DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells

T cells show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to provide a renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains a significant challenge. Here, we report an efficient serum- and feeder-free system for differentiating human PSCs into hematopoietic progenitors and T cells. This fully-defined approach allowed us to study the impact of individual proteins on blood emergence and differentiation. Providing DLL4 and VCAM1 during the endothelial-to-hematopoietic transition enhanced downstream progenitor T cell output by ~80-fold. These two proteins synergised to activate notch signalling in nascent hematopoietic stem and progenitor cells and VCAM1 additionally promoted an inflammatory transcriptional program. We also established optimised media formulations that enabled efficient and chemically defined maturation of functional CD8aß+, CD4-, CD3+, TCRaß+ T cells with a diverse TCR repertoire. Overall design: CD34+ cells derived from IPSCs were plated on one of four coating conditions (Uncoated plates, VCAM1 coated, DLL4 coated, or DLL4+VCAM1 coated plates) and were differentiated for 5 days in EHT media. We collected non-adherent cells at Day 5 of this process from each of the conditions and analyzed using scRNAseq.