating front transiently up-regulate fetal-specific genes, including Afp and Cdh17, as they reprogram to a pericentral state. Zonated endothelial, hepatic-stellate cell (HSC) and macrophage populations are differentially involved in immune recruitment, proliferation and matrix remodeling. We observe massive transient infiltration of myeloid cells, yet stability of lymphoid cell abundance, in accordance with global decline in antigen presentation. Our study provides a resource for understanding the coordinated programs of zonal liver regeneration.

scholarly journals Fragmentation of macrophages during isolation confounds analysis of single cell preparations from mouse hematopoietic tissues

2021 ◽  
Author(s):  
Susan M Millard ◽  
Ostyn Heng ◽  
Khatora S Opperman ◽  
Anuj Sehgal ◽  
Katharine M Irvine ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Cell Types ◽  
Mouse Bone Marrow ◽  
Bone Homeostasis ◽  
Systematic Strategy ◽  
Stem And Progenitor Cells ◽  
Adhesive Interactions ◽  
Macrophage Populations

SummaryMouse hematopoietic tissues contain abundant and heterogeneous populations of tissue-resident macrophages attributed trophic functions in control of immunity, hematopoiesis and bone homeostasis. A systematic strategy to characterise macrophage subsets in mouse bone marrow (BM), spleen and lymph node, unexpectedly revealed macrophage surface marker staining typically emanated from membrane-bound subcellular remnants associated with unrelated cell types. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters and mRNA were all detected in non-macrophage cell populations including isolated stem and progenitor cells. The profile of macrophage remnant association reflects adhesive interactions between macrophages and other cell types in vivo. Applying this knowledge, reduced macrophage remnant attachment to BM granulocytes in Siglec1 deficient mice was associated with compromised emergency granulocytosis, revealing a function for Siglec1-dependent granulocyte-macrophage interactions. Analysis of published RNA-seq data for purified macrophage and non-macrophage populations indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single cell analysis of disaggregated tissues.

Looking at the Developing Lung in Single-cell Resolution

2020 ◽  
Author(s):  
Ivana Mižíková ◽  
Bernard Thébaud
Keyword(s):  
Single Cell ◽  
Lung Development ◽  
Cell Types ◽  
Lineage Tracing ◽  
Cell Lineages ◽  
Heterogeneous Nature ◽  
Cell Transcriptome ◽  
Tracing Techniques ◽  
Postnatal Lung Development ◽  
Single Cell Transcriptome

Lung development is a complicated and delicate process, facilitated by spatially and temporarily coordinated crosstalk of up to 40 cell types. Developmental origin and heterogeneity of lung cell lineages in context of lung development have been a focus of research efforts for decades. Bulk RNA and protein measurements, RNA and protein labelling, and lineage tracing techniques have been traditionally employed. However, the complex and heterogeneous nature of lung tissue presents a particular challenge when identifying subtle changes in gene expression in individual cell types. Rapidly developing single-cell RNA sequencing (scRNA-seq) techniques allow for unbiased and robust assessment of complex cellular dynamics during biological processes in unprecedented ways. Discovered a decade ago, scRNA-seq has been applied in respiratory research to understand lung cellular composition and to identify novel cell types. Still, very few studies to date have addressed the single-cell transcriptome in healthy or aberrantly developing lung. In this mini-review, we discuss principal discoveries with scRNA-seq in the field of prenatal and postnatal lung development. In addition, we examine challenges and expectations, and propose future steps associated with the use of scRNA-seq to study developmental lung diseases.

Ultrastructure of pulmonary microvasculature in acute endotoxemia

1978 ◽  
Vol 36 (2) ◽  
pp. 470-471
Author(s):  
R. G. Gerrity ◽  
M. Richardson
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Alveolar Epithelium ◽  
Endothelial Damage ◽  
Capillary Endothelium ◽  
Type Ii Cells ◽  
Type Ii ◽  
Interstitial Edema ◽  
Pulmonary Capillaries ◽  
Lung Ultrastructure ◽  
Fibrin Thrombi

Dogs were injected intravenously with E_. coli endotoxin (2 mg/kg), and lung samples were taken at 15 min., 1 hr. and 24 hrs. At 15 min., occlusion of pulmonary capillaries by degranulating platelets and polymorphonuclear leukocytes (PML) was evident (Fig. 1). Capillary endothelium was intact but endothelial damage in small arteries and arterioles, accompanied by intraalveolar hemorrhage, was frequent (Fig. 2). Sloughing of the surfactant layer from alveolar epithelium was evident (Fig. 1). At 1 hr., platelet-PML plugs were no longer seen in capillaries, the endothelium of which was often vacuolated (Fig. 3). Interstitial edema and destruction of alveolar epithelium were seen, and type II cells had discharged their granules into the alveoli (Fig. 4). At 24 hr. phagocytic PML's were frequent in peripheral alveoli, while centrally, alveoli and vessels were packed with fibrin thrombi and PML's (Fig. 5). In similar dogs rendered thrombocytopenic with anti-platelet serum, lung ultrastructure was similar to that of controls, although PML's were more frequently seen in capillaries in the former (Fig. 6).

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