Abstract Rationale Mucormycosis, caused by Mucorales fungi, is a rapidly progressive and often fatal pulmonary infection in immunocompromised individuals. Mortality exceeds 50% and approaches 100% in disseminated disease. Current in vitro models use immortalized cancer-derived cell lines (e.g., A549), which lack the structural and regenerative features of native alveolar epithelium. To overcome this limitation, we developed a human induced pluripotent stem cell (hiPSC)-derived alveolar model containing either alveolar type 1 (AT1) or type 2 (AT2) cells cultured at the air-liquid interface (ALI), a physiologically relevant system not previously applied to mucormycosis research. Methods Differentiation was performed using well-established protocols. Epithelial identity was confirmed by flow cytometry for SFTPC (AT2) and AGER (AT1). Infection and injury assays, including 51Cr release, transepithelial electrical resistance (TEER), and immunostaining, were conducted in fully differentiated ALI cultures following apical infection with Rhizopus delemar. Proteomic profiling identified fungal-binding receptors. In immunosuppressed mice, RNA sequencing was performed at early (Day 4) and late (Day 8) infection to assess alveolar epithelial responses and altered cell populations. Results AT1 cells exhibited significant cytotoxicity by 12 hours post-infection, while AT2 cells showed delayed injury at 24 hours; in contrast, A549 cells displayed damage only after 36 hours. TEER declined within 6 hours in AT1 and 12 hours in AT2 ALI cultures, whereas A549 cells failed to establish measurable tight junctions. Proteomic profiling identified fungal-binding receptors unique to hiPSC-derived AT2 cells, including α-actinin and annexin A2 (absent in A549 cells). To examine structural integrity and repair dynamics, ZO-1 immunostaining revealed early tight-junction fragmentation beginning at 6 hours post-infection, accompanied by a sharp decline in Ki-67 proliferation marker expression by 1.5 hours and near-complete loss by 9 hours, indicating that barrier disruption was coupled with impaired epithelial repair. These findings were confirmed and recapitulated in an immunosuppressed murine model, where R. delemar infection caused persistent alveolar injury even after liposomal amphotericin B therapy. RNA sequencing revealed upregulation of genes associated with Damage-Associated Transient Progenitor (DATP) populations (Krt8, Lgals3, Cldn4) and basal-like cells (Krt17) at late infection (Day 8) only. Conclusion This hiPSC-derived ALI model captures early epithelial injury and regenerative failure observed in vivo, providing a physiologically relevant platform for studying host-fungal interactions and testing antifungal and regenerative therapies in pulmonary mucormycosis. The model identifies unique AT2-specific fungal-binding mechanisms and demonstrates the temporal dynamics of epithelial damage and repair, offering a novel tool for mechanistic and therapeutic studies. This abstract is funded by: CIRM (EDUC4-12837) and P01 (1P01AI186818-01)
Youssef et al. (Fri,) studied this question.