Ap2a2 Is Crucial for LT-HSC Quiescence

Despite the relative rarity of haematopoietic stem cells (HSCs) within the blood system, functional heterogeneity is paramount to their ability to sustain lifelong blood production. The quiescent HSC sits at the functional apex possessed with self-renewal properties and the greatest repopulation output. We previously identified the gene, Ap2a2 as an enhancer of HSC function and its protein as a potential cell fate determinant in HSC asymmetric cell divisions (Ting SB et al., Blood 2012). Mechanistically, we hypothesise Ap2a2 induces a state of HSC quiescence. Using the Tet-On histone H2B-GFP mouse model (Foudi et al., Nat Biotech 2009), we have shown Ap2a2 to be highly and differentially expressed in the predominantly, G0 dormant CD150⁺48^-LSK GFP^high as opposed to the more cycling GFP^low HSC subpopulation. Competitive transplantation of Ap2a2- versus empty vector-transduced H2B-GFP HSCs results in a three-fold increase of the CD150⁺48^-LSK GFP^high HSC subpopulation. To further confirm the importance of Ap2a2 in haematopoiesis, we have constructed Ap2a2-LacZ reporter and constitutive Ap2a2 knockout (KO) mouse lines. The Ap2a2 LacZ reporter with b-galactosidase flow cytometry staining of bone marrow subpopulations confirmed high endogenous Ap2a2 expression in the CD150⁺48^-LSK long-term (LT-) versus CD150^-48^-LSK short-term (ST-) repopulating HSCs. Interim analyses of the constitutive Ap2a2 KO mice have revealed two obvious phenotypes: 14% of Ap2a2-null mice termed "non-survivors" are smaller, paler with failure of fetal liver (FL) development and die between E18.5 and weaning, whilst the remaining 11% are adult viable "survivors". However, at E14.5, Ap2a2-null compared to Ap2a2-wild type fetal livers showed less absolute total FL cells but increased CD150⁺48^-LSM FL HSCs. This was quantitatively correlated via limiting dilution assay assessed at 16 weeks post-transplant with a two-fold increase in Ap2a2-null HSC numbers (1 in 78,917 versus 1 in 150,891, p=0.027). This suggests Ap2a2 has a role in FL HSC differentiation and/or fate with potential impairment of symmetrical versus asymmetrical HSC divisions currently being studied. When E14.5 FL cells were competitively transplanted, the Ap2a2-null HSC had impaired donor reconstitution function measured at 16 weeks post-transplant (19.8% versus 48.6%, p=0.015). Ap2a2-null versus wild-type E14.5 FL cells showed equivalent numbers of primary in vitro methylcellulose colony assays but loss of secondary colonies upon re-plating indicative of loss of in-vitro HSC self-renewal. Importantly, although the Ap2a2 adult "survivors" exhibited normal quantities of bone marrow HSC subpopulations, when functionally assessed, Ap2a2-null adult "survivor" HSCs showed loss of in-vivo HSC self-renewal in secondary transplantation assays. To investigate potential cellular mechanisms, we studied the cell cycle state of Ap2a2-null and wild-type E14.5 FL cells and identified that Ap2a2-null "non-survivors" had a relative loss of quiescent G0, specifically in the LT-HSC (and not seen in the ST-HSC) subpopulation throughout all of (E14.5 to E18.5) FL development. In contrast, the LT-HSC subpopulation in FLs of Ap2a2-null "survivors" had an initial loss of G0 at E14.5 but a compensatory increase in LT-HSC G0 by E18.5. Our preliminary data suggests Ap2a2 is a crucial factor for the quiescent LT-HSC subpopulation, and we propose that both during the highly proliferative fetal liver stage of haematopoiesis and adult HSCs under stress that Ap2a2 maintains a critical balance of dormant ("deep-sleeper") HSCs to ensure global HSC function.