Myelo-lymphoid lineage restriction occurs in the human haematopoietic stem cell compartment before lymphoid-primed multipotent progenitors

Capturing where and how multipotency is lost is crucial to understand how blood formation is controlled. Blood lineage specification is currently thought to occur downstream of multipotent haematopoietic stem cells (HSC). Here we show that, in human, the first lineage restriction events occur within the CD19(-)CD34(+)CD38(-)CD45RA(-)CD49f(+)CD90(+) (49f(+)) HSC compartment to generate myelo-lymphoid committed cells with no erythroid differentiation capacity. At single-cell resolution, we observe a continuous but polarised organisation of the 49f(+) compartment, where transcriptional programmes and lineage potential progressively change along a gradient of opposing cell surface expression of CLEC9A and CD34. CLEC9A(hi)CD34(lo) cells contain long-term repopulating multipotent HSCs with slow quiescence exit kinetics, whereas CLEC9A(lo)CD34(hi) cells are restricted to myelo-lymphoid differentiation and display infrequent but durable repopulation capacity. We thus propose that human HSCs gradually transition to a discrete lymphoid-primed state, distinct from lymphoid-primed multipotent progenitors, representing the earliest entry point into lymphoid commitment.