Lineage tracing of both quiescent G0 and active Hoxb5+ LT-HSCs that actively contribute to homeostatic mouse hematopoiesis.

Studying the lineage commitment and differentiation potential of long-term hematopoietic stem cells (LT-HSCs) is important to understand the dynamics of hematopoiesis. A central question concerns which hematopoietic stem and progenitor cell populations are responsible for sustaining steady-state hematopoiesis in vivo without conditioning. Noninvasive HSC fate-mapping strategies to address this question require specific labeling of LT-HSCs only. In this study, we selectively labeled a subset of Hoxb5+ LT-HSCs-excluding short-term HSCs (ST-HSCs) and multipotent progenitors (MPPs)-to track the progeny of these cells. Hoxb5+ LT-HSCs comprise ~1 in 100,000 bone marrow cells. MPPs were not labeled until several months post-induction, indicating their derivation from LT-HSCs. At no time were MPPs labeled and LT-HSCs not, consistent with the origin and maintenance of MPPs from LT-HSCs. Hoxb5+ LT-HSCs are the principal contributors to steady-state in situ hematopoiesis, but only a fraction of LT-HSCs were labeled by the Cre/LoxP conversion to a lineage-tracing color. We tested whether quiescent HSCs could have incised the DNA at loxp sites, but did not finish the rearrangement. Analysis of phosphorylated H2AX (γ-H2AX) revealed that quiescent LT-HSCs retain Cre/LoxP-induced DNA incisions, which are repaired upon cell cycle entry, leading to the appearance of newly labeled LT-HSCs at later time points, mainly of the myeloid-biased HSC. Moreover, most LT-HSCs exhibit marked expansion in response to hematopoietic stress. With the age-related shift of blood formation from balanced to myeloid biased, the myeloid-biased HSCs expand preferentially after 6 mo of tracking.