Notch activation during early mesoderm induction modulates emergence of the T/NK cell lineage from human iPSCs

A robust method of producing mature T cells from iPSCs is needed to realize their therapeutic potential. NOTCH1 is known to be required for the production of hematopoietic progenitor cells with T cell potential in vivo. Here we identify a critical window during mesodermal differentiation when Notch activation robustly improves access to definitive hematopoietic progenitors with T/NK cell lineage potential. Low-density progenitors on either OP9-hDLL4 feeder cells or hDLL4-coated plates favored T cell maturation into TCRab⁺CD3⁺CD8⁺ cells that express expected T cell markers, upregulate activation markers, and proliferate in response to T cell stimulus. Single-cell RNAseq shows Notch activation yields a 6-fold increase in multi-potent hematopoietic progenitors that follow a developmental trajectory toward T cells with clear similarity to post-natal human thymocytes. We conclude that early mesodermal Notch activation during hematopoietic differentiation is a missing stimulus with broad implications for producing hematopoietic progenitors with definitive characteristics.