Article

A Novel Method for Isolating Individual Cellular Components from the Adult Human Distal Lung

Department of Advanced Preventive Medicine for Infectious Disease, Tohoku University Graduate School of Medicine, Aobaku, Sendai, Japan.
American Journal of Respiratory Cell and Molecular Biology (Impact Factor: 3.99). 10/2011; 46(4):422-30. DOI: 10.1165/rcmb.2011-0172OC
Source: PubMed

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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Available from: Hiroshi Kubo, Jan 21, 2016
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    • "Here, we mainly based our ATI-cell-like definition on the expression of T1α along with typical morphological changes in the AT phenotype over culture. Although other ATI cell markers have been described, including aquaporin 5, HOP homeobox (Hopx1) or receptor for advanced glycosylation end products (RAGE), T1α has been established as a robust ATI cell marker conserved over species (Flozak et al., 2010;Zhao et al., 2013;Marconett et al., 2013;Barkauskas et al., 2013;Treutlein et al., 2014), has been associated with an ATI cell phenotype in vivo (Yee et al., 2006), and, most importantly, this has been confirmed in human lung tissue (Fujino et al., 2012;Ghaedi et al., 2014;Barkauskas et al., 2013;Marconett et al., 2013). Although ATII cell plasticity and progenitor cell function is welldescribed in vitro and in vivo, data on ATI cell function and plasticity remain sparse. "

    Full-text · Article · Aug 2015 · Development
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    • "Here, we mainly based our ATI-cell-like definition on the expression of T1α along with typical morphological changes in the AT phenotype over culture. Although other ATI cell markers have been described, including aquaporin 5, HOP homeobox (Hopx1) or receptor for advanced glycosylation end products (RAGE), T1α has been established as a robust ATI cell marker conserved over species (Flozak et al., 2010;Zhao et al., 2013;Marconett et al., 2013;Barkauskas et al., 2013;Treutlein et al., 2014), has been associated with an ATI cell phenotype in vivo (Yee et al., 2006), and, most importantly, this has been confirmed in human lung tissue (Fujino et al., 2012;Ghaedi et al., 2014;Barkauskas et al., 2013;Marconett et al., 2013). Although ATII cell plasticity and progenitor cell function is welldescribed in vitro and in vivo, data on ATI cell function and plasticity remain sparse. "
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    ABSTRACT: The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI) and type II (ATII) cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis, and functional studies in primary murine ATII cells to identify novel proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2) and an increase in enolase 1 (ENO1) and protein disulfide isomerase associated 3 (PDIA3) protein expression during ATII to ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α, exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, while CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with SFTPC, whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified novel proteins involved in ATII to ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair. © 2015. Published by The Company of Biologists Ltd.
    Full-text · Article · May 2015 · Disease Models and Mechanisms
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    • "A previous study showing numerous differences in gene expression between freshly isolated rat AT1 cells and monolayers from primary rat AT2 cells cultured for 7 days to an “AT1-like” cell phenotype has led to an assumption that AT2 cells “de-differentiate” in vitro rather than transition into AT1 cells [20]. However, Fujino, et al., recently reported a FACS-based method for separating multiple viable cell populations from the adult human distal lung [47] using EpCAM, T1α, and VE-cadherin antibodies; immunofluorescence of the EpCAM+/T1α– subpopulation revealed this fraction to be nearly pure hAT2 cells, with 94% of cells expressing pro-SP-C. That the FACS-isolated hAT2 sub-population, cultured on collagen 1-coated slides for IF, continued to express pro-SP-C on Day 2 after seeding (the equivalent of our Day 1) and by Day 7 was expressing both AQP5 and T1α suggests that collagen 1 promotes transdifferentiation. "
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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.
    Full-text · Article · Apr 2014 · PLoS ONE
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