An iPSC-based in vitro model recapitulates human thymic epithelial development and multi-lineage specification.

Thymic epithelial cells (TEC) are crucial in supporting T cell development, but their high heterogeneity and difficulty of isolation pose obstacles to their study in humans. Particularly, how diverse TEC lineages arise from a common progenitor remains poorly understood. To address this, here we establish a human iPSC-based model of thymus organogenesis capable of deriving these lineages in vitro. Through controlled retinoid signaling followed by self-directed differentiation, we obtain FOXN1+ TEC progenitor-like cells and diverse mature MHCII+ populations resembling cortical and medullary TECs, allowing us to infer their developmental trajectories. Upon thymocyte co-culture, induced TECs support the generation of naïve T cells with diverse TCR repertoires and further develop into AIRE+ and mimetic TEC subpopulations. Our system provides a fully in vitro model of human TEC differentiation from early fate specification to late-stage maturation, offering new insights into human thymus development and potential regenerative applications for congenital thymic disorders.