Advancements in In Vitro Human Thymus Development Using Induced Pluripotent Stem Cells

Researchers led by Professor Yoko Hamazaki and Assistant Professor Yann Pretemer from Kyoto University's Department of Life Science Frontiers have successfully established an in vitro model that accurately simulates the development of human thymic epithelial cells (TECs) utilizing induced pluripotent stem (iPS) cells. Published in Nature Communications, this groundbreaking work offers significant insights into thymus organogenesis and immune system development.

The thymus is a vital organ responsible for educating T cells to distinguish between self and foreign antigens, a process orchestrated by TECs. However, understanding TEC heterogeneity and isolating these cells from human tissues—especially during embryonic stages—has been challenging. Traditional models like mouse systems and primary human TEC cultures face limitations due to species differences, limited tissue access, and short-term culture viability.

To overcome these obstacles, the research team developed a precise, chemically defined protocol guiding iPS cells through key developmental stages: anterior foregut endoderm, pharyngeal endoderm, and the third pharyngeal pouch, the embryonic origin of the thymus. By fine-tuning retinoic acid signaling pathways, they induced the formation of FOXN1-positive TEC progenitor-like cells capable of differentiating into various TEC subtypes, including cortical and medullary TECs essential for T cell selection and deletion of self-reactive T cells.

A FOXN1 fluorescent reporter system enabled real-time tracking of TEC differentiation processes. The induced TECs (iTECs) expressed critical functional markers such as IL7, DLL4, and MHC class II, forming distinct niches reminiscent of the thymic cortex and medulla. When co-cultured with human thymocytes, these iTECs supported the generation of naive CD4+ and CD8+ T cells with diverse TCR repertoires, overcoming previous hurdles faced with cell line-based approaches.

Moreover, this co-culture system facilitated the emergence of AIRE-positive cells and mimetic TECs that mimic extrathymic tissues like neurons and secretory cells, indicating the model's capability to replicate full TEC development. Single-cell RNA sequencing confirmed the transcriptional similarities between iTECs and primary pediatric TECs, validating the model's biological relevance.

Advanced analyses such as trajectory and RNA velocity studies uncovered how TEC subtypes diverge from a common progenitor, highlighting potential transcriptional regulators like ELF3, GRHL3, and EHF in TEC differentiation. The presence of mesenchymal-like cells suggests intricate epithelial-mesenchymal interactions that may support long-term TEC maintenance.

This fully in vitro system provides a robust platform for exploring human thymus development, studying congenital thymic disorders, and screening drugs aimed at thymic regeneration. It also paves the way for clinical applications, including generating patient-specific T cells and TECs for immune reconstitution in conditions such as DiGeorge syndrome and other thymic deficiencies, advancing the fields of immunology and regenerative medicine.