In Vitro and In Vivo Development of the Human Airway at Single-Cell Resolution

Abstract

Basal stem cells (basal cells), located in the bronchi and trachea of the human lung epithelium, play a critical role in normal airway homeostasis and repair, and have been implicated in the development of diseases such as cancer [1][2][3][4] . Additionally, basal-like cells contribute to alveolar regeneration and fibrosis following severe injury [5][6][7][8] . However, the developmental origin of basal cells in humans is unclear. Previous work has shown that specialized progenitor cells exist at the tips of epithelial tubes during lung branching morphogenesis, and in mice, give rise to all alveolar and airway lineages 9,10 . These 'bud tip progenitor cells' have also been described in the developing human lung [11][12][13] , but the mechanisms controlling bud tip differentiation into specific cell lineages, including basal cells, are unknown. Here, we interrogated the bud tip-to-basal cell transition using human tissue specimens, bud tip progenitor organoid cultures 11 , and single-cell transcriptomics. We used single-cell mRNA sequencing (scRNAseq) of developing human lung specimens from 15-21 weeks gestation to identify molecular signatures and cell states in the developing human airway epithelium. We then inferred differentiation trajectories during bud tip-to-airway differentiation, which revealed a previously undescribed transitional cell state ('hub progenitors') and implicated SMAD signaling as a regulator of the bud tip-to-basal cell transition. We used bud tip progenitor organoids to show that TGFβ1 and BMP4 mediated SMAD signaling robustly induced the transition into functional basal-like cells, and these in vitro-derived basal cells exhibited clonal expansion, self-renewal and multilineage differentiation. This work provides a framework for deducing and validating key regulators of cell fate decisions using single cell transcriptomics and human organoid models. Further, the identification of SMAD signaling as a critical regulator .

Differentiation of human intestinal organoids with endogenous vascular endothelial cells

“…Validation of the organ-specific EC genes and gene signatures identified by bulk RNAseq (Figure 3) was carried out using both scRNAseq and fluorescent in situ hybridization (FISH) (Figure 4). We used scRNAseq to profile human fetal lung (Miller et al, 2020), intestine (Czerwinski et al, 2020), and kidney across 7 specimens spanning 13.5-19 weeks of gestation (Figure S4A). In total, our analyses included 62,046 cells across these three organs (Figure 3A and S3A).…”

“…De-identified human fetal lung, intestine, and kidney tissue was obtained from the University of Washington Laboratory of Developmental Biology. Tissue was shipped overnight in Belzer-UW Cold Storage Solution (ThermoFisher, NC0952695) with cold packs, as previously published (Menon et al, 2018; Miller et al, 2020). A list of tissue specimens can be found in the key resources table.…”

Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells

“…3a). These included 6 published datasets [63][64][65][66][67][68] and 16 datasets that are not yet published [69][70][71][72][73] . In the case of unpublished data, we only obtained singlecell expression counts for the three genes, as well as the total UMI counts per cell, cell identity annotations, and the relevant anonymous clinical variables (age and sex, as well as smoking status when ascertained).…”

“…As reports suggest that most infants and young children cases do not display severe disease 11,74 , we inspected the subsets of studies of human development and pediatric samples from our integrated analysis (Supplementary Table 2). These included cells from 12 first trimester samples (8 donors) of fetal lung (8.5 weeks post conception; wpc), 29 fetal lung samples (26 donors) from the second trimester (13-24 weeks) 68 , 13 lung samples (10 donors) spanning from third trimester premature births (n=4), full term newborns (n=1), ~3-month old (n=4), 3-year-old (n=3), and 10-year-old (n=1) children. Because the number of samples here is small, all observations must be interpreted with caution.…”

Cell-Type-Specific Expression of Renin-Angiotensin-System Components in the Human Body and Its Relevance to SARS-CoV-2 Infection

“…The first step in the analysis was to generate averaged expression profiles for the individual cell types using scRNA-seq datasets. For this purpose, the metadata containing cell type assignments to the barcodes and the raw UMI (unique molecular barcodes) count matrices were obtained for various tissues: ileum, colon, and rectum 7 , and fetal lung 6 . For other tissues such as adult lung, esophagus, and spleen published R object containing metadata and raw counts were obtained 5 .…”