Transcriptional Characterization Suggests Accelerated Epithelial Differentiation in Trisomy 21 Lungs

Abstract

RationaleDown Syndrome (DS), from trisomy 21 (T21), is the most common human chromosomal anomaly in the United States. Although DS can affect many organ systems, lung and heart disease are the leading causes of death. We recently reported that structural and molecular changes originate at the late pseudoglandular/early canalicular stages of development in T21 lungs. The initial, most striking, observation was dilatation of the terminal airways/acinar tubules. Thus, our goal was to better understand cellular heterogeneity at the transcriptional level that could be contributing to this phenotypic anomaly.MethodsSingle cell RNA sequencing (scRNAseq) was used to generate transcriptomic profiles of individual human lung cells in tissue obtained from T21 (n=5) and non‐T21 (n=4) prenatal lungs. Clustering, cluster marker identification, differential expression analysis, and UMAP representation was performed in Seurat. Cell populations were annotated using Toppfun. Spatial differences in cellular phenotypes were examined using immunofluorescent staining (IF) and fluorescent in situ hybridization (FISH).ResultsAll the major cell lineages were identified in both the T21 and non‐T21 lungs, with mesenchymal cells representing the largest fraction (63%), followed by epithelial (16%), immune (13%), and endothelial (8%) cells. Each cluster displayed a relatively equal distribution of cells between T21 and non‐T21 (Figure 1). Having previously observed structural changes in the airways, we focused on re‐clustering the epithelial cell population. This resulted in 12 different epithelial sub‐clusters, with 4 distal bud clusters analyzed separately. We noted differentially expressed genes within each epithelial sub‐cluster between the T21 and non‐T21 samples. 332 genes were significantly different in T21 in one or more cell clusters at FDR < 0.05. SOX2 expression was significantly down in two of the distal bud clusters, which was confirmed by IF. Additionally, immunohistochemical staining showed a trend for an increase in the number of SCGB1A1 and FOXJ1 positive cells within the proximal airways of T21 lungs as compared to non‐T21. Simultaneously, multiple distal epithelial sub‐populations demonstrated significant increases in gene expression associated with surfactant synthesis in T21, with pathway analysis displaying the activation of surfactant metabolism. Our sequencing data demonstrated differentially increased SFTPC within the AT2 and distal bud populations, which was confirmed by FISH.ConclusionOur data demonstrate that T21 is associated with increased expression of markers of differentiation in the prenatal lung, which may be an indication of accelerated epithelial cell differentiation.