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

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^mCherryRUNX1^GFP, in which the endogenous expression of RUNX1 and TCF7 are illustrated by the GFP and mCherry fluorescence, respectively. Utilizing TCF7^mCherryRUNX1^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.