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

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.

Fig. 1.

Detection and enrichment of pediatric lung cell populations by fluorescence-activated cell sorting. Schematic representation of the lung cell sorting strategy (A) and sorting template (B) are shown. 7-AAD, 7-aminoactinomycin D. A dead cell (7-AAD⁺) and erythrocyte (CD235a⁺) gate was determined on control heat-killed cells (green dots) to exclude these from the total cell populations (B, i and ii). CD45⁺ mixed immune cells (MICs) were collected from RBC-depleted live cell populations (Biii). The CD31⁺CD144⁺ endothelial cells (ENDs) were enriched from remaining CD45⁻ cells (Biv). From the leukocyte and endothelial cell depleted cells, EpCAM⁺ cells (EPI) were collected as epithelial cells (Bv). Residual live cells lacking any of the above membrane proteins were collected as nonendothelial mesenchymal cells (MESs). Cells unexposed to antibody (unstained, blue contours) are compared by overlay to antibody-incubated cells (red contour plots). In these presort plots, percentages of gated cell populations are provided as compared with total mixed cells. Viability and percentages of identified EPI, END, MES, and MIC populations were consistent in mixed-cell aliquots of the same donors, arranged left to right by age, sorted independently over time (C). CD, cluster of differentiation; EpCAM, epithelial cell adhesion molecule; D, donor; RBC, red blood cell; T1α, lung type I cell membrane-associated glycoprotein (podoplanin).

Fig. 2.

Flow cytometry on postsort samples confirm highly enriched four major subpopulations of dissociated lung cells. A: postsort density plots show enrichment of EpCAM⁺ (EPI), endothelial (END), nonendothelial mesenchymal (MES), and mixed immune (MIC) cells. B: bar graphs show mean postsort viability (closed bars) and purity (open bars) from multiple sorting experiments (n = 10, data shown as means ± SE). C: RNA integrity numbers (RINs) were >7 in all isolated-cell RNA. D: RNA yield (ng) per million cells is demonstrated. Significantly higher RNA yield was obtained from MICs compared with other cell types. C and D represent median, 95% of median and range; *P < 0.05 and **P < 0.01 by t-test, n = 11 per cell type. 7-AAD, 7-aminoactinomycin D; CD, cluster of differentiation; EpCAM, epithelial cell adhesion molecule; PMX, antibody-stained presort mixed cells; T1α, lung type I cell membrane-associated glycoprotein (podoplanin).

Fig. 3.

RNA sequencing (RNAseq) quality and purity. The total number of sequencing reads (A), proportion of uniquely mapped (B), total transcript count per sample (C), and proportion of the genome detected as expressed transcripts (D) for samples grouped by cell type. Normalized expression intensity of standard, relatively cell-specific marker transcripts for each of the four cell types is shown (E). Columns represent individual samples clustered by cell type. Each cell type is arranged in ascending order by age of donor (left to right). Expression patterns for individual genes are shown in rows. High expression is shown in red and low in green as indicated by the scale bar. END, endothelial cell; EPI, epithelial cell; MES, mesenchymal cell; MIC, mixed immune cell.