Mesenchymal stromal cells from neonatal tracheal aspirates demonstrate a pattern of lung-specific gene expression

Abstract

We have previously isolated mesenchymal stromal cells (MSCs) from the tracheal aspirates of premature neonates with respiratory distress. Although isolation of MSCs correlates with the development of bronchopulmonary dysplasia, the physiologic role of these cells remains unclear. To address this, we further characterized the cells, focusing on the issues of gene expression, origin, and cytokine expression. Microarray comparison of early passage neonatal lung MSC gene expression to cord blood MSCs and human fetal and neonatal lung fibroblast lines demonstrated that the neonatal lung MSCs differentially expressed 971 gene probes compared with cord blood MSCs, including the transcription factors Tbx2, Tbx3, Wnt5a, FoxF1, and Gli2, each of which has been associated with lung development. Compared with lung fibroblasts, 710 gene probe transcripts were differentially expressed by the lung MSCs, including IL-6 and IL-8/CXCL8. Differential chemokine expression was confirmed by protein analysis. Further, neonatal lung MSCs exhibited a pattern of Hox gene expression distinct from cord blood MSCs but similar to human fetal lung fibroblasts, consistent with a lung origin. On the other hand, limiting dilution analysis showed that fetal lung fibroblasts form colonies at a significantly lower rate than MSCs, and fibroblasts failed to undergo differentiation along adipogenic, osteogenic, and chondrogenic lineages. In conclusion, MSCs isolated from neonatal tracheal aspirates demonstrate a pattern of lung-specific gene expression, are distinct from lung fibroblasts, and secrete pro-inflammatory cytokines.

FIG. 1.

Immunophenotypic analysis of mesenchymal stromal cells (MSCs) by flow cytometry. Cells were stained for CD73, CD90, CD105, and Stro-1, each of which identifies MSCs. CD34 is mildly positive, and the endothelial and hematopoietic cell markers CD31 and CD45 are negative. Control reactions were performed with irrelevant isotype IgG controls (gray lines). Typical results from individual subjects are shown. The percentage of positive cells is shown for each marker (mean±SD, n=3).

FIG. 2.

Concordance of gene probe detection P value; P≤0.05 between tracheal aspirate MSCs, cord blood MSCs, and fetal lung fibroblasts. Four MSC isolates, 3 fibroblast isolates, and 2 cord blood isolates were studied. Thin black lines signify 1.5-fold change based on average probe signal.

FIG. 3.

Neonatal lung MSCs express higher levels of lung-specific transcription factors than cord blood MSCs. (A) mRNA expression of FoxF1, HoxA2, HoxA4, HoxA5, HoxB5, Gli2, and Wnt5A was assessed by qPCR. Data were normalized to GAPDH. (B–H). Immunocytochemistry of neonatal lung MSCs, fetal lung fibroblasts, and cord blood MSCs. Cells were probed for Wnt5a (red), FoxF1 (green), and Tbx3 (blue). Colocalization is white. (B) Neonatal lung MSCs. (C) MRC-5 fetal lung fibroblasts. (D) Cord blood MSCs. (E–G) Individual channels for Wnt5a, FoxF1, and Tbx3. (H) MRC-5 fetal lung fibroblasts stained with labeled control IgGs.

FIG. 4.

Cytokine secretion by neonatal lung MSCs and fetal lung fibroblasts. Passage 3 neonatal lung MSCs secreted greater amounts of CXCL1/GRO-α, CXCL-8/IL-8, and IL-6 (n=9) compared with the 3 isolates of fetal/neonatal lung fibroblasts tested (*P<0.05, unpaired t-test). There was no statistical difference in the secretion of CCL2/MCP-1, hepatocyte growth factor, or vascular endothelial growth factor.

FIG. 5.

Differentiation capability of neonatal lung MSCs and fetal lung fibroblasts. Under adipogenic conditions, lipid droplets are revealed with staining by with oil red O (A–C). Osteogenic growth conditions create calcium deposits seen after staining with alizarin red (D–F). Chondrogenic differentiation promotes production of glycosaminoglycans, which are detected by staining with Alcian blue (G–I). Neonatal lung MSCs, but not fetal lung fibroblasts, differentiate along these lineages. We analyzed 3 MSC isolates, 3 fibroblast isolates, and 2 cord blood MSC isolates, and all yielded the same results.