Single cell reconstruction of human basal cell diversity in normal and IPF lung (single-cell RNAseq)

Declining lung function in patients with interstitial lung disease is accompanied by epithelial remodeling and progressive scarring of the gas-exchange region. There is a need to better understand the contribution of basal cell hyperplasia and associated mucosecretory dysfunction to the development of idiopathic pulmonary fibrosis (IPF). Single cell RNA sequencing was used to map epithelial cell types of the normal and IPF human airway. Organoid and ALI cultures were used to investigate functional properties of basal cell subtypes. We confirmed that Notch2 maintains undifferentiated basal cells and restrict basal-to-ciliated differentiation, and present evidence that Notch3 functions to restrain secretory differentiation. When characterizing single cell transcriptomes of the IPF lung we found a bias towards accumulation of the secretory primed basal cell subset. Overall design: Single cell RNA-seq using 10x chromium system on isolated epithelial cells (DAPI-,CD45-,CD31-,CD326+) from donor trachea, and IPF patients lung explant tissue. Due to the sample processing timing, samples are processed either v2 or v3 three prime mRNA kit. Similarly, sample was aligned either with cellranger 2 or cellranger 3. Thus in detail, sample dd09, ipf01, ipf02, ifp04, ifp07 and ipf1883 were processed using cellranger2, others are processed using cellranger3.For cc05, trachea, extralobar bronchi and dissected proximal intra-lobar bronchi with minimal connective distal tissue. TotalSeq-A human hashing antibodies (BioLegend, 394605 and 394607) are used to label cells from trachea, extralobar bronchi (TotalSeq-A0253, 394605) and proximal intra-lobar bronchi (TotalSeq-A0254, 394607) followed standard protocol.