The thymus plays a central role in shaping the immune system by educating T cells to distinguish between self and non-self. TECs form the structural and functional core of this process, guiding T cell selection and maturation through complex interactions. However, TECs are highly heterogeneous and difficult to isolate from human tissue, especially during embryonic development, leaving many gaps in our understanding of thymus organogenesis. Existing models, including murine systems and primary human TECs, have provided valuable insights but are limited by species differences, tissue accessibility, and short-term culture viability.

To overcome these challenges, the research team established a chemically defined protocol to direct iPS cells through key developmental stages—anterior foregut endoderm, pharyngeal endoderm, and the third pharyngeal pouch, the embryonic origin of the thymus. By precisely modulating retinoic acid signaling, they induced FOXN1⁺ TEC progenitor-like cells capable of differentiating into diverse TEC subtypes. The use of a FOXN1 fluorescent reporter system allowed real-time tracking of TEC development and revealed the emergence of both cortical and medullary TEC-like populations, two crucial TEC subtypes responsible for selecting antigen-recognizing T cells and deleting self-reactive T cells, respectively.

These induced TECs (iTECs) expressed functional markers including IL7, DLL4, and MHC class II molecules, and formed spatially distinct niches resembling the thymic cortex and medulla. When co-cultured with human thymocytes, iTECs supported the generation of naïve CD4⁺ and CD8⁺ T cells with diverse T cell receptor (TCR) repertoires, which has been highly challenging with existing technologies such as delta-like ligand-expressing cell lines. Notably, this co-culture also facilitated the emergence of AIRE ⁺ cells and mimetic TECs that imitate extrathymic tissues such as neurons and secretory cells, indicating that iTECs are capable of faithfully recapitulating human TEC development up to terminal differentiation.

Single-cell RNA sequencing further confirmed that iTECs share transcriptional profiles with primary TECs from pediatric donors, validating the biological relevance of the model. Trajectory and RNA velocity analyses revealed how TEC subtypes diverge from a common progenitor, shedding light on the origins of TEC heterogeneity. The study also identified transcription factors such as ELF3, GRHL3, and EHF as potential regulators of mimetic TEC development. Moreover, mesenchymal-like cells present in the culture were found to contribute to epithelial-mesenchymal interactions, potentially supporting long-term TEC maintenance.

This new fully in vitro system offers a stable and reproducible platform for studying human thymus organogenesis, modeling congenital thymic disorders, and screening drugs that promote thymic regeneration. It also holds promise for clinical applications, such as generating patient-specific T cells or TECs for immune reconstitution in conditions like DiGeorge syndrome and other forms of athymia. By enabling the production of diverse TEC subtypes and functional T cells, the model provides a powerful tool for advancing both basic immunology and regenerative medicine.

induced TECs (iTECs)
(green: HLA-DR, red: FOXN1^mCherry, blue: DAPI)

• doi: 10.1038/s41467-025-62523-1