scholarly journals Single-cell transcriptomic assessment of cellular phenotype stability in human precision-cut lung slices

2021 ◽  
Author(s):  
Nichelle I. Winters ◽  
Chase J. Taylor ◽  
Christopher S. Jetter ◽  
Jane E. Camarata ◽  
Austin J. Gutierrez ◽  
...  
Keyword(s):  
Single Cell ◽  
Disease Modeling ◽  
Immune Cell ◽  
Human Lung ◽  
Ex Vivo ◽  
Alveolar Epithelial Cell ◽  
Cellular Phenotype ◽  
Human Lung Tissue ◽  
Alveolar Epithelial ◽  
Precision Cut Lung Slices

ABSTRACTPrecision-cut lung slices (PCLS) are increasingly utilized for ex vivo disease modeling, but a high-resolution characterization of cellular phenotype stability in PCLS has not been reported. Comparing the single-cell transcriptomic profile of human PCLS after five days of culture to freshly isolated human lung tissue, we found striking changes in endothelial cell and alveolar epithelial cell programs, reflecting both injury and pathways activated in static culture, while immune cell frequencies and programs remained largely intact and similar to the native lung. These cellular dynamics should be considered when utilizing PCLS as a model of the human lung.

scholarly journals An integrated multiomic and quantitative label-free microscopy-based approach to study pro-fibrotic signalling in ex vivo human precision-cut lung slices

2020 ◽  
pp. 2000221
Author(s):  
Muzamil Majid Khan ◽  
Daniel Poeckel ◽  
Aliaksandr Halavatyi ◽  
Joanna Zukowska-Kasprzyk ◽  
Frank Stein ◽  
...  
Keyword(s):  
Immune Cell ◽  
Ex Vivo ◽  
Second Harmonic ◽  
Integrated Approach ◽  
Fibrillar Collagen ◽  
Collagen Deposition ◽  
Label Free ◽  
Human Lung Tissue ◽  
Ecm Proteins ◽  
Precision Cut Lung Slices

Fibrosis can affect any organ resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by expansion of connective tissue due to excessive deposition of extracellular matrix proteins (ECM), including the fibrillar forms of collagen. A significant limitation for discovering cures for fibrosis is the availability of suitable human models and techniques to quantify mature fibrillar collagen deposition as close as possible to human physiological conditions. Here we have extensively characterised an ex vivo cultured human lung tissue-derived, precision-cut lung slices model (hPCLS) using label-free second harmonic (SHG) light microscopy to quantify fibrillar collagen deposition and mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint of hPCLS in ex vivo culture.We demonstrate that hPCLS are viable and metabolically active with mesenchymal, epithelial, endothelial, and immune cell types surviving for at least 2 weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed a strong induction of pulmonary fibrosis-related ECM proteins upon TGFß1 stimulation. This upregulation of ECM proteins was not translated into an increased deposition of fibrillar collagen. In support of this observation, we revealed the presence of a pro-ECM degradation activity in our ex vivo cultures of hPCLS, inhibition of which by metalloproteinase inhibitor resulted in increased collagen deposition in response to TGFß1 stimulation. Together the data show that an integrated approach of measuring soluble pro-fibrotic markers alongside quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing antifibrotic agents.

Preparation of Single Cell Suspension from Human Lung Tissue v1

2021 ◽  
Author(s):  
Steven B. Wells ◽  
Peter A. Szabo ◽  
Basak Ural ◽  
Maya M.L. Poon
Keyword(s):  
Epithelial Cells ◽  
Cell Suspension ◽  
Single Cell ◽  
Lung Tissue ◽  
Immune Cells ◽  
Human Lung ◽  
Dilution Factor ◽  
Single Cell Suspension ◽  
Human Lung Tissue ◽  
Defined Media

This protocol describes a method for the isolation of the immune cells, structural and epithelial cells, and progenitors from human lung sections of about two grams. By providing defined media formulations, volumes at each step, and a defined dilution factor for density centrifugation, it yields consistent single-cell suspensions across samples.

Host and viral determinants for efficient SARS-CoV-2 infection of the human lung

2020 ◽  
Author(s):  
Hin Chu ◽  
Bingjie Hu ◽  
Xiner Huang ◽  
Yue Chai ◽  
Yixin Wang ◽  
...  
Keyword(s):  
Human Lung ◽  
Ex Vivo ◽  
Sialic Acids ◽  
Lung Epithelial Cells ◽  
Human Lung Tissue ◽  
Human Lungs ◽  
Lung Epithelial ◽  
Viral Determinants ◽  
Attachment Factor ◽  
Human Lung Epithelial Cells

Abstract SARS-CoV-2 has affected over 9 million patients with more than 460,000 deaths in about 6 months. Understanding the factors that contribute to efficient SARS-CoV-2 infection of human cells, which are not previously reported, may provide insights on SARS-CoV-2 transmissibility and pathogenesis, and reveal targets of intervention. Here, we reported key host and viral determinants that were essential for efficient SARS-CoV-2 infection in the human lung. First, we identified heparan sulfate as an important attachment factor for SARS-CoV-2 infection. Second, we demonstrated that while cell surface sialic acids significantly restricted SARS-CoV infection, SARS-CoV-2 could largely overcome sialic acid-mediated restriction in both human lung epithelial cells and ex vivo human lung tissue explants. Third, we demonstrated that the inserted furin-like cleavage site in SARS-CoV-2 spike was required for efficient virus replication in human lung but not intestine tissues. Overall, these findings contributed to our understanding on efficient SARS-CoV-2 infection of human lungs.

scholarly journals Characterization of Early Stages of Human Alveolar Infection by the Q Fever AgentCoxiella burnetii

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Amanda L. Dragan ◽  
Richard C. Kurten ◽  
Daniel E. Voth
Keyword(s):  
Epithelial Cells ◽  
Lung Tissue ◽  
Alveolar Macrophages ◽  
Human Lung ◽  
Q Fever ◽  
Cell Types ◽  
Alveolar Epithelial Cells ◽  
Human Lung Tissue ◽  
Content Type ◽  
Alveolar Epithelial

ABSTRACTHuman Q fever is caused by the intracellular bacterial pathogenCoxiella burnetii. Q fever presents with acute flu-like and pulmonary symptoms or can progress to chronic, severe endocarditis. After human inhalation,C. burnetiiis engulfed by alveolar macrophages and transits through the phagolysosomal maturation pathway, resisting the acidic pH of lysosomes to form a parasitophorous vacuole (PV) in which to replicate. Previous studies showed thatC. burnetiireplicates efficiently in primary human alveolar macrophages (hAMs) inex vivohuman lung tissue. AlthoughC. burnetiireplicates in most cell typesin vitro, the pathogen does not grow in non-hAM cells of human lung tissue. In this study, we investigated the interaction betweenC. burnetiiand other pulmonary cell types apart from the lung environment.C. burnetiiformed a prototypical PV and replicated efficiently in human pulmonary fibroblasts and in airway, but not alveolar, epithelial cells. Atypical PV expansion in alveolar epithelial cells was attributed in part to defective recruitment of autophagy-related proteins. Further assessment of theC. burnetiigrowth niche showed that macrophages mounted a robust interleukin 8 (IL-8), neutrophil-attracting response toC. burnetiiand ultimately shifted to an M2-polarized phenotype characteristic of anti-inflammatory macrophages. Considering our findings together, this study provides further clarity on the uniqueC. burnetii-lung dynamic during early stages of human acute Q fever.

scholarly journals Silver Nanoparticles Alter Cell Viability Ex Vivo and in Vitro and Induce Proinflammatory Effects in Human Lung Fibroblasts

Nanomaterials ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1868
Author(s):  
Anna Löfdahl ◽  
Andreas Jern ◽  
Samuel Flyman ◽  
Monica Kåredal ◽  
Hanna L Karlsson ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Cell Viability ◽  
Human Lung ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Lung Fibroblasts ◽  
Human Lung Fibroblasts ◽  
Metabolic Activities ◽  
Precision Cut Lung Slices

Silver nanoparticles (AgNPs) are commonly used in commercial and medical applications. However, AgNPs may induce toxicity, extracellular matrix (ECM) changes and inflammatory responses. Fibroblasts are key players in remodeling processes and major producers of the ECM. The aims of this study were to explore the effect of AgNPs on cell viability, both ex vivo in murine precision cut lung slices (PCLS) and in vitro in human lung fibroblasts (HFL-1), and immunomodulatory responses in fibroblasts. PCLS and HFL-1 were exposed to AgNPs with different sizes, 10 nm and 75 nm, at concentrations 2 µg/mL and 10 μg/mL. Changes in synthesis of ECM proteins, growth factors and cytokines were analyzed in HFL-1. Ag10 and Ag75 affected cell viability, with significantly reduced metabolic activities at 10 μg/mL in both PCLS and HFL-1 after 48 h. AgNPs significantly increased procollagen I synthesis and release of IL-8, prostaglandin E2, RANTES and eotaxin, whereas reduced IL-6 release was observed in HFL-1 after 72 h. Our data indicate toxic effects of AgNP exposure on cell viability ex vivo and in vitro with altered procollagen and proinflammatory cytokine secretion in fibroblasts over time. Hence, careful characterizations of AgNPs are of importance, and future studies should include timepoints beyond 24 h.

scholarly journals Gene transfer to adult human lung tissue ex vivo

Gene Therapy ◽  
2000 ◽  
Vol 7 (8) ◽  
pp. 675-678 ◽  
Author(s):  
S McBride ◽  
D Rannie ◽  
D J Harrison
Keyword(s):  
Gene Transfer ◽  
Lung Tissue ◽  
Human Lung ◽  
Ex Vivo ◽  
Human Lung Tissue ◽  
Adult Human ◽  
Adult Human Lung

scholarly journals Highly selective and rapidly activatable fluorogenic Thrombin sensors and application in human lung tissue

2017 ◽  
Vol 15 (20) ◽  
pp. 4344-4350 ◽  
Author(s):  
Alicia Megia-Fernandez ◽  
Bethany Mills ◽  
Chesney Michels ◽  
Sunay V. Chankeshwara ◽  
Kevin Dhaliwal ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Human Lung ◽  
Ex Vivo ◽  
Human Lung Tissue ◽  
Fluorogenic Probe

A fast and selective fluorogenic probe for Thrombin is reported and applied in ex vivo fibrotic human lung tissue.

Measurement of histamine release from human lung tissue ex vivo by microdialysis technique

1998 ◽  
Vol 47 (12) ◽  
pp. 501-505 ◽  
Author(s):  
D. Nissen ◽  
L. J. Petersen ◽  
H. Nolte ◽  
H. Permin ◽  
N. Melchior ◽  
...  
Keyword(s):  
Histamine Release ◽  
Lung Tissue ◽  
Human Lung ◽  
Ex Vivo ◽  
Human Lung Tissue

scholarly journals Cross-talk between LAM cells and fibroblasts may influence alveolar epithelial cell behavior in Lymphangioleiomyomatosis

2021 ◽  
Author(s):  
Debbie Clements ◽  
Suzanne Miller ◽  
Roya Babaei-Jadidi ◽  
Mike Adam ◽  
S. Steven Potter ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cross Talk ◽  
Ex Vivo ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelial Cells ◽  
Dependent Manner ◽  
Epithelial Migration ◽  
Alveolar Epithelial

Lymphangioleiomyomatosis (LAM) is a female specific cystic lung disease in which TSC2 deficient LAM cells, LAM-Associated Fibroblasts (LAFs) and other cell types infiltrate the lungs. LAM lesions can be associated with type II alveolar epithelial cells (AT2 cells). We hypothesised that the behaviour of AT2 cells in LAM is influenced locally by LAFs. We tested this hypothesis in patient samples and in vitro. In human LAM lung, nodular AT2 cells show enhanced proliferation when compared to parenchymal AT2 cells, demonstrated by increased Ki67 expression. Further, nodular AT2 cells express proteins associated with epithelial activation in other disease states including Matrix Metalloproteinase 7, and Fibroblast Growth Factor 7 (FGF7). In vitro, LAF conditioned medium is mitogenic and positively chemotactic for epithelial cells, increases the rate of epithelial repair and protects against apoptosis. In vitro, LAM patient-derived TSC2 null cells cocultured with LAFs upregulate LAF expression of the epithelial chemokine and mitogen FGF7, which is a potential mediator of fibroblast-epithelial crosstalk, in an mTOR dependent manner. In a novel in vitro model of LAM, ex vivo cultured LAM lung-derived microtissues promote both epithelial migration and adhesion. Our findings suggest that AT2 cells in LAM display a proliferative, activated phenotype and that fibroblast accumulation following LAM cell infiltration into the parenchyma contributes to this change in AT2 cell behaviour. Fibroblast-derived FGF7 may contribute to the cross-talk between LAFs and hyperplastic epithelium in vivo, but does not appear to be the main driver of the effects of LAFs on epithelial cells in vitro.

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