siRNA-mediated perturbations in human precision-cut lung slices coupled to time-resolved single-nuclei RNA-seq

Analyzing mechanisms of regeneration and fibrogenesis directly in human lung tissue has been hampered by the lack of organotypic models and analytical techniques. In previous work, we coupled ex vivo pro-fibrogenic cytokine treatments of peritumor-derived human precision-cut lung slices (hPCLS) with single-cell RNA-seq, revealing a multilineage circuit of fibrogenic cell states in hPCLS (Lang et al, Sci Transl Med. 2023 Dec 6). In this work, we aim to study the differentiation trajectories and their associated gene regulatory networks in lung fibrogenesis. We performed a time-resolved experiment in which peritumor-derived hPCLS were treated with pro-fibrogenic cytokines and collected at different time points (12, 24, 48, 72, and 144 hours after treatments) for profiling via multiplexed single-nuclei RNA-seq, yielding 100k single cell profiles. Using the time-resolved snRNA-seq data we delineate the cellular trajectories giving rise to fibrogenic cell states in the stromal, epithelial, endothelial, and immune cell lineages. For instance, we find that KRT17+/TP63+/KRT5- aberrant basaloid cells appear 48 hours after cytokine treatments. TP63 is upregulated in two distinct types of alveolar AT2 cells and the AT2 cellular program is gradually lost over time. We are currently dissecting this process of basaloid differentiation of AT2 cells using siRNA-mediated depletion of proteins in the basaloid differentiation trajectory and will show preliminary data at the conference. In summary, our work provides an experimental framework for scalable perturbational single-cell genomics directly in human lung tissue, enabling analysis of tissue homeostasis, regeneration, and pathology.