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A high-yield isolation and enrichment strategy for human lung microvascular endothelial cells.

ABSTRACT: Vasculopathies, characterized by the formation of fragile and abnormal microvessels, are associated with the severity of many chronic lung diseases, including pulmonary fibrosis, emphysema/chronic obstructive pulmonary disease, systemic sclerosis, and hypertension. However, the study of human lung vasculature has been limited by the ability to isolate generous quantities of microvascular endothelial cells (MVEC) free from mesenchymal contamination. Expansion and passaging of primary human MVEC in vitro typically results in loss of a traditional phenotype in favor of an intermediate mesenchymal one, as early as passage five. Here we provide a detailed protocol for the selection of large quantities of enriched primary human lung MVEC based upon differential adherence from mesenchyme and simple magnetic separation, which decreases the need for excessive passaging, in order to obtain sufficient cell numbers to successfully freeze stock cultures. Additional protocols are provided for Ac-di-LDL selection, characterization, and a sandwich angiogenesis method of functional tube formation. The complete protocol including cell isolation and characterization takes approximately six weeks to complete.

SUBMITTER: Gaskill C

PROVIDER: S-EPMC5448535 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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A high-yield isolation and enrichment strategy for human lung microvascular endothelial cells.

Gaskill Christa C Majka Susan M SM

Pulmonary circulation 20170327 1

Vasculopathies, characterized by the formation of fragile and abnormal microvessels, are associated with the severity of many chronic lung diseases, including pulmonary fibrosis, emphysema/chronic obstructive pulmonary disease, systemic sclerosis, and hypertension. However, the study of human lung vasculature has been limited by the ability to isolate generous quantities of microvascular endothelial cells (MVEC) free from mesenchymal contamination. Expansion and passaging of primary human MVEC i ...[more]

PMID: 28680570

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Project description:Telocytes (TCs) are described as a particular type of cells of the interstitial space (www.telocytes.com). Their main characteristics are the very long telopodes with alternating podoms and podomers. Recently, we performed a comparative proteomic analysis of human lung TCs with fibroblasts, demonstrating that TCs are clearly a distinct cell type. Therefore, the present study aims to reinforce this idea by comparing lung TCs with endothelial cells (ECs), since TCs and ECs share immunopositivity for CD34. We applied isobaric tag for relative and absolute quantification (iTRAQ) combined with automated 2-D nano-ESI LC-MS/MS to analyse proteins extracted from TCs and ECs in primary cell cultures. In total, 1609 proteins were identified in cell cultures. 98 proteins (the 5th day), and 82 proteins (10th day) were confidently quantified (screened by two-sample t-test, P < 0.05) as up- or down-regulated (fold change >2). We found that in TCs there are 38 up-regulated proteins at the 5th day and 26 up-regulated proteins at the 10th day. Bioinformatics analysis using Panther revealed that the 38 proteins associated with TCs represented cellular functions such as intercellular communication (via vesicle mediated transport) and structure morphogenesis, being mainly cytoskeletal proteins and oxidoreductases. In addition, we found 60 up-regulated proteins in ECs e.g.: cell surface glycoprotein MUC18 (15.54-fold) and von Willebrand factor (5.74-fold). The 26 up-regulated proteins in TCs at 10th day, were also analysed and confirmed the same major cellular functions, while the 56 down-regulated proteins confirmed again their specificity for ECs. In conclusion, we report here the first extensive comparison of proteins from TCs and ECs using a quantitative proteomics approach. Our data show that TCs are completely different from ECs. Protein expression profile showed that TCs play specific roles in intercellular communication and intercellular signalling. Moreover, they might inhibit the oxidative stress and cellular ageing and may have pro-proliferative effects through the inhibition of apoptosis. The group of proteins identified in this study needs to be explored further for the role in pathogenesis of lung disease.

Dataset Information

A high-yield isolation and enrichment strategy for human lung microvascular endothelial cells.

Publications

A high-yield isolation and enrichment strategy for human lung microvascular endothelial cells.

Similar Datasets

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