Emergence of Disease-relevant Aberrant Epithelial Cell States in Human Alveolar Organoids

Dysregulated alveolar epithelial repair is a central aspect of Idiopathic Pulmonary Fibrosis but it has proven challenging to reliably model alveolar epithelial cell biology in-vitro. We previously reported persistent activation of Hypoxia-inducible Factor 2 (HIF2) is a hallmark of aberrant epithelial cell phenotypes in IPF. We hypothesized that HIF2 activation primes AECs for aberrant differentiation. In these studies, we investigated how primary human alveolar epithelial organoids respond to a commonly-used commercial alveolar differentiation media intended to facilitate AT2->AT1 differentiation (ADM; which includes human serum and withdrawal of multiple growth factors/inhibitors) in isolation and following biased HIF activation. Alveolar organoids from donor lungs were expanded and transitioned to ADM with pharmacologic modulators to establish HIF-biased signaling. Multimodal analysis integrating scRNA-seq, quantitative label-free proteomics, and Cell Painting revealed that ADM induces expression of several AT1 markers, yet these conditions are insufficient to generate mature AT1-like cells but drives the emergence of KRT17+/KRT5- "aberrant basaloid" -like cells which demonstrate high transcriptional similarity to populations in end-stage IPF lungs (p = 3x10-286). HIF2-biased activation significantly exacerbated these aberrant transitions. Gene module co-expression analysis (hdWGCNA) linked ADM to cytoskeletal modulation and HIF2-biased signaling to metabolic shifts and cytoskeletal rearrangement. Proteomics reinforced protein-level induction of aberrant markers (e.g., GDF15) and revealed novel signatures, including increased complement production. Orthogonal transmission electron microscopy confirmed ultrastructural remodeling, including lysosomal inclusions and mitochondrial modulation. Collectively, these data establish that ADM models disease-emergent transitional states rather than homeostatic AT1 differentiation. These data further position HIF2 as a critical potentiator of aberrant alveolar epithelial cell states in PF.