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

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.

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

10.1101/868802 ◽

2019 ◽

Cited By ~ 1

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.

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Lung tissue volume changes induced by hypertonic NaCl: morphometric evaluation

Journal of Applied Physiology ◽

10.1152/jappl.1981.51.6.1443 ◽

1981 ◽

Vol 51 (6) ◽

pp. 1443-1450 ◽

Cited By ~ 10

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.

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An automated workflow for label-free and multiplexed single cell proteomics sample preparation at unprecedented sensitivity.

10.1101/2021.04.14.439828 ◽

2021 ◽

Author(s):

David Hartlmayr ◽

Claudia Ctortecka ◽

Anjali Seth ◽

Sasha Mendjan ◽

Guilhem Tourniaire ◽

...

Keyword(s):

Sample Preparation ◽

Single Cell ◽

Global Analysis ◽

Single Cells ◽

Direct Injection ◽

Cell Types ◽

Mrna Levels ◽

Label Free ◽

Final Sample ◽

Main Driver

The analysis of single cell proteomes has recently become a viable complement to transcript and genomics studies. Proteins are the main driver of cellular functionality and mRNA levels are often an unreliable proxy of such. Therefore, the global analysis of the proteome is essential to study cellular identities. Both multiplexed and label-free mass spectrometry-based approaches with single cell resolution have lately attributed surprising heterogeneity to believed homogenous cell populations. Even though specialized experimental designs and instrumentation have demonstrated remarkable advances, the efficient sample preparation of single cells still lacks behind. Here, we introduce the proteoCHIP, a universal option for single cell proteomics sample preparation at surprising sensitivity and throughput. The automated processing using a commercial system combining single cell isolation and picoliter dispensing, the cellenONE®, allows to reduce final sample volumes to low nanoliters submerged in a hexadecane layer simultaneously eliminating error prone manual sample handling and overcoming evaporation. With this specialized workflow we achieved around 1,000 protein groups per analytical run at remarkable reporter ion signal to noise while reducing or eliminating the carrier proteome. We identified close to 2,000 protein groups across 158 multiplexed single cells from two highly similar human cell types and clustered them based on their proteome. In-depth investigation of regulated proteins readily identified one of the main drivers for tumorigenicity in this cell type. Our workflow is compatible with all labeling reagents, can be easily adapted to custom workflows and is a viable option for label-free sample preparation. The specialized proteoCHIP design allows for the direct injection of label-free single cells via a standard autosampler resulting in the recovery of 30% more protein groups compared to samples transferred to PEG coated vials. We therefore are confident that our versatile, sensitive, and automated sample preparation workflow will be easily adoptable by non-specialized groups and will drive biological applications of single cell proteomics.

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

10.1101/790162 ◽

2019 ◽

Cited By ~ 1

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.

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

10.1101/2021.12.23.473945 ◽

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.

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Single cell RNAseq provides a molecular and cellular cartography of changes to the human endometrium through the menstrual cycle

10.1101/350538 ◽

2018 ◽

Cited By ~ 5

Author(s):

Wanxin Wang ◽

Felipe Vilella ◽

Pilar Alama ◽

Inmaculada Moreno ◽

Marco Mignardi ◽

...

Keyword(s):

Menstrual Cycle ◽

Single Cell ◽

Cell Types ◽

Human Endometrium ◽

Cellular Heterogeneity ◽

Cell Type ◽

Stromal Fibroblasts ◽

Unique Type ◽

Ciliated Epithelial Cell ◽

Human Menstrual Cycle

SummaryIn a human menstrual cycle, the endometrium undergoes remodeling, shedding, and regeneration, all of which are driven by substantial gene expression changes in the underlying cellular hierarchy. Despite its importance in human fertility and regenerative biology, mechanistic understanding of this unique type of tissue homeostasis remains rudimentary. We characterized the transcriptomic transformation of human endometrium at single cell resolution, dissecting the multidimensional cellular heterogeneity of this tissue across the entire natural menstrual cycle. We profiled the behavior of 6 endometrial cell types, including a previously uncharacterized ciliated epithelial cell type, during four major phases of endometrial transformation, and found characteristic signatures for each cell type and phase. We discovered that human window of implantation opens with an abrupt and discontinuous transcriptomic activation in the epithelia, accompanied with widespread decidualized feature in the stromal fibroblasts. These data reveal signatures in the luminal and glandular epithelia during epithelial gland reconstruction, and suggest a mechanism for adult gland formation.

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

10.1101/2021.02.23.432590 ◽

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.

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The spatio-temporal program of liver zonal regeneration

10.1101/2021.08.11.455924 ◽

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.

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

10.1101/2021.04.28.441876 ◽

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.

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Looking at the Developing Lung in Single-cell Resolution

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00385.2020 ◽

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.

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