T cell development from expanded hematopoietic progenitors reveals progression control by Lmo2 , Erg , Spi1 , Hoxa9 , and Meis1

To gain access to the earliest stages of T cell development, we adapted a serum-free culture system that expands hematopoietic stem and progenitor-like cells. These expanded cells efficiently undergo normal T-cell differentiation in vivo and in vitro , verified by early gene expression trajectories from single-cell RNA sequencing, though their absolute differentiation speed is slower than that of fresh progenitors and can be modulated with cytokine priming. Leveraging this expansion system to observe the first T-lineage events, we revealed that initial Notch activation immediately induces chromatin opening and transcriptional activation of the TCR-Cβ locus. Additionally, acute CRISPR knockouts confirmed T-lineage entry requirements for Ikzf1 , Hes1 , Gabpa , and Myb while revealing that Lmo2 , Erg, Spi1, Hoxa9 , and Meis1 retard developmental progression with differing effects on proliferation. Endogenous expression of the stem, progenitor, and leukemia-associated factor Lmo2 markedly restrains initiation of the T cell program, with Lmo2 knockout greatly accelerating germline TCRβ locus transcription and expression of Tcf7, Gata3, Runx family, and E protein genes and their targets.