C70-33 NKX2-1-high Basal Cells With Hybrid Secretory Features Define a Progenitor Population for Distal Airway-specific Secretory Cells

Rationale

Lung epithelial development is patterned along the proximal-distal airway axis by region-specific transcriptional programs established during embryogenesis. Whether and how these programs are maintained in adult human airways to regulate epithelial identity, progenitor fate, and mucosal defense remain poorly defined. In the human distal airway epithelium, both basal cells and distal airway-specific secretory cells (DASCs), characterized by expression of SFTPB and SCGB3A2, have been proposed to function as epithelial progenitors. However, the upstream cellular origin of the DASC lineage has not been defined. Given the relevance of DASCs to muco-obstructive lung diseases, we sought to identify a specific progenitor population responsible for DASC differentiation.

Methods

Primary human large and small airway epithelial (LAE and SAE) progenitor cells were isolated from previously healthy transplant-rejected donor lungs, expanded by conditional reprogramming, and differentiated under air-liquid interface conditions. Basal cells and DASCs were isolated by fluorescence-activated cell sorting (FACS) using the basal cell marker NGFR in combination with a lentiviral EGFP reporter driven by the human SCGB3A2 promoter. Sorted populations were profiled by bulk RNA sequencing and assessed for progenitor function using 3D organoid assays with quantitative cell-type composition analyses. Spatial localization of candidate DASC progenitor populations was examined in human bronchiolar tissue sections.

Results

LAE cultures yielded NGFR⁺EGFP⁻ basal and NGFR⁻EGFP⁻ non-basal cells. In contrast, SAE cultures additionally contained NGFR⁻EGFP⁺ DASCs and a distinct NGFR⁺EGFP⁺ hybrid basal-DASC population. In organoid assays, NGFR+ populations exhibited robust clonogenic capacity, whereas DASCs exhibited limited proliferative potential, and NGFR⁻EGFP⁻ non-basal/DASC populations formed few colonies. Notably, EGFP+ DASCs arose most frequently from the SAE NGFR⁺EGFP⁺ hybrid basal-DASC population and less frequently from pre-existing DASCs, but were rare or absent in colonies derived from LAE or SAE NGFR⁺EGFP⁻ basal cells. Organoids derived from the hybrid basal-DASC population uniquely expressed SFTPB protein and showed enrichment of DASC marker genes, while all NGFR+ basal populations retained multilineage differentiation capacity, generating TP63+ basal, MUC5B+ secretory, and ciliated cells. This hybrid basal-DASC population exhibited elevated NKX2-1 expression and increased transcriptional activity predicted by regulatory network analysis of bulk RNA sequencing data. NKX2-1-high hybrid basal-DASC cells were identified in human distal bronchioles, with spatial distribution correlating to DASC abundance.

Conclusions

We identify a distal airway-specific NKX2-1-high basal cell population with hybrid basal-secretory features as a dedicated progenitor for DASCs. These findings define a previously unrecognized distal airway progenitor hierarchy and establish NKX2-1-dependent transcriptional programs as key regulators of distal airway epithelial differentiation.

This abstract is funded by: Cystic Fibrosis Foundation