Hematopoietic transcription factor mrna in the absence of hematopoietic cytokines yields ipsc-cd34 cells with increased t cell potential

Generation of T cells from pluripotent stem cells (PSCs) is a crucial aim of regenerative hematology. While there has been some success in generating T cells from iPSCs with a prearranged TCR, more feeder-free methods need to be established for iPSC lines derived from somatic origins. Previously using a dermal fibroblast-derived iPSC line, we reported a novel 2D monolayer differentiation protocol that incorporated transient expression of hematopoietic transcription factors ERG, HOXA5, HOXA9, HOXA10, and RUNX1 by mRNA delivery (TF-mRNA) at the hemogenic endothelial stage and resulted in the production of iPSC-CD34 cells with increased in vitro T cell potential. To further demonstrate the effectiveness of the TF-mRNA, we removed hematopoietic cytokines (VEGF, SCF, IL-3, IL-6, TPO, SR-1) from the iPSC-CD34 differentiation protocol and achieved 51% CD34+CD45+ expression in the TF-mRNA Minimal Cytokine condition compared to only 27% in the GFP-mRNA Minimal Cytokine condition. Remarkably, the TF-mRNA Minimal Cytokine condition resulted in increased expression of CD34+CD117+ cells compared to the TF-mRNA All Cytokine condition (51% vs 4%). Supernatant cells were collected from all four conditions and seeded into a feeder-free T cell differentiation culture. After 14 days, the amount of CD45+CD5+CD7+ T cell progenitors in both the All Cytokine and Minimal Cytokine conditions was 7x greater than GFP mRNA controls. Extended T cell differentiation resulted in 20% of the cells expressing CD4+CD8+CD3+TCRab+ for both TF-mRNA conditions compared to less than 2% for GFP-mRNA controls. While phenotype was similar, the TF-mRNA Minimal Cytokine produced nearly two-fold more cells than the TF-mRNA All Cytokine condition. These findings demonstrate the effectiveness of the hematopoietic TF-mRNA and provide a new method for the production of iPSC-CD34 cells with enhanced T cell potential. Generation of T cells from pluripotent stem cells (PSCs) is a crucial aim of regenerative hematology. While there has been some success in generating T cells from iPSCs with a prearranged TCR, more feeder-free methods need to be established for iPSC lines derived from somatic origins. Previously using a dermal fibroblast-derived iPSC line, we reported a novel 2D monolayer differentiation protocol that incorporated transient expression of hematopoietic transcription factors ERG, HOXA5, HOXA9, HOXA10, and RUNX1 by mRNA delivery (TF-mRNA) at the hemogenic endothelial stage and resulted in the production of iPSC-CD34 cells with increased in vitro T cell potential. To further demonstrate the effectiveness of the TF-mRNA, we removed hematopoietic cytokines (VEGF, SCF, IL-3, IL-6, TPO, SR-1) from the iPSC-CD34 differentiation protocol and achieved 51% CD34+CD45+ expression in the TF-mRNA Minimal Cytokine condition compared to only 27% in the GFP-mRNA Minimal Cytokine condition. Remarkably, the TF-mRNA Minimal Cytokine condition resulted in increased expression of CD34+CD117+ cells compared to the TF-mRNA All Cytokine condition (51% vs 4%). Supernatant cells were collected from all four conditions and seeded into a feeder-free T cell differentiation culture. After 14 days, the amount of CD45+CD5+CD7+ T cell progenitors in both the All Cytokine and Minimal Cytokine conditions was 7x greater than GFP mRNA controls. Extended T cell differentiation resulted in 20% of the cells expressing CD4+CD8+CD3+TCRab+ for both TF-mRNA conditions compared to less than 2% for GFP-mRNA controls. While phenotype was similar, the TF-mRNA Minimal Cytokine produced nearly two-fold more cells than the TF-mRNA All Cytokine condition. These findings demonstrate the effectiveness of the hematopoietic TF-mRNA and provide a new method for the production of iPSC-CD34 cells with enhanced T cell potential.