Here, we show that the Wnt5a -haploinsufficient niche regenerates dysfunctional HSCs, which do not successfully engraft in secondary recipients. RNA sequencing of the regenerated donor Lin − SCA-1 + KIT + (LSK) cells shows dysregulated expression of ZEB1-associated genes involved in the small GTPase-dependent actin polymerization pathway. Misexpression of DOCK2, WAVE2, and activation of CDC42 results in apolar F-actin localization, leading to defects in adhesion, migration and homing of HSCs regenerated in a Wnt5a -haploinsufficient microenvironment. Moreover, these cells show increased differentiation in vitro, with rapid loss of HSC-enriched LSK cells. Our study further shows that the Wnt5a -haploinsufficient environment similarly affects BCR-ABL p185 leukemia-initiating cells, which fail to generate leukemia in 42% of the studied recipients, or to transfer leukemia to secondary hosts. Thus, we show that WNT5A in the bone marrow niche is required to regenerate HSCs and leukemic cells with functional ability to rearrange the actin cytoskeleton and engraft successfully.

Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1–CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1–CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1–CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis.

Long-term hematopoietic stem cells (HSCs [LT-HSCs]) are well known to display unpredictable differences in their clonal expansion capacities after transplantation. Here, by analyzing the cellular output after transplantation of stem cells differing in surface expression levels of the Kit receptor, we show that LT-HSCs can be systematically subdivided into two subtypes with distinct reconstitution behavior. LT-HSCs expressing intermediate levels of Kit receptor (Kit int ) are quiescent in situ but proliferate extensively after transplantation and therefore repopulate large parts of the recipient’s hematopoietic system. In contrast, metabolically active Kit hi LT-HSCs display more limited expansion capacities and show reduced but robust levels of repopulation after transfer. Transplantation into secondary and tertiary recipient mice show maintenance of efficient repopulation capacities of Kit int but not of Kit hi LT-HSCs. Initiation of differentiation is marked by the transit from Kit int to Kit hi HSCs, both of which precede any other known stem cell population.