Porcine iPSC-Derived Intestinal Cells as a High-Fidelity Platform for PEDV Pathogenesis

Porcine epidemic diarrhea virus (PEDV) continues to cause devastating economic losses in the swine industry. However, the lack of species-specific in vitro models and reliance on costly animal models hinders development of effective interventions. We established a high-fidelity platform using porcine induced pluripotent stem cells (piPSCs; line VSMUi001-C) via a 28-day stepwise differentiation protocol. Successful lineage commitment was validated by OCT4 downregulation and robust FOXA2/SOX17 expression at day 5. By day 28, the piPSC-derived intestinal epithelial cells (piPSC-IECs) exhibited organized LGR5+ niches, MUC2+ goblet cells, and Villin+ enterocytes, mirroring native tissue architecture. Barrier functionality was confirmed by the continuous distribution of the tight junction protein zonula occludens-1 (ZO-1). Our results demonstrate that piPSC-IECs are permissive to PEDV and support productive infection, encompassing the complete viral life cycle from entry to release of infectious progeny. Productive replication was confirmed by direct immunodetection of viral antigen within inoculated cells. Viral challenge induced characteristic cytopathic effects and a specific pro-inflammatory elevation of tumor necrosis factor-alpha (TNF-α). Notably, infection fragmented ZO-1, providing a mechanistic explanation for the leaky gut phenotype observed in vivo. This platform serves as a robust system for investigating viral kinetics and host–pathogen interactions with high species-specific relevance.