SRP407809 PRJNA901444 GSE217945 Myeloid, B, and T lineage regeneration from pluripotent stem cells by defined transcription factors Generating prolonged multi-lineage hematopoiesis from pluripotent stem cells (PSCs), an unlimited cell source, is a crucial aim for regenerative hematology. In this study, we used a gene-edited PSC line and revealed that simultaneous expression of three transcription factors, Runx1, Hoxa9, and Hoxa10, drove robust emergence of induced hematopoietic progenitor cells (iHPCs). The iHPCs engrafted successfully in wild type animals and repopulated abundant and complete myeloid, B, and T lineage mature cells in primary and secondary C57BL/6 recipients. The regenerative multi-lineage hematopoiesis distributed normally in multiple organs, persisted over six months, and eventually declined over time with no leukemogenesis. Transcriptome characterization of regenerative myeloid, B, and T cells at the single cell resolution further projected their identities to natural cell counterparts. Thus, we provide strong genetic evidence that co-expression of exogenous Runx1, Hoxa9, and Hoxa10 simultaneously leads to long-term reconstitution of myeloid, B, and T lineages using PSC-derived iHPCs as cell source. Overall design: mRNA profiles of 76 single iHPCs. Single iHPCs were sorted from the iHPCs in vitro and were treated according to kit instructions of Discover-sc WTA Kit V2 (Vazyme) and TruePrepTM DNA Library Prep Kit V2 (Vazyme) GSE217945 pubmed 36801005