Optimizing in vitro T cell differentiation by using induced pluripotent stem cells with GFP‐RUNX1 and mCherry‐TCF7 labelling

Abstract In vitro T‐cell differentiation from pluripotent stem cells (PSCs) could potentially provide an unlimited source of T cells for cancer immunotherapy, which, however is still hindered by the inefficient obtaining functionally‐matured, terminally‐differentiated T cells. Here, we established a fluorescence reporter human induced pluripotent stem cell (iPSC) line termed TCF7 mCherry RUNX1 GFP , in which the endogenous expression of RUNX1 and TCF7 are illustrated by the GFP and mCherry fluorescence, respectively. Utilizing TCF7 mCherry RUNX1 GFP , we defined that the feeder cells incorporating CXCL12‐expressing OP9 cells with DL4‐expressing OP9 cells at a 1:3 ratio (OP9‐C1D3) significantly enhanced efficiency of CD8 + T cell differentiation from PSCs. Additionally, we engineered a chimeric antigen receptor (CAR) targeting EGFR into iPSCs. The CAR‐T cells differentiated from these iPSCs using OP9‐C1D3 feeders demonstrated effective cytotoxicity toward lung cancer cells. We anticipate this platform will help the in vitro HSPC and T cell differentiation optimization, serving the clinical demands of these cells.