Scalable generation of T lymphocytes from human pluripotent stem cells in suspended organoids

Human pluripotent stem cells (hPSCs) are a promising cell source for producing T cells for regenerative medicine and immunotherapy. However, it is challenging to develop a scalable suspension culture system for generating functional T lymphocytes from hPSCs. Here, we developed a Matrigel-based artificial thymic organoid (Gel-ATO) system by encapsulating hPSC-derived induced hematopoietic progenitor cells (iHPCs) with MS5-hDLL4 stromal cells. The resulting Gel-ATOs produced functional iT cells capable of antigen-specific cytotoxicity in vitro and tumor suppression in vivo. Building on this system, we further established a rotating suspension culture to robustly generate iT lymphocytes with nearly 20-fold enhancement of iT cells production compared to stationary suspension culture. Importantly, these rotating suspension culture-derived iT cells were functional, exhibiting cytokine secretion, proliferation, and cytotoxicity. By addressing the dual requirements of scalability and function, our work paves the way for future clinical production of T cells for T cell-based cell therapy.