Qi Lei, Yudi Miao, Feng Zheng, Ruoqi Cheng, Yating Mo, Weifeng Lai, Zhengyang Zhou, Lejun Lv, Yuanyuan He, Huangfan Xie, Chenyi Dai, Yingfeng Zhang, Zixin Zhao, Mengxin Liu, Lemei Jia, Jun Xu, Cheng Li, Hongkui Deng, Chengyan Wang
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.