Pulmonary fibrosis (PF) is the shared central pathomechanism across multiple entities of interstitial lung diseases. Studying mechanisms of regeneration and fibrogenesis directly in human tissue has been hampered by the lack of organotypic models and analytical techniques.

Here, we coupled ex vivo cytokine and drug perturbations of human precision-cut lung slices (hPCLS) with single-cell RNA sequencing to study early lung fibrogenesis directly in human tissue.

Comparing the hPCLS perturbation data against an in vivo multi-cohort single-cell atlas from PF patients revealed the induction of a multi-lineage circuit of conserved fibrogenic cell states in hPCLS. Using micro-CT staged patient tissues, we characterized the appearance and interaction of CTHRC1+ myofibroblasts, an ectopic PLVAP+/VWA1+ endothelial cell state and KRT17+/KRT5- basaloid epithelial cells in the thickened alveolar septum of early-stage PF. Induction of these states in the ex vivo model provides evidence that the basaloid cell state originated from alveolar type-2 cells, whereas the ectopic endothelial cell state emerged from capillary cell plasticity. Cell-cell communication routes in patients were largely conserved in the hPCLS model, allowing us to identify established and novel drug-induced cell state changes and dissect drug-specific cellular communication patterns.

In summary, we provide a framework for perturbational single-cell genomics directly in human lung tissue that enables spatiotemporal analysis of early lung fibrogenesis. We further demonstrate that hPCLS offer novel avenues for high-resolution drug testing to accelerate anti-fibrotic drug development and translation.