A novel method for isolating individual cellular components from the adult human distal lung.
ABSTRACT A variety of lung diseases, such as pulmonary emphysema and idiopathic pulmonary fibrosis, develop in the lung alveoli. Multiple cell types are localized in the alveoli, including epithelial, mesenchymal, and endothelial cells. These resident cells participate in the pathogenesis of lung disease in various ways. To elaborate clearly on the mechanisms of these pathologic processes, cell type-specific analyses of lung disease are required. However, no method exists for individually isolating the different types of cells found in the alveoli. We report on the development of a FACS-based method for the direct isolation of individual cell types from the adult human distal lung. We obtained human lung tissue from lung resections, and prepared single-cell suspension. After depleting CD45-positive cells, a combination of antibodies against epithelial cell adhesion molecule (EpCAM), T1α, and vascular endothelial (VE)-cadherin as used to delineate alveolar cell types. Alveolar Type II cells were highly purified in the EpCAM(hi)/T1α(-) subset, whereas the EpCAM(+)/T1α(-/low) subset contained a mixed epithelial population consisting of alveolar Type I and bronchiolar epithelial cells. The EpCAM(-)/T1α(-) subset included both microvascular endothelial and mesenchymal cells, and these were separated by immunoreactivity to VE-cadherin. Lymphatic endothelial cells existed in the EpCAM(-)/T1α(hi) subset. Isolated cells were viable, and further cell culture studies could be performed. These results suggest that this novel method enables the isolation of different cellular components from normal and diseased lungs, and is capable of elucidating phenotypes specific to certain alveolar cell types indicative of lung disease.
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ABSTRACT: Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality each year. There is a paucity of information regarding the mechanisms necessary for ARDS resolution. Foxp3(+) regulatory T cells (Foxp3(+) Treg cells) have been shown to be an important determinant of resolution in an experimental model of lung injury. We demonstrate that intratracheal delivery of endotoxin (lipopolysaccharide) elicits alveolar epithelial damage from which the epithelium undergoes proliferation and repair. Epithelial proliferation coincided with an increase in Foxp3(+) Treg cells in the lung during the course of resolution. To dissect the role that Foxp3(+) Treg cells exert on epithelial proliferation, we depleted Foxp3(+) Treg cells, which led to decreased alveolar epithelial proliferation and delayed lung injury recovery. Furthermore, antibody-mediated blockade of CD103, an integrin, which binds to epithelial expressed E-cadherin decreased Foxp3(+) Treg numbers and decreased rates of epithelial proliferation after injury. In a non-inflammatory model of regenerative alveologenesis, left lung pneumonectomy, we found that Foxp3(+) Treg cells enhanced epithelial proliferation. Moreover, Foxp3(+) Treg cells co-cultured with primary type II alveolar cells (AT2) directly increased AT2 cell proliferation in a CD103-dependent manner. These studies provide evidence of a new and integral role for Foxp3(+) Treg cells in repair of the lung epithelium.Mucosal Immunology advance online publication, 21 May 2014; doi:10.1038/mi.2014.33.Mucosal Immunology 05/2014; · 7.54 Impact Factor
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ABSTRACT: Genetically engineered mouse models of lung adenocarcinoma have proven invaluable for understanding mechanisms of tumorigenesis, therapy response, and drug resistance. However, mechanistic studies focused on studying these processes in tumor-bearing mouse lungs are confounded by the fact that, in most cases, relevant signaling pathways are analyzed in whole lung preparations, which are composed of a heterogeneous mixture of cells. Given our increasing knowledge about the roles played by different subpopulations of cells in the development of lung adenocarcinoma, separating the major cellular compartments of the tumor microenvironment is recommended to allow for a precise analysis of relevant pathways in each isolated cell type. In this study, we optimized magnetic and fluorescence-based isolation protocols to segregate lung epithelial (CD326/EpCAM positive), endothelial (CD31 positive), and immune cells (CD45 positive), with high purity, from the lungs of transgenic mice with mutant EGFR-induced lung adenocarcinomas. This approach, which can potentially be extended to additional lung adenocarcinoma models, enables delineation of the molecular features of individual cell types that can be used to gain insight into their roles in lung adenocarcinoma initiation, progression, and response to therapy.American Journal of Respiratory Cell and Molecular Biology 10/2014; · 4.15 Impact Factor
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ABSTRACT: It is generally accepted that the surfactant-producing pulmonary alveolar epithelial type II (AT2) cell acts as the progenitor of the type I (AT1) cell, but the regulatory mechanisms involved in this relationship remain the subject of active investigation. While previous studies have established a number of specific markers that are expressed during transdifferentiation from AT2 to AT1 cells, we hypothesized that additional, previously unrecognized, signaling pathways and relevant cellular functions are transcriptionally regulated at early stages of AT2 transition. In this study, a discovery-based gene expression profile analysis was undertaken of freshly isolated human AT2 (hAT2) cells grown on extracellular matrix (ECM) substrata known to either support (type I collagen) or retard (Matrigel) the early transdifferentiation process into hAT1-like cells over the first three days. Cell type-specific expression patterns analyzed by Illumina Human HT-12 BeadChip yielded over 300 genes that were up- or down-regulated. Candidate genes significantly induced or down-regulated during hAT2 transition to hAT1-like cells compared to non-transitioning hAT2 cells were identified. Major functional groups were also recognized, including those of signaling and cytoskeletal proteins as well as genes of unknown function. Expression of established signatures of hAT2 and hAT1 cells, such as surfactant proteins, caveolin-1, and channels and transporters, was confirmed. Selected novel genes further validated by qRT-PCR, protein expression analysis, and/or cellular localization included SPOCK2, PLEKHO1, SPRED1, RAB11FIP1, PTRF/CAVIN-1 and RAP1GAP. These results further demonstrate the utility of genome-wide analysis to identify relevant, novel cell type-specific signatures of early ECM-regulated alveolar epithelial transdifferentiation processes in vitro.PLoS ONE 04/2014; 9(4):e93413. · 3.53 Impact Factor