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Am J Physiol Lung Cell Mol Physiol. 2018 Oct 1;315(4):L576-L583. doi: 10.1152/ajplung.00041.2018. Epub 2018 Jul 5.

Dissociation, cellular isolation, and initial molecular characterization of neonatal and pediatric human lung tissues.

Author information

1
Division of Neonatology, Department of Pediatrics, University of Rochester Medical Center , Rochester, New York.
2
Program in Pediatric Molecular and Personalized Medicine, Department of Pediatrics, University of Rochester Medical Center, Rochester, New York.
3
University of Rochester Genomics Research Center, University of Rochester Medical Center , Rochester, New York.
4
University of Rochester Flow Cytometry Core Facility, University of Rochester Medical Center , Rochester, New York.
5
University of Rochester Biocomputational Center, University of Rochester Medical Center , Rochester, New York.
6
Department of Pathology, University of Rochester Medical Center , Rochester, New York.
7
Department of Pathology, Seattle Children's Hospital, University of Washington , Seattle, Washington.
8
Division of Neonatology, Perinatal and Pulmonary Biology Cincinnati Children's Hospital Medical Center , Cincinnati, Ohio.

Abstract

Human lung morphogenesis begins by embryonic life and continues after birth into early childhood to form a complex organ with numerous morphologically and functionally distinct cell types. Pulmonary organogenesis involves dynamic changes in cell proliferation, differentiation, and migration of specialized cells derived from diverse embryonic lineages. Studying the molecular and cellular processes underlying formation of the fully functional lung requires isolating distinct pulmonary cell populations during development. We now report novel methods to isolate four major pulmonary cell populations from pediatric human lung simultaneously. Cells were dissociated by protease digestion of neonatal and pediatric lung and isolated on the basis of unique cell membrane protein expression patterns. Epithelial, endothelial, nonendothelial mesenchymal, and immune cells were enriched by fluorescence-activated cell sorting. Dead cells and erythrocytes were excluded by 7-aminoactinomycin D uptake and glycophorin-A (CD235a) expression, respectively. Leukocytes were identified by membrane CD45 (protein tyrosine phosphatase, receptor type C), endothelial cells by platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial cadherin (CD144), and both were isolated. Thereafter, epithelial cell adhesion molecule (CD326)-expressing cells were isolated from the endothelial- and immune cell-depleted population to enrich epithelial cells. Cells lacking these membrane markers were collected as "nonendothelial mesenchymal" cells. Quantitative RT-PCR and RNA sequencing analyses of population specific transcriptomes demonstrate the purity of the subpopulations of isolated cells. The method efficiently isolates major human lung cell populations that we announce are now available through the National Heart, Lung, and Blood Institute Lung Molecular Atlas Program (LungMAP) for their further study.

KEYWORDS:

fluorescence-based cell sorting; human lung cell markers; human pediatric lung cell populations

PMID:
29975103
PMCID:
PMC6230879
[Available on 2019-10-01]
DOI:
10.1152/ajplung.00041.2018

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