Derivation of functional thymic epithelial organoid lines from adult murine thymus.

Thymic epithelial cells (TECs) orchestrate T cell development by imposing positive and negative selection on thymocytes. Current studies on TEC biology are hampered by the absence of long-term ex vivo culture platforms, while the cells driving TEC self-renewal remain to be identified. Here, we generate long-term (>2 years) expandable 3D TEC organoids from the adult mouse thymus. For further analysis, we generated single and double FoxN1-P2A-Clover, Aire-P2A-tdTomato, and Cldn4-P2A-tdTomato reporter lines by CRISPR knockin. Single-cell analyses of expanding clonal organoids reveal cells with bipotent stem/progenitor phenotypes. These clonal organoids can be induced to express Foxn1 and to generate functional cortical- and Aire -expressing medullary-like TECs upon RANK ligand + retinoic acid treatment. TEC organoids support T cell development from immature thymocytes in vitro as well as in vivo upon transplantation into athymic nude mice. This organoid-based platform allows in vitro study of TEC biology and offers a potential strategy for ex vivo T cell development. [Display omitted] • Adult thymus-derived 3D organoids exhibit long-term expansion capacity • Thymus organoids exhibit cortical and medullary epithelial cell differentiation • Thymus organoids support T cell development in vitro and in vivo Lim et al. establish a condition to generate 3D thymic epithelial cell (TEC) organoids from the adult murine thymus. The TEC organoids allow long-term expansion and differentiation to functional mTECs and cTECs in vitro. TEC organoids successfully support T cell development in vitro and in nude mice in vivo after transplantation. [ABSTRACT FROM AUTHOR]