Fibrotic cocktail treated human precision lung slices replicate the cellular diversity of the IPF lung - 20/03/24

Pulmonary fibrosis (PF) is a chronic irreversible and deadly lung disease with extensive changes in lung cellular repertoire including the emergence of aberrant and ectopic cellular populations. Fibrotic cocktail treated human precision-cut lung slices (FC-PCLS) have been described as a novel ex vivo translational model of pulmonary fibrosis. We sought to determine whether FC-PCLS mimic the aberrant cellular repertoire seen in the IPF lung (Adams et al., Science Advances 2018).

PCLS were generated from donor lung explant and treated with or without fibrotic cocktail (FC), containing TGFb, TNFa, PDGF and LPA, for 5 days, as previously described 1. The PCLS were then harvested and snap frozen. Single nuclei suspensions were barcoded using the 10x Chromium Single Cell platform, cDNA libraries generated and sequenced on Illumina platform. The sequencing reads were aligned with human Genome using StarSolo and trimmed using cutadapt. All the samples were integrated together using reciprocal PCA. To build the heatmap we calculate the average mean expression of gene of interest within each cell type and across the two conditions, control or FC. We also estimated and compared the proportion of each cell types between the two experimental conditions.

In all, 160,000 single nuclei transcriptomes from 8 samples obtained from donors (4 PCLS per condition) were analyzed. All the major lung cell populations were identified on the UMAP, except for airway epithelial cells, adventitial fibroblasts and lymphoid immune cells. Across the epithelial and mesenchymal cellular populations, we identified aberrant basaloid cells, characterized by an enrichment of basal markers, EMT markers and senescent markers (Fig. 1A and B) as well as fibrotic fibroblasts characterized by an enrichment of COL1A1, COL3A1, CTHRC1. The proportions of these cells were significantly increased in FC-PCLS compared to control cocktail (Fig. 1B and C). FC-PCLS further exhibited a significant decreased of AT1 and AT2 cells proportions.

These results suggest that stimulation of PCLS, obtained from human control lung, by fibrotic cocktail mimics several of the features described in the IPF lung including the emergence of aberrant basaloid cells and CTHRC1 fibroblasts. We’re currently generating a time course of the same experiment to decipher the origin of these fibrotic cells and testing whether known fibrotic drugs can affect their emergence.