TY  - JOUR
TI  - Generation of Functional Patient-Specific Thymus Organoids From Human Pluripotent Stem Cells (hPSCs) Using Air–Liquid Interface Culture
AU  - Ramos, Stephan A.
AU  - Russ, Holger A.
VL  - 16
IS  - 9
PY  - 2026
DA  - 2026/05/05
SP  - e5684
C1  - Bio-protocol 2026;16:e5684
DO  - 10.21769/BioProtoc.5684
UR  - https://doi.org/10.21769/BioProtoc.5684
AB  - The thymus is critical for the establishment of a functional and self-tolerant adaptive immune system, but it involutes with age, resulting in reduced naive T-cell output. Generation of a functional human thymus from human pluripotent stem cells (hPSCs) is an attractive regenerative medicine strategy. Direct differentiation of thymic epithelial progenitors (TEPs) from hPSCs has been demonstrated in vitro, but functional thymic epithelial cells (TECs) develop only after transplantation of TEPs in vivo. Functional human reaggregated thymic organoid cultures (RTOCs) and artificial thymic organoids (ATOs) cultured at the air–liquid interface support T-cell development in vitro and in vivo and permit the interrogation of human thymic function and T-cell development. However, these approaches require access to primary human tissues or murine bone marrow stromal cells, are allogeneic, and do not support negative selection. Recently, we reported the directed differentiation of induced PSCs (iPSCs) to functional thymic epithelial progenitors (TEPs) that support murine T-cell development after transplantation in nude mice. Here, we combined hPSC-derived TEPs, hematopoietic progenitor cells (HPCs), and mesenchymal cells, differentiated from the same hPSC line, and generated functional isogenic stem cell–derived thymic organoids (sTOs). Our revised protocol improves our TEP differentiation process and allows the generation of functional isogenic, patient-specific thymic organoids in vitro.
KW  - Stem cell–derived thymic organoids
KW  - Thymic epithelial cells
KW  - Human pluripotent stem cells
KW  - Human T-cell development
KW  - Positive and negative selection
KW  - Cell therapy
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False